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https://openalex.org/W3157696129
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https://www.nature.com/articles/s41598-021-94886-y.pdf
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English
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Does the Osseodensification Technique Allow for the use of a Healing Chamber with Primary Stability in Low-Density Bone? An in Vivo Study
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Research Square (Research Square)
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Osseodensification enables bone
healing chambers with improved
low‑density bone site primary
stability: an in vivo study
OPEN Rafael Coutinho Mello‑Machado1,2,11, Suelen Cristina Sartoretto3,4,5,11,
Jose Mauro Granjeiro5,6, José de Albuquerque Calasans‑Maia7,
Marcelo Jose Pinheiro Guedes de Uzeda3,5, Carlos Fernando de Almeida Barros Mourão5,
Bruna Ghiraldini8, Fabio Jose Barbosa Bezerra9, Plinio Mendes Senna10 &
Mônica Diuana Calasans‑Maia5* Primary implant stability is a prerequisite for successful implant osseointegration. The
osseodensification technique (OD) is a non-subtractive drilling technique that preserves the bone
tissue, increases osteotomy wall density, and improves the primary stability. This study aimed to
investigate the hypothesis that OD, through a wider osteotomy, produces healing chambers (HCs) at
the implant-bone interface without impacting low-density bone primary stability. Twenty implants
(3.5 × 10 mm) with a nanohydroxyapatite (nHA) surface were inserted in the ilium of ten sheep. Implant beds were prepared as follows: (i) 2.7-mm-wide using subtractive conventional drilling (SCD)
technique (n = 10); (ii) 3.8-mm-wide using an OD bur system (n = 10). The sheep were randomized to
two groups, with samples collected at either 14-(n = 5) or 28-days (n = 5) post-surgery and processed
for histological and histomorphometric evaluation of bone-implant contact (BIC) and bone area
fraction occupancy (BAFO). No significant group differences were found with respect to final insertion
torque and implant stability quotient (p > 0.050). BIC values were higher for SCD after 14 and 28 days
(p < 0.050); however, BAFO values were similar (p > 0.050). It was possible to conclude that the OD
technique allowed a wider implant bed preparation without prejudice on primary stability and bone
remodeling. Dental implants failure may be caused by local (low-density bone, compromised bone volume, and immediate
implant placement) and systemic factors (systemic diseases, titanium allergy and tobacco use)1–3. Remodeling
at the bone-implant interface requires the mechanical engagement of the dental implant with bone at the point
of insertion, clinically defined as primary stability4. Several factors may influence this primary stability such as
material biocompatibility, bone type and volume at the host site, loading conditions, surface technology (micro-
nano topography and chemical composition), macrogeometry (implant body and thread design), and surgical
preparation of the implant site5.h p p
p
The use of biomimetic surfaces was observed in the early 1990s, particularly with hydroxyapatite (HA)
coatings6, to improve the osteoconductive property of titanium for enhanced connection with the bone tissue7,8. However, a systematic review of clinical trials found similar long-term survival between HA-coated and uncoated 1Graduate Program, Dentistry School, Universidade Federal Fluminense, Niterói, Brazil. www.nature.com/scientificreports www.nature.com/scientificreports Osseodensification enables bone
healing chambers with improved
low‑density bone site primary
stability: an in vivo study
OPEN 2Implantology
Department, Universidade Iguaçu, Nova Iguaçu, RJ, Brazil. 3Oral Surgery Department, Universidade Iguaçu,
Nova Iguaçu, RJ, Brazil. 4Post‑Graduation Program in Dentistry, Universidade Veiga de Almeida, Rio de Janeiro,
RJ, Brazil. 5Clinical Research Laboratory, Dentistry School, Universidade Federal Fluminense, Rua Mario Santos
Braga, 28/4 Floor, Niterói, RJ, Brazil. 6National Institute of Metrology, Quality and Technology (INMETRO), Duque
de Caxias, RJ, Brazil. 7Orthodontics Department, Dentistry School, Universidade Federal Fluminense, Niterói, RJ,
Brazil. 8Dental Research Division, Dentistry School, Universidade Paulista, São Paulo, SP, Brazil. 9Laboratory of
Bioassays and Cell Dynamics, IBB-UNESP, Botucatu, São Paulo, Brazil. 10Implantology Department, UNIGRANRIO,
Duque de Caxias, RJ, Brazil. 11These authors contributed equally: Rafael Coutinho Mello-Machado and Suelen
Cristina Sartoretto. *email: monicacalasansmaia@gmail.com | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ titanium implants9. In fact, the use of plasma- sprayed HA coating may lead to implant failure caused by a rupture
of the HA-titanium interface, leading to increased bacterial adhesion, and peri-implantitis10. With the development of nanotechnology, biomimetic surfaces have migrated to the nanometric level. As a
result, the emergence of nanohydroxyapatite (nHA) coatings allow for the use of HA to induce a chemical bond
to the bone without the complications associated with plasma-sprayed11. Because bone tissue deposition on the
surface of implant devices is strongly dependent on cellular interactions with the surface12,13, the nHA coating
may accelerate osseointegration because it creates a hydrophilic surface with nanostructures-resembling the
extracellular matrix of the bone tissue with respect to the size, shape, and crystallinity— which provides more
substantial anchoring points at the surface for bone cells14,15.hi g p
The osseodensification (OD) concept was introduced to improve the primary stability of implants placed
in low-density bone sites16. OD is a non-excavation osteotomy preparation method. In contrast to traditional
standard drilling, OD compacts and auto-grafts bone in its plastic deformation phase17. It is a surgical instru-
mentation technique where the bone is compacted into open marrow spaces during drilling, increasing implant
insertion torque through the preservation and densification of osteotomy site walls16,18–23. Because more bone
particles will be present at the bone-implant interface when the implant bed is prepared, the use of OD maintains
and conserves bone density, creates more bone-implant contact, and accelerates bone healing, consequently,
enabling faster osseointegration21,24. g
g
Due to the potential of biomimetic surfaces, creating a space between the implant surface and the bone tissue
is recommended when drilling the implant bed as it facilitates the deposition of new bone at the interface25. This
space, referred to as a healing chamber (HD),is accomplished by using a final drill with a diameter larger than the
implant’s core diameter, but smaller diameter than the implant thread. Consequently, at the same time as the OD
removes the necrotic bone layer created by the surgical instrumentation, the created space allows coagulum to
accumulate at the interface, recruiting bone cells for faster bone formation26,27. www.nature.com/scientificreports/ However, the creation of an HC
reduces the bone-to-implant contact (BIC), causing lower primary stability; hence, this procedure is generally
not recommended for low-density bone.h y
The present study hypothesizes that the instrumentation when using the OD technique promotes a wider
implant bed in low-density bone, enabling primary stability for implants with nHA coatings without impaired
osseointegration. This study reports an in vivo biomechanical, histological, and histomorphometric analysis of
nHA-coated dental implants. Resultsh This study was conducted on the ilium of sheep to evaluate two instrumentation techniques for nano-sized HA
coatings dental implant installation. SEM micrographs of the implants surface showed a homogeneous topography of the implant surface at
50 ×, 1000 ×, 5000 ×, and 15,000 ×, and the energy-dispersive X-ray spectroscopy (EDS) showed the presence
of calcium (Fig. 1).ih In the present research, SCD and OD groups presented final IT values above 60 N-cm. The implant stab
quotient was above 76, and the median for both groups was 75. (Table 1).hi g
The histological analyses of non-decalcified sections allowed the assessment of the biological response to the
tested surgical techniques, the area of interest for BAFO and BIC evaluation was determined and drawn, from
the first thread of the implant to the fourth thread’s beginning (Fig. 2a). Both groups indicated peri-implant
bone regeneration. After 14 days of surgical procedure, the SCD group presented newly formed bone around the
implants’ threads demonstrating an evident bone–implant contact (Fig. 2b). The OD group presented a similar
reaction after 14 days, presenting new bone trabeculae islands surrounded by connective tissue permeating the
implant surface (Fig. 2d). After 28 days, in both groups, newly formed bone around the implants was clearly
apparent, and several areas of direct BIC were observed in a time-dependent fashion. The SCD presented exten-
sive remodeling around the implant with a larger area and advanced degree of bone maturity (Fig. 2c), when
compared to the previous period. The bone remodeling pattern in the OD group also presented more organized
and compact bone tissue showing larger trabeculae of newly formed bone compared to the first period (Fig. 2e). p
g
g
y
pi
p
g
A time-dependent increase of BIC and BAFO was observed in SCD (p = 0.002) and OD (p = 0.006) groups
(Fig. 3); however, no significant between-group differences were identified (p > 0.05). After 14 days of healing,
BIC values were 66.09% (CI: 49.80– 82.37) and 55.97% (CI: 43.63–68.31) for SCD and OD (Fig. 3a), respectively,
whereas BAFO yielded 47.96% (CI: 41.29–54.64) and 49.99% (CI: 41.52–58.46) (Fig. 3b). After 28 days of healing,
BIC values were 82.27% (CI: 78.08–86.47) and 74.30% (CI: 67.99–80.61) for SCD and OD (Fig. 3a), respectively,
whereas BAFO yielded 65.53% (CI: 57.80–73.27) and 61.76% (CI: 56.24–67.29) (Fig. | https://doi.org/10.1038/s41598-021-94886-y Scientific Reports | (2021) 11:15436 www.nature.com/scientificreports/ Resultsh 3b).hif y
g
The results of the between-group comparison revealed no statistically significant difference with respec
the amount of osteoid, woven bone, and vessels (Fig. 4a,b).t After the Shapiro–Wilk test, the groups were submitted to statistical analysis of One-way ANOVA and Tukey
post-test (p < 0.05) to evaluate the differences between groups and time points according to the presence of woven
bone, osteoid and connective tissue percentage. The woven bone increased from 14 to 28 days while the volume
of connective tissue decreased (p < 0.005). There was no difference between SCD and OD in the osteoid, wave
bone and connective tissue percentage (p > 0.05).h The neovascularization was evaluated using the scores system from ISO 10993-6/2016: no capillary prolifera-
tion (0); minimal capillary proliferation, focal 1–3 buds (1); groups of 4–7 capillaries with supporting fibroblastic
structure (2); broad band of capillaries with supporting structures (3); extensive band of capillaries with support-
ing fibroblastic structures (4). The values are presented as median ± confidence interval (n = 5). After Mann–Whit-
ney test, no statistical difference was observed between groups and periods after 14 and 28 days (p > 0.05). Scientific Reports | (2021) 11:15436 | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ (S
)
h
f
A
( )
S
l
h Figure 1. Scanning electron microscopy (SEM) micrographs of HAnano. (a) EDS results showing the
peak of Ca and P; (b) implant geometry at 50 × magnification (scale bar = 2 mm); (c) implant surface at
1000 × magnification (scale bar = 100 µm); (d) implant surface at 5000 × magnification (scale bar = 20 µm); (e)
implant surface at 15,000 × magnification (scale bar = 5 µm). Figure 1. Scanning electron microscopy (SEM) micrographs of HAnano. (a) EDS results showing the
peak of Ca and P; (b) implant geometry at 50 × magnification (scale bar = 2 mm); (c) implant surface at
1000 × magnification (scale bar = 100 µm); (d) implant surface at 5000 × magnification (scale bar = 20 µm); (e)
implant surface at 15,000 × magnification (scale bar = 5 µm). Table 1. Final insertion torque and implant stability quotient values for both surgical techniques: subtractive
conventional (SCD) and the osseodensification (OD) drilling groups. *Results represent average of five samples
calculated for each group for IT and implant stability in minimum, median, and maximum. Resultsh The values
are presented as mean ± confidence interval (n = 5). (b) The neovascularization was evaluated using the scores
system from ISO 10993-6/2016. There was no difference between SCD and OD in the osteoid, wave bone,
connective tissue percentage, and neovascularization (p > 0.05). www.nature.com/scientificreports/ ntificreports/
Figure 3. (a) The bone-to-implant contact (BIC) and (b) bone area fraction occupancy (BAFO) values of
implants installed after subtractive conventional drilling (SCD) and osseodensification drilling (OD). * and **
indicates significant difference between the evaluation period (p < 0.05). The data are presented as mean and
confidence interval (CI) at 95% of confidence. Figure 3. (a) The bone-to-implant contact (BIC) and (b) bone area fraction occupancy (BAFO) values of
implants installed after subtractive conventional drilling (SCD) and osseodensification drilling (OD). * and **
indicates significant difference between the evaluation period (p < 0.05). The data are presented as mean and
confidence interval (CI) at 95% of confidence. Figure 4. (a) Woven bone, osteoid and connective tissue percentage in the SCD and OD groups. The values
are presented as mean ± confidence interval (n = 5). (b) The neovascularization was evaluated using the scores
system from ISO 10993-6/2016. There was no difference between SCD and OD in the osteoid, wave bone,
connective tissue percentage, and neovascularization (p > 0.05). Figure 4. (a) Woven bone, osteoid and connective tissue percentage in the SCD and OD groups. The values
are presented as mean ± confidence interval (n = 5). (b) The neovascularization was evaluated using the scores
system from ISO 10993-6/2016. There was no difference between SCD and OD in the osteoid, wave bone,
connective tissue percentage, and neovascularization (p > 0.05). Figure 4. (a) Woven bone, osteoid and connective tissue percentage in the SCD and OD groups. The values
are presented as mean ± confidence interval (n = 5). (b) The neovascularization was evaluated using the scores
system from ISO 10993-6/2016. There was no difference between SCD and OD in the osteoid, wave bone,
connective tissue percentage, and neovascularization (p > 0.05). Resultsh Figure 5 shows the presence of osteoid matrix limited by osteoblasts; the different colors of osteoid matrix
(light blue) and osteoblast (blue navy) allowed the quantification of osteoid matrix in both groups, which showed
no differences between the groups.hh Figure 5 shows the presence of osteoid matrix limited by osteoblasts; the different colors of osteoid matrix
(light blue) and osteoblast (blue navy) allowed the quantification of osteoid matrix in both groups, which showed
no differences between the groups.hh f
The histological events at the HC are summarized in Fig. 6. The cascade of cellular events that occur between
the biological environment and the implant surface initially involves the presence of blood clotting with a thin
layer of serum protein, which progresses to granulation tissue, followed by immature woven bone. The bone
formation begins early, during the first week, through the promotion of osteoblast differentiation and the produc-
tion of osteogenic factors, cytokines, and growth factors. The primary bone that includes trabecular of woven
bone is substituted by parallel-fibered and/or lamellar bone and marrow. Between weeks 1 and 2, the bone tissue
responsible for primary mechanical stability of the implant, immediately lateral to the implant region, is resorbed,
and substituted by newly formed bone. After 4 weeks, secondary stability is established with a substantial number
of osteocytes, as illustrated in Fig. 6. Resultsh Procedure
Insertion torque (N/cm)*
Implant stability quotient (ISQ)*
Minimum
Median
Maximum
Minimum
Median
Maximum
SCD
60
70
80
69
75
76
OD
60
80
80
70
75
76 Procedure
Insertion torque (N/cm)*
Implant stability quotient (ISQ)*
Minimum
Median
Maximum
Minimum
Median
Maximum
SCD
60
70
80
69
75
76
OD
60
80
80
70
75
76 Table 1. Final insertion torque and implant stability quotient values for both surgical techniques: subtractive
conventional (SCD) and the osseodensification (OD) drilling groups. *Results represent average of five samples
calculated for each group for IT and implant stability in minimum, median, and maximum. Figure 2. Illustration of histomorphometry methodology. (a) the area of interest for BAFO and BIC evaluation
was determined from the first thread of the implant to the fourth thread’s beginning (dashed rectangle). The red
line delimitation was used to determine the BIC value, which was later transformed into a percentage. The bone
area fraction occupancy (BAFO) analysis was calculated after replication the design line of the implant profile
270 µm away from this profile. (b) SCD group after 14 days; (c) SCD group after 28 days; (d) OD group after
14 days, and (e) OD group after 28 days. Stain: Toluidine Blue and Acid Fuchsin stained. Scale bar: 100 μm. Figure 2. Illustration of histomorphometry methodology. (a) the area of interest for BAFO and BIC evaluation
was determined from the first thread of the implant to the fourth thread’s beginning (dashed rectangle). The red
line delimitation was used to determine the BIC value, which was later transformed into a percentage. The bone
area fraction occupancy (BAFO) analysis was calculated after replication the design line of the implant profile
270 µm away from this profile. (b) SCD group after 14 days; (c) SCD group after 28 days; (d) OD group after
14 days, and (e) OD group after 28 days. Stain: Toluidine Blue and Acid Fuchsin stained. Scale bar: 100 μm. https://doi.org/10.1038/s41598-021-94886-y Scientific Reports | (2021) 11:15436 | scientificreports/
Figure 3. (a) The bone-to-implant contact (BIC) and (b) bone area fraction occupancy (BAFO) values of
implants installed after subtractive conventional drilling (SCD) and osseodensification drilling (OD). * and **
indicates significant difference between the evaluation period (p < 0.05). The data are presented as mean and
confidence interval (CI) at 95% of confidence. Figure 4. (a) Woven bone, osteoid and connective tissue percentage in the SCD and OD groups. Discussionh The focus of the current study was to evaluate the primary stability and osseointegration of an nHA coated
implant inserted using OD with a wider implant bed; the use of SCD was chosen as control. The sheep ilium
model characterized by low-density bone was selected for its use in this study. The results revealed no between-
group differences with respect to BIC and BAFO values, confirming the null hypothesis of the study. Scientific Reports | (2021) 11:15436 | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ Figure 5. Representative photomicrographs of wound healing to different surgical drill techniques. The dashed
area is the magnification of the square, which allows the visualization of the vessels (yellow arrow), woven bone
(red asterisk), osteoblasts paving (red arrow) and below, the osteoid matrix (black asterisk). SCD (a, b) and OD
(c, d) 14 and 28 days after implantation, respectively. The area corresponds to the third most coronal thread. Stain: Toluidine Blue and Acid Fuchsin. Magnification: 20 × and 40 × ; Scale bar: 100 µm and 50 µm. Figure 5. Representative photomicrographs of wound healing to different surgical drill techniques. The dashed
area is the magnification of the square, which allows the visualization of the vessels (yellow arrow), woven bone
(red asterisk), osteoblasts paving (red arrow) and below, the osteoid matrix (black asterisk). SCD (a, b) and OD
(c, d) 14 and 28 days after implantation, respectively. The area corresponds to the third most coronal thread. Stain: Toluidine Blue and Acid Fuchsin. Magnification: 20 × and 40 × ; Scale bar: 100 µm and 50 µm. Figure 6. Graphical representation of timeline of osseointegration of dental implants concerning cellular events
from day 1 to 28 after implantation. It is possible to see the presence of a well-defined bone-implant interface
after 28 days of implantation. The cellular information of days 128 and 729 was based on previous studies. The
authors acknowledge Dr. Helder Valiense for the help in making the schematic figure. Figure 6. Graphical representation of timeline of osseointegration of dental implants concerning cellular events
from day 1 to 28 after implantation. It is possible to see the presence of a well-defined bone-implant interface
after 28 days of implantation. The cellular information of days 128 and 729 was based on previous studies. The
authors acknowledge Dr. Helder Valiense for the help in making the schematic figure. www.nature.com/scientificreports/ demonstrated in vivo study in dogs that milling with a final diameter 0.2 mm narrower than
the implant produces a bone–implant contact similar to subtractive conventional osteotomy, with a final diameter
0.8 mm smaller than the implant40. When SCD is compared to OD at same final diameter, previous studies, using
the same animal model, have shown that this technique improved the bone volume around dental implants19,49,50. In contrast, other studies did not find any healing impairment related to the instrumentation47. Unfortunately, in
the current study design, the BIC was not assessed at the time of implant insertion, which would have allowed
for assessment of the histological variation in 0 to 14 days of healing.i g
y
g
Following histomorphometric analysis of BAFO, the implant–profile design was then duplicated and aligned
at 270 µm in the long axis of implant, thus completing the total area of interest. This methodology was based on a
previous study that used 200 µm; in our study, 270 µm was used to enlarge the area of interest51,52. When BAFO
is observed, it is possible to note that a faster bone healing in the OD group again compensates for the larger
osteotomy. The bone interface was furthest from the implant surface in the OD group at the moment of implant
insertion, and after 14 days of bone healing, BAFO values were similar to the SCD group. In contrast, previous
data reported similar bone healing when using OD drills in subtractive (clockwise rotation) and densification
(counterclockwise rotation) modes47,49. Because a 3.8 mm-wide implantation bed without OD group was not
evaluated, it was not possible to conclude whether the faster healing would be a consequence of the HCs at the
interface, as proposed by other authors24,49, or because there was a denser bone–implant interface.tt After 28 days of healing, BIC and BAFO values were similar in both SCD and OD groups. After the same
period, a higher between-groups difference was reported in a previous study using a pig model, in which BIC
results were obtained for implants inserted in beds prepared with OD (62.5%) and compared to implants inserted
in beds prepared by the osteotome technique (31.4%) in the mandibular crest45. However, the initial interlocking
due to the implant geometry was higher in the present study, which can overlap the benefit of OD in improving
bone density at the interface. www.nature.com/scientificreports/ In the OD group, the final implant bed was 3.3 mm, whereas in the SCD group, it was 2.7 mm. The
wider implant bed in the OD group allowed for the creation of an HC.hii The first prerequisite for osseointegration is adequate final insertion torque and the implant’s primary
stability13,41. Both surgical techniques in the present study demonstrated adequate primary stability in the low-
density bone, reflecting the special design of the implant to boost the primary stability42. Comparing the surgical
techniques, similar IT and ISQ were identified for both groups, demonstrating that the densification of the inter-
face promoted by OD compensates the use of an implant–implant bed discrepancy of 0.6 mm. The improvement
of primary stability when using OD was also observed in previous studies16,21,26,37,43–46, regardless of the implant’s i The first prerequisite for osseointegration is adequate final insertion torque and the implant’s primary
stability13,41. Both surgical techniques in the present study demonstrated adequate primary stability in the low-
density bone, reflecting the special design of the implant to boost the primary stability42. Comparing the surgical
techniques, similar IT and ISQ were identified for both groups, demonstrating that the densification of the inter-
face promoted by OD compensates the use of an implant–implant bed discrepancy of 0.6 mm. The improvement
of primary stability when using OD was also observed in previous studies16,21,26,37,43–46, regardless of the implant’s
design or the implant surface20,22,47. Nevertheless, previous studies demonstrated that the densification of the
bone interface does not guarantee adequate primary stability ex-vivo18 and in vivo48 because of high interfacial
stresses that caused fractures and triggered a prolonged period of bone resorption. However, it was the first
time that such a small implant–implant bed difference had been evaluated, and no fibrous tissue formation was
identified at the bone–implant interface.f design or the implant surface20,22,47. Nevertheless, previous studies demonstrated that the densification of the
bone interface does not guarantee adequate primary stability ex-vivo18 and in vivo48 because of high interfacial
stresses that caused fractures and triggered a prolonged period of bone resorption. However, it was the first
time that such a small implant–implant bed difference had been evaluated, and no fibrous tissue formation was
identified at the bone–implant interface.f i
As expected for a wider implantation bed, BIC results at 14 days indicated no statistical differences. In con-
trast, Pantani et al. www.nature.com/scientificreports/ involving dental implants, an absence of postoperative morbidity36 and no need to euthanize the animals38. Once
the bone quality was characterized (type IV), the anatomical region in the jaws was not considered because the
presence of low-quality bone may be related to the individual physiology of each patient and not to the anatomi-
cal position in the jaws. involving dental implants, an absence of postoperative morbidity36 and no need to euthanize the animals38. Once
the bone quality was characterized (type IV), the anatomical region in the jaws was not considered because the
presence of low-quality bone may be related to the individual physiology of each patient and not to the anatomi-
cal position in the jaws. p
j
All previous animal studies aimed to investigate the bone–implant contact and bone area fraction occupancy
at different time points and macro/micro geometries. Furthermore, two clinical trials4,39 aimed to compare
the insertion torque and implant stability of implants placed via OD and SCD. The present study evaluated for
the first-time histological events around nanohydroxyapatite dental implant coating using different drilling
techniques.h q
The clinical approach for the installation of dental implants in low-density bone, such as the posterior region
of the maxilla, typically consists of an underprepared implantation site to improve the implant’s primary stability
so that osseointegration can proceed. On the other hand, implants presenting biomimetic surfaces, which can
enhance the bone healing process, may benefited when HCs are present at the surface40. Furthermore, when
the osteotomy is performed to allow the presence of the HCs at the bone–implant interface, it reduces the BIC. However, there is a gap of evidence concerning the ability of the biomimetic surfaces to overcome the inadequate
primary stability induced by the production of the HCs. Hence, it is worth exploring whether a hydrophilic sur-
face with nanostructures and associated with a wider osteotomy technique, producing HCs at the implant–bone
interface, would produce sufficient bone to provide satisfactory initial implant stability.h fi
Therefore, the present study was designed to verify whether the OD technique could be a viable approach
for standardization of the HC in low-density bone when using dental implants with a biomimetic surface. The
implants used in this study have a bioactive surface with nanostructured hydroxyapatite and the same macro-
geometry, diameter, and length; therefore, the only difference between the groups was the instrumentation
technique. Discussionh Whereas bone healing in many animal species is recognized to be faster than in humans, sheep are considered
to have a bone healing rate similar to humans30 and have been previously established as useful models for human
bone turnover and remodeling activity31. Sheep present advantages, compared with other experimental models,
for evaluating orthopedic and dental implant systems, such as acceptance by the animal research society and
easy management32,33, similarity to humans with respect to body weight34, composition, metabolism, and bone
remodeling and regeneration time32,35. Furthermore they have bones with dimensions suitable for the deployment
of implant systems and devices of bone fixation that are designed for humans32, and enable evaluation of up to
12 implants (in the final size for marketing) per animal34. Early osseointegration is still considered a challenge in
areas with the most trabeculated bone (bone type IV). The sheep ilium was used as the animal model because this
region is considered to be a low-density bone21,36,37, it had already been used in other histomorphometric studies https://doi.org/10.1038/s41598-021-94886-y Scientific Reports | (2021) 11:15436 | www.nature.com/scientificreports/ Methods
D
l i Dental implants and scanning electron microscopy. This study used 20 titanium dental implants
(3.5 mm in diameter and 10 mm in length) with a nano-sized crystalline nHA coating (Epikut Plus, S.I.N. Implant System, Sao Paulo, SP, Brazil). High-resolution scanning electron microscopy (SEM) images obtained
with an FEI-Quanta 450 (Thermo Fisher Scientific, Waltham, MA, USA) revealed the surface topography of the
implants, at an accelerating voltage of 10 kV; focal width of 3.0; and magnifications of 50 ×, 1000 ×, 5000 ×, and
15,000 ×. Energy-dispersive X-ray spectroscopy analysis determined each surface’s chemical composition at an
acceleration voltage of 20 kV and focal width of 40 using an EDAX detector equipped with a dual-beam elec-
tron microscope (AMETEK Materials Analysis Division, Mahwah, NJ, USA) and the Genesis software program
(EDAX, LLC, Mahwah, NJ, USA). Animal model. This in vivo study was approved by the Institutional Animal Care and Use Committee from
Federal Fluminense University (protocol # 9531061119) following the Animal Research: Reporting of In Vivo
Experiments (ARRIVE) and Planning Research and Experimental Procedures on Animals: Recommendations
for Excellence (PREPARE) guidelines57,58. The animals were kept and operated at the Federal Fluminense Uni-
versity Farm School and were accompanied by a veterinarian with more than 20 years of experience. All experi-
ments were performed between March and July of 2020.h p
y
The sample size was calculated using a priori power analysis based on the results from a previous study, which
evaluated BIC in the same experimental animal model, to estimate the effect size36. Considering a type 1 error
of 0.05 and power of 0.95, the two-tailed t-test determined a sample size of 5 implants per group/time point. In
compliance with the reduction, refinement and replacement program59, the animals were also used for another
study36. None of the animals were euthanized after the end of the present study. yt
p
y
Ten adult female Santa Ines sheep aged 2–4 years, with an average body weight of 37.05 kg (range 31–42 kg),
were randomly allocated using the coin-toss method into two experimental periods (14 or 28 days of healing). Each animal received two implants (one for each group) in the ilium, a low-density bone, with a wide bone
area that simultaneously allowed the installation of multiple implants. The bone blocks can be collected without
any morbidity for the animals in terms of locomotion and health. Methods
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l i The implant position was randomly defined
using the sealed envelope method, a surgical map of implant positioning inserted in the selected animal, ensuring
a similar distribution into bone tissue for both groups. The inter-implant distance was at least 5 mm.t h
Before beginning the study, all animals presented good general health and physical condition after clinical
examination by an experienced veterinarian. In the preoperative period, the animals received food composed of
the pastures and, during the postoperative period, in addition to the aforementioned pastures, nutritional sup-
plementation appropriate for sheep. Salt mineral water ad libitum was available during the entire experimental
period. The animals were transferred from the field to the research center two weeks before the surgeries to avoid
stress. The animals fasted for eight hours before the surgery. Surgical procedure and implants installation. The animals were given 0.05 mg/kg of aceproma-
zine intravenously (Acepran; Vetnil, Louveira, Sao Paulo, SP, Brazil) and 0.2 mg/kg of diazepam intravenously
(Diazepan; Teuto, Anapolis, GO, Brazil), as well as 0.4 mg/kg of morphine intramuscularly (Dimorf; Cristalia,
Itapira, Sao Paulo, SP, Brazil) for premedication. After orotracheal intubation and ventilation, 4 mg/kg of propo-
fol intravenously (Propofol Baxter; Baxter Hospitalar LTDA; São Paulo, SP, Brazil) was provided and sustained
using 1% isoflurane (Cristalia, Itapira, SP, Brazil). Meanwhile, 4 mg/kg of lidocaine (Xylestesin; Cristalia, Itapira,
SP, Brazil) and 0.1 mg/kg of morphine (Dimorf; Cristalia, Itapira, SP, Brazil) were used for epidural. block. The
edges of the iliac crests were exposed through a horizontal skin incision of 5 cm in length. The skin and fascial
layers were opened separately using a scalpel handle no. 3 (Bard Parker; Aspen Surgical, Caledonia, MI, USA)
and blade no. 15 (Solidor; Lamedid, Osasco, Sao Paulo, SP, Brazil).f Two different instrumentation techniques for the preparation of the implantation bed were used: control
group, subtractive conventional drilling (SCD) according to the implant manufacturer instructions for low-
density bone (lance bur, 2.0 and 2.7 mm diameter tapered burs); and experimental group, OD drilling using
multi-fluted tapered burs (2.0 mm pilot, 2.5, 3.0, and 3.3 conical burs) (Densah Bur; Versah, Jackson, MI, USA),
with a final diameter larger than the implant core diameter. Drilling was performed with clockwise rotation for
SCD and counterclockwise rotation for OD group at 1200 rpm under saline irrigation for both groups. Drilling
for both groups was performed by the same operator (M.D.C-M.). www.nature.com/scientificreports/ After 28 days, it was possible to observe the secondary bone and connective tissue
around the implants; hence, the longest experimental period in this study was 28 days. p
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According to Trisi et al.53, immediate implant loading can be recommended when IT is at least 45 N-cm,
and ISQ is at least 6853,54. Here the median for IT was higher than 70 N-cm and the ISQ was 75 for both groups,
allowing for both instrumentation techniques for the immediate implant loading.h The OD concept can be applied in clinical practice in situations such as promoting bone ridge expansion with
enhanced primary stability and higher insertion torque values, minimizing implant dehiscence, fenestrations, and
can be used for crestal sinus lift in a simple, safe, and predictable way with reduced morbidity55. Previous clinical
studies have demonstrated improved of osseointegration56 and higher IT and RFA17,55,56. The present pre-clinical
study adds new evidence through a histomorphometric analysis of the bone-implant interface occurring after
14 and 28 days of healing, quantifying the osteoid, woven bone, connective tissue, and vascular neoformation. Scientific Reports | (2021) 11:15436 | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ Altogether, the results improve understanding of the osseointegration process through two different instrumenta-
tion techniques on a bioactive nano-hydroxyapatite surface.i Despite this study finding that a wider implant bed using OD is a viable approach in low-density bone, it is
important to highlight that only one implant geometry was used. Therefore, the extrapolation of the results of
the present study to other implant systems should be done with care. Although the ability of the biomimetic
nHA implant surface together with the HC allows for adequate primary stability, extrapolation of the current
results to other implant surfaces should be done with caution and long-term analyses are required to better
understand the effect of OD with another implant surface on implant survival. Long-term analyses for assessing
bone saucerization as a function of the osteotomy technique are strongly recommended. Within the limitations
of this study, it can be concluded that OD technique performed with a wider surgical bed provided comparable
levels of initial implant stability, BIC, and BAFO to the conventional subtractive under-drilling procedure without
impairing the osseointegration. Methods
D
l i The dehydration of samples in ascending alcohol solutions of 60%, 70%, 90%, and
100% was performed under agitation and was subsequently infiltrated through daily changes of ascending grades
of alcohol/resin (Technovit 7200 VLC; Kultzer, Wehrheim, Hesse, Germany): 70/30, 50/50, 30/70, and 100%
resin. Thereafter, embedding of specimens into resin (Technovit 7200; Kulzer, Wehrheim, Hesse, Germany) was
performed using a light polymerization unit (EXAKT 520; Exakt System, Norderstedt, Hamburg, Germany) in 2
steps by different wavelengths (white/blue light) 8 h for each light. The bone blocks were cut in the mid-axial and
apical-coronal planes using a macro-scale cutting and grinding technique (Exakt 310 CP series; Exakt System,
Norderstedt, Hamburg, Germany). The obtained slices were ground and polished to a final thickness of 30 to 40
µm38. Finally, the slices were stained with toluidine blue to differentiate newly formed bone, and acid fuchsin was
used to contrast the background. Light microscopy at 10 × and 20 × magnifications (Olympus BX43; Olympus
Corporation, Tokyo, Japan) supported the analysis of the slices, with images acquired using Olympus Cellsens
(cellSens software; Olympus Corporation, Tokyo, Japan).hh t
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The histomorphometric analysis was conducted from reconstructions of the implant and adjacent bone. These
images were obtained from captured photomicrographs with 10 × magnification in sequenced fields to scan and
reconstruct. After the reconstruction of all images, the area of interest for BAFO evaluation was determined and
drawn, from the first thread of the implant to the fourth thread’s beginning. This line delimitation was used to
determine the BIC value, which was later transformed into a percentage. The implant-profile design was then
duplicated and aligned at 270 µm in the long axis of the implant, thus completing the total area of interest. Image
J software (National Institutes of Health, Bethesda, MD, USA) manually determined the bone area fraction occu-
pancy (BAFO), which was later transformed into a percentage36. Using the reconstructed images, the presence
of osteoid, woven bone, connective tissue, and vessels around the implants was also quantified. For this analysis,
toluidine blue staining was used, as it allows for the color identification of osteoid and mineralized bone. The area
of interest was drawn with a 200 µm box around the implant. Methods
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All implants were installed with the aid of a handpiece coupled to a drilling unit (BLM 600 plus; K Driller,
Sao Paulo, SP, Brazil) under a profuse 0.9% sodium chloride solution (Sterile Saline Solution; Eurofarma, Rio de Scientific Reports | (2021) 11:15436 | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ Janeiro, RJ, Brazil) and in low rotation (24 rpm) to avoid tissue necrosis due to overheating. The final insertion
torque (IT) was recorded for each implant by the drilling unit. When the IT value was higher than 50 N-cm,
an analogic wrench was used (S.I.N. Implant System, Sao Paulo, Brazil). The minimum, median, and maxi-
mum values for IT of the five samples were calculated for each group. The implant stability quotient (ISQ) was
determined with an Osstell IDx device (Ostell/Integration Diagnostics; Gothenburg, Västra Götaland, Sweden),
simulating mesiodistal and buccolingual measurements, and the average was recorded60. The minimum, median,
and maximum values for ISQ of the five samples were calculated for each group.ti i
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After surgical procedures, all animals received 4 mg/kg of the analgesic Tramal (Tramadol; Pfizer, New York,
NY, USA) and 0.5 mg/kg of the anti-inflammatory meloxicam (Meloxivet; Duprat, Rio de Janeiro, RJ, Brazil)
over five days. Antibiotic therapy by intramuscular injection of 0.1 mL/kg of oxytetracycline (Terramicina; Pfizer,
New York, NY, USA) was also used every 24 h for three days, including the day of the surgery. Oxytetracycline
spray with hydrocortisone (Terra-Cortril Spray; Zoetis, Sao Paulo, SP, Brazil), and zinc oxide ointment with
cresylic acid (Unguento Chemitec; Chemitec, Sao Paulo, SP, Brazil) together with silver spray (Aerocid Total;
Agener União, Araçoiaba da Serra, SP, Brazil) was applied daily at the wound site to support healing and prevent
local infection. Histological procedures. The sheep were submitted to the same anesthetic procedures following 14 and
28 days of healing. The bone blocks were collected with a 5-mm internal diameter trephine drill (S.I.N. Implant
System, São Paulo, SP, Brazil). Anesthetic and surgical procedures were followed according to the protocol
reported above, and all sheep were subsequently returned to the farm, where they completely recovered after
the biopsies.tif p
Immediately after the collection, the samples containing bone and implants were fixed in 4% neutral-buffered
formalin solution for 48 h. Methods
D
l i For vessel quantification, scores from 0 to 4 from
ISO 10993-6/2016 were used, where 0: no neovascularization; 1: minimal capillary proliferation focal 1–3 buds;
2: groups of 4–7 capillaries with supporting fibroblastic structures; 3: broad band of capillaries with supporting
structures; and 4: extensive band of capillaries with supporting fibroblastic structures. p
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One single and experienced observer conducted the histologic and histomorphometric evaluations. All
samples were coded, and the examiner evaluated the slides blindly with respect to the experimental group and
endpoints. Statistical analysis. A Shapiro–Wilk test was used to check data distribution. The log transformation of
ISQ was used to conform to normality. Fitting a normal distribution, the groups and the healing time points
were compared using a t-test considering a significance level of 0.05. All analyses were accomplished using Prism
Graph Pad 8.3 software (GraphPad Software, San Diego, CA, USA). The values for IT are presented in minimum,
median, and maximum, and the data for BIC and BAFO are presented in mean plus confidence interval at 95%
of significance. Data availabilityh y
The datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request. Received: 25 April 2021; Accepted: 18 July 2021 Received: 25 April 2021; Accepted: 18 July 2021 Scientific Reports | (2021) 11:15436 | https://doi.org/10.1038/s41598-021-94886-y www.nature.com/scientificreports/ References Apatite coating on ceramics, metals and polymers utilizing a biological process. J. Mater. Sci
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52. de Jesus, R. N. R. et al. Histomorphometric evaluation of a dual acid‐etched vs. a chemically modified hydrophilic dual acid‐etched
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3. Trisi, P., Carlesi, T., Colagiovanni, M. & Perfetti, G. Implant stability quotient (ISQ) vs direct in vitro measurement of primary
stability (micromotion): Effect of bone density and insertion torque. J. Osteol. Biomat. 1, 141–149 (2010). p
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53. Trisi, P., Carlesi, T., Colagiovanni, M. & Perfetti, G. Implant stability quotient (ISQ) vs direct in vitro measurement
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stability (micromotion): Effect of bone density and insertion torque. J. Osteol. Biomat. 1, 141–149 (2010). 3. Trisi, P., Carlesi, T., Colagiovanni, M. & Perfetti, G. Implant stability quotient (ISQ) vs direct in vitro measurement of primary
stability (micromotion): Effect of bone density and insertion torque. J. Osteol. Biomat. 1, 141–149 (2010). f
4. Atsumi, M., Park, S. H. & Wang, H. L. Methods used to assess implant stability: current status. Int. J. Oral Maxillofac. Implants 22
743–754 (2007).i 55. Author contributions M.D.C.-M.; F.J.B.B. and B.G..: Study design and preparation of the manuscript. M.D.C.-M.; S.C.S.; M.J.G.P.U.;
B.G.: Surgical Intervention and preparation and submission of the manuscript. J.A.C.-M.; M.D.C.-M; S.C.S.:
Histological and histomorphometric evaluation; P.S.; C.F.A.B.M.; R.C.M.M.: interpretation of the results and
preparation of the materials. J.M.G. and M.D.C.-M.: Supervision, interpretation of the results, and preparation
of the manuscript. All authors have read, edited, and approved the final manuscript. References Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of
Biomaterials 34, 9960–9968. https://doi.org/10.1016/j.biomaterials.2013.09.011 (2013). 2. Cipitria, A. et al. Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae
Biomaterials 34, 9960–9968. https://doi.org/10.1016/j.biomaterials.2013.09.011 (2013). Biomaterials 34, 9960–9968. https://doi.org/10.1016/j.biomaterials.2013.09.011 (2013). 33. Turner, A. S. Experiences with sheep as an animal model for shoulder surgery: Strengths and shortcomings. J. Shoulder Elbow Biomaterials 34, 9960–9968. https://doi.org/10.1016/j.biomaterials.2013.09.011 (2013). 33. Turner, A. S. Experiences with sheep as an animal model for shoulder surgery: Strengths and shortcomings. J. Shoulder Elbow
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34. Med, M. S. C. B. V. Code of Practice for the Care & Handling of Sheep: Review of Scientific Research on Priority Issues (Nova Scotia
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Coop. Between Eur. Found. Osteoporos. Natl. Osteoporos. Found. USA 16(Suppl 2), S129-138. https://doi.org/10.1007/s00198-005-
1859-7 (2005).hl 6. Sartoretto, S. C. et al. The influence of nanostructured hydroxyapatite surface in the early stages of osseointegration: A multipa-
rameter animal study in low-density bone. Int. J. Nanomed. 15, 8803 (2020).hfif y
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37. Lahens, B. et al. The effect of osseodensification drilling for endosteal implants with different surface treatments: A study in sheep. J. Biomed. Mater. Res. Part B Appl. Biomater. 107, 615–623, https://doi.org/10.1002/jbm.b.34154 (2019). J
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38. Sartoretto, S. C. et al. Accelerated healing period with hydrophilic implant placed in Sheep Tibia. Braz. Dent. J. 28, 559–565. https://
doi.org/10.1590/0103-6440201601559 (2017). https://doi.org/10.1038/s41598-021-94886-y Scientific Reports | (2021) 11:15436 | Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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The edible seaweed Laminaria japonica contains cholesterol analogues that inhibit lipid peroxidation and cyclooxygenase enzymes
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The edible seaweed Laminaria japonica contains cholesterol analogues that
inhibit Lipid Peroxidation and Cyclooxygenase Enzymes The edible seaweed Laminaria japonica contains cholesterol analogues that
inhibit Lipid Peroxidation and Cyclooxygenase Enzymes The edible seaweed Laminaria japonica contains cholesterol analogues that
inhibit Lipid Peroxidation and Cyclooxygenase Enzymes • Author names and affiliations Xingyu Lua, Amila A. Dissanayakeb, Chuqiao Xiaoc, d, Jie Gaoa, b *, Mouming Zhaoc,
Muraleedharan G. Nairb Xingyu Lua, Amila A. Dissanayakeb, Chuqiao Xiaoc, d, Jie Gaoa, b *, Mouming Zhaoc,
Muraleedharan G. Nairb
a School of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
b Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State
University, East Lansing, 48824, MI, USA
c School of Food Science and Engineering, South China University of Technology Guangzhou 510640,
China
d Chaozhou Branch of Chemistry and Chemical Engineering Guangdong Laboratory, Chaozhou 521031,
China
• Corresponding author
Jie Gao
Tel /Fax: +86 0771 3236100; E mail: gaojie@gxu edu cn a School of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
b Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State
University, East Lansing, 48824, MI, USA b Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State
University, East Lansing, 48824, MI, USA c School of Food Science and Engineering, South China University of Technology Guangzhou 510640,
China c School of Food Science and Engineering, South China University of Technology Guangzhou 510640,
China d Chaozhou Branch of Chemistry and Chemical Engineering Guangdong Laboratory, Chaozhou 521031,
China .
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bioRxiv preprint Jie Gao Tel./Fax: +86 0771-3236100; E-mail: gaojie@gxu.edu.cn Present/permanent address No.100, Daxue East Road, Xixiangtang District, Nanning 530004, Guangxi Province, China No.100, Daxue East Road, Xixiangtang District, Nanning 530004, Guangxi Province, China 1 1 . CC-BY 4.0 International license
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bioRxiv preprint 1
Abstract It is made available under a
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Abstract 2
In this study, 5 sterols were isolated and purified from Laminaria japonica, commonly
3
known as edible brown seaweed, and their structures were identified based on detailed chemical
4
methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-
5
Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol, saringosterol (24-vinyl-cholest-5-ene-3β,24-diol),
6
24-methylenecholesterol, fucosterol (stigmasta-5,24-diene-3β-ol), and 24-Hydroperoxy-24-
7
vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO)
8
and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest
9
COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-
10
methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the
11
other compounds. In addition, the results of molecular docking revealed that the 5 sterols were
12
located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin
13
methine achieved the highest binding energy (-7.85 and -9.02 kcal/mol) through hydrophobic
14
interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica
15
and its significant anti-inflammatory and antioxidant activity. 2
In this study, 5 sterols were isolated and purified from Laminaria japonica, commonly
3
known as edible brown seaweed, and their structures were identified based on detailed chemical
4
methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-
5
Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol, saringosterol (24-vinyl-cholest-5-ene-3β,24-diol),
6
24-methylenecholesterol, fucosterol (stigmasta-5,24-diene-3β-ol), and 24-Hydroperoxy-24-
7
vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO)
8
and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest
9
COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-
10
methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the
11
other compounds. In addition, the results of molecular docking revealed that the 5 sterols were
12
located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin
13
methine achieved the highest binding energy (-7.85 and -9.02 kcal/mol) through hydrophobic
14
interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica
15
and its significant anti-inflammatory and antioxidant activity. 16
Keywords: Laminaria japonica; sterols; structure elucidation; anti-inflammatory activity;
17
molecular docking 16
Keywords: Laminaria japonica; sterols; structure elucidation; anti-inflammatory activity;
17
molecular docking 2 2 . CC-BY 4.0 International license
perpetuity. 18
1. Introduction ;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint 40
investigations revealed the hexane extract from L. japonica exerts anti-inflammatory
41
effects.[15] Nevertheless, limited studies on the purification of hexane extracts from L. 42
japonica and structure characterization of the pure compounds have been performed
43
and, to the best of our knowledge, anti-inflammatory and antioxidant activities of these
44
pure compounds have not been reported. Herein, 5 sterols from hexane extracts of L. 45
japonica have been purified by VLC (vacuum liquid chromatography), multi-step
46
MPLC (medium-pressure liquid chromatography), Prep-TLC (preparative thin-layer
47
chromatography) and HPLC (high pressure liquid chromatography), and characterized
48
by high-resolution electrospray ionization mass spectrometry (HRESITOFMS),
49
nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-
50
MS) analyses. Lipid peroxidation and cyclooxygenase enzyme inhibitory of these
51
sterols were determined by using lipid peroxidation (LPO) and cyclooxygenase
52
enzymes (COX-1 and -2) inhibitory assays according to our previous study.[16-21]
53
Moreover, molecular docking analysis was performed to investigate the structure-
54
activity relationships of these sterols. 40
investigations revealed the hexane extract from L. japonica exerts anti-inflammatory
41
effects.[15] Nevertheless, limited studies on the purification of hexane extracts from L. 42
japonica and structure characterization of the pure compounds have been performed
43
and, to the best of our knowledge, anti-inflammatory and antioxidant activities of these
44
pure compounds have not been reported. Herein, 5 sterols from hexane extracts of L. 45
japonica have been purified by VLC (vacuum liquid chromatography), multi-step
46
MPLC (medium-pressure liquid chromatography), Prep-TLC (preparative thin-layer
47
chromatography) and HPLC (high pressure liquid chromatography), and characterized
48
by high-resolution electrospray ionization mass spectrometry (HRESITOFMS),
49
nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-
50
MS) analyses. Lipid peroxidation and cyclooxygenase enzyme inhibitory of these
51
sterols were determined by using lipid peroxidation (LPO) and cyclooxygenase
52
enzymes (COX-1 and -2) inhibitory assays according to our previous study.[16-21]
53
Moreover, molecular docking analysis was performed to investigate the structure-
54
activity relationships of these sterols. 18
1. Introduction 19
Laminaria japonica, a commercial edible brown seaweed, is widely cultivated and
20
consumed as a health food resource in China, Japan, and Korea. Over the past decades,
21
L. japonica is one of the most important marine medicinal foodstuffs and a rich source
22
of various functional compounds, which have been widely investigated by both in vitro
23
and in vivo experiments.[1-4] Numerous phytochemical investigations on L. japonica
24
revealed the presence of polyphenols, fatty acids, carotenoids, steroidal ketones, amine,
25
sterols and tocopherol.[5-13] Fucoxanthin, one of the most abundant carotenoids and
26
the major functional pigment present in L. japonica, has been reported to have
27
antioxidative, cytoprotective, anti-inflammatory and anti-obese properties.[6-9] Butyl-
28
isobutyl-phthalate, extracted from L. japonica, exhibited hypoglycemic effect in vivo
29
and non-competitive inhibition of α-glucosidase in vitro.[10, 11] Six isolates from
30
EtOAc extracts of L. japonica have been determined as 2,6-dibromo-4-(2-
31
(methylamino)ethyl)phenol,
6-bromo-1H-indole-3-carbaldehyde,
1H-indole-3-
32
carbaldehyde, 4-bromobenzoic aldehyde, 4-bromobenzoic acid and 4-hydroxybenzoic
33
acid.[12] Four steroidal ketones including ergosta-4,24(28)-diene-3-one, ergosta-
34
4,24(28)-diene-3,6-dione, stigmasta-4,24(28)-diene-3-one and stigmasta-4,24(28)-
35
diene-3,6-dione have also been isolated from L. japonica.[13]
36
O id ti
t
ti
t
i t
f t
i ti
f
t
hi h l
d t
th 36
Oxidative stress can activate a variety of transcription factors, which lead to the
37
differential expression of some genes involved in inflammatory pathways.[14] The
38
oxidative stress machinery and inflammatory signaling are not only interrelated, but
39
their impairment can lead to many chronic diseases. Previous phytochemical 36
Oxidative stress can activate a variety of transcription factors, which lead to the
37
differential expression of some genes involved in inflammatory pathways.[14] The
38
oxidative stress machinery and inflammatory signaling are not only interrelated, but
39
their impairment can lead to many chronic diseases. Previous phytochemical 3 . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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this version posted October 11, 2021. 56
2.1 General experimental procedures 57
Waters 2010 HPLC (high pressure liquid chromatography) system (Waters Corp.,
58
Milford, MA, USA) equipped with XTerra Prep MS C-18 column (10 µm, 19 x 250
59
mm) (Waters Corporation, Milford, MA, USA) was used for the HPLC purification and
60
all solvents used for HPLC purification were of ACS reagent grade (Sigma-Aldrich
61
Chemical Company, St. Louis, MO, USA). The NMR (Nuclear magnetic resonance) 4 . CC-BY 4.0 International license
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bioRxiv preprint 62
spectra were obtained using Agilent DirectDrive2 500 MHz (Agilent Technologies,
63
Palo Alto, CA, USA) in deuterated chloroform (CDCl3) purchased from Cambridge
64
Isotope Laboratories, Inc. The 1H and 13C chemical shift values were expressed in ppm,
65
based on the residual chemical shift values, for CDCl3 at δH 7.24 and δC 77.2 ppm,
66
respectively. The COX-1 enzyme was prepared from ram seminal vesicles purchased
67
from Oxford Biomedical Research, Inc. (Oxford, MI). The COX-2 enzyme was
68
prepared from insect cells cloned with human PGHS-2 enzyme. Positive controls for
69
the COX and LPO assays aspirin, naproxen, Celebrex®, ibuprofen, TBHQ (t-
70
butylhydroquinone), BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene)
71
and cholesterol were purchased from Sigma-Aldrich Chemical Company (St. Louis,
72
MO, USA) and arachidonic acid was purchased from Oxford Biomedical Research, Inc
73
(Oxford Biomedical Research, Inc., Oxford, MI, USA). The fluorescent probe, 3-[p-(6-
74
phenyl)-1,3,5-hexatrienyl] phenylpropionic acid was purchased from Molecular Probes
75
(Eugene, OR, USA) and 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine (SLPC)
76
was purchased from Avanti Polar Lipids (Alabaster, AL, USA). 78
L. japonica (GS-02-004-2013) used in this study were cultivated at Yantai
79
(Shandong province, China) and harvested in July 2018. Fresh raw L. japonica was
80
dried immediately after harvest under sun light and then shipped to the lab. The raw L. 56
2.1 General experimental procedures 81
japonica in the lab was first washed with running tap water and deionized water, and
82
then dried in an oven at 50 °C. The dry sample was further pulverized with a blender to
83
obtain the dry L. japonica power through a 50-mesh screen. 78
L. japonica (GS-02-004-2013) used in this study were cultivated at Yantai
79
(Shandong province, China) and harvested in July 2018. Fresh raw L. japonica was
80
dried immediately after harvest under sun light and then shipped to the lab. The raw L. 81
japonica in the lab was first washed with running tap water and deionized water, and
82
then dried in an oven at 50 °C. The dry sample was further pulverized with a blender to
83
obtain the dry L. japonica power through a 50-mesh screen. 5 5 . CC-BY 4.0 International license
perpetuity. It is made available under a
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bioRxiv preprint 84
2.3 Extraction and isolation 85
The dry L. japonica power (5 kg) was extracted with Hexane (10 L, 12 h×3) and
86
evaporated under vacuum to afford hexanes extract HE (22 g). An aliquot of HE (13 g)
87
was pretreated with activated charcoal and then fractionated using silica gel VLC by
88
eluting with hexanes-ethyl acetate (4:1, 2:1 and 1:1, v/v), followed by methanol (100%),
89
to yield fractions A (855 mg), B (4 g), C (1.44 g), D (374 mg), E (205 mg), and F (3 g),
90
respectively. An aliquot of fractions A (68 mg) was fractionated by Pre-TLC with
91
hexanes-acetone (10: 1, v/v) to yield the main fraction A-1(31 mg). An aliquot of
92
fractions B (76 mg) was fractionated by Pre-TLC with hexanes-acetone (8: 1, v/v) to
93
yield the main fraction B-1(50 mg), which was characterized as fatty acid and hence
94
were kept aside. Fraction D (374 mg) fractionated by MPLC with hexanes-acetone (2:
95
1, and 1: 1, v/v) and Pre-TLC with chloroform: methanol (40: 1, v/v) yielded three main
96
fractions D-1(24 mg, 86B), D-2(2 mg, 91C) and D-3(20 mg, 72B), which were the same
97
compounds that from fraction C. And fraction E and F were found to be salt and put
98
away. 85
The dry L. japonica power (5 kg) was extracted with Hexane (10 L, 12 h×3) and
86
evaporated under vacuum to afford hexanes extract HE (22 g). An aliquot of HE (13 g)
87
was pretreated with activated charcoal and then fractionated using silica gel VLC by
88
eluting with hexanes-ethyl acetate (4:1, 2:1 and 1:1, v/v), followed by methanol (100%),
89
to yield fractions A (855 mg), B (4 g), C (1.44 g), D (374 mg), E (205 mg), and F (3 g),
90
respectively. An aliquot of fractions A (68 mg) was fractionated by Pre-TLC with
91
hexanes-acetone (10: 1, v/v) to yield the main fraction A-1(31 mg). An aliquot of
92
fractions B (76 mg) was fractionated by Pre-TLC with hexanes-acetone (8: 1, v/v) to
93
yield the main fraction B-1(50 mg), which was characterized as fatty acid and hence
94
were kept aside. 84
2.3 Extraction and isolation Fraction D (374 mg) fractionated by MPLC with hexanes-acetone (2:
95
1, and 1: 1, v/v) and Pre-TLC with chloroform: methanol (40: 1, v/v) yielded three main
96
fractions D-1(24 mg, 86B), D-2(2 mg, 91C) and D-3(20 mg, 72B), which were the same
97
compounds that from fraction C. And fraction E and F were found to be salt and put
98
away. 99
Fraction C (1.44 g) was further purified with silica gel MPLC by eluting with
100
hexanes-acetone (4: 1, 2:1 and 1: 1, v/v), to yield fractions G (301 mg), H (599 mg), I
101
(137 mg), J (160 mg), and K (166 mg), respectively. Fraction G had been put away for
102
it was too complicated. 99
Fraction C (1.44 g) was further purified with silica gel MPLC by eluting with
100
hexanes-acetone (4: 1, 2:1 and 1: 1, v/v), to yield fractions G (301 mg), H (599 mg), I
101
(137 mg), J (160 mg), and K (166 mg), respectively. Fraction G had been put away for
102
it was too complicated. 103
An aliquot of the main fraction H (215 mg) was fractionated by silica gel VLC
104
and eluted under gradient conditions using chloroform: methanol (100:1 and 4.1 v/v) to
105
yield fractions LJ-1 (161 mg) and LJ-2 (51 mg). An aliquot of fraction LJ-1 (160 mg), 103
An aliquot of the main fraction H (215 mg) was fractionated by silica gel VLC
104
and eluted under gradient conditions using chloroform: methanol (100:1 and 4.1 v/v) to
105
yield fractions LJ-1 (161 mg) and LJ-2 (51 mg). An aliquot of fraction LJ-1 (160 mg), 6 . CC-BY 4.0 International license
perpetuity. It is made available under a
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The copyright holder for this
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doi:
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this version posted October 11, 2021. 84
2.3 Extraction and isolation ;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint 106
purified by HPLC by eluting with acetonitrile: methanol 50:50 v/v, 4 mL/min under
107
isocratic conditions yielded fraction LJ-1(a) (62 mg), compound 3 (39 mg, 42 min, 24-
108
methylenecholesterol, Figure S1 SI File) and compound 4 (58 mg, 51 min, fucosterol,
109
Figure S1 SI File).[22-24] Repeated HPLC purification of the fraction LJ-1(a) (40 mg)
110
eluting with acetonitrile:methanol 95:5 v/v, 3 mL/min under isocratic conditions
111
yielded compound 1 (12 mg, 46 min, 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol,
112
Figure S1 SI File) and compound 2 (22 mg, 51 min, saringosterol Figure S1 SI
113
File).[22] 106
purified by HPLC by eluting with acetonitrile: methanol 50:50 v/v, 4 mL/min under
107
isocratic conditions yielded fraction LJ-1(a) (62 mg), compound 3 (39 mg, 42 min, 24-
108
methylenecholesterol, Figure S1 SI File) and compound 4 (58 mg, 51 min, fucosterol,
109
Figure S1 SI File).[22-24] Repeated HPLC purification of the fraction LJ-1(a) (40 mg)
110
eluting with acetonitrile:methanol 95:5 v/v, 3 mL/min under isocratic conditions
111
yielded compound 1 (12 mg, 46 min, 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol,
112
Figure S1 SI File) and compound 2 (22 mg, 51 min, saringosterol Figure S1 SI
113
File).[22] 106
purified by HPLC by eluting with acetonitrile: methanol 50:50 v/v, 4 mL/min under
107
isocratic conditions yielded fraction LJ-1(a) (62 mg), compound 3 (39 mg, 42 min, 24-
108
methylenecholesterol, Figure S1 SI File) and compound 4 (58 mg, 51 min, fucosterol,
109
Figure S1 SI File).[22-24] Repeated HPLC purification of the fraction LJ-1(a) (40 mg)
110
eluting with acetonitrile:methanol 95:5 v/v, 3 mL/min under isocratic conditions
111
yielded compound 1 (12 mg, 46 min, 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol,
112
Figure S1 SI File) and compound 2 (22 mg, 51 min, saringosterol Figure S1 SI
113
File).[22]
114
Fraction I (137 mg) was purified by Prep-TLC on silica gel with chloroform-
115
Methanol (40: 1) as the mobile phase, yielded fraction L (33 mg). An aliquot of fraction
116
L (20 mg) was further separated by recrystallization from hexane: acetone to afford
117
compound 5 (17 mg, 24-hydroperoxy-24-vinyl-cholesterol).[22]
118
Pre-TLC with chloroform: methanol (60: 1, v/v) of fraction J (51 mg) produced
119
J-1 (15 mg, 87B) and J-2 (8 mg, 87D). Fraction K (75 mg) fractionated by Pre-TLC
120
with hexanes-acetone (8: 1, v/v) afforded K-1 (43 mg, 69B). 84
2.3 Extraction and isolation It is made available under a
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bioRxiv preprint 128
Inc.) at 37 °C according to the published procedure.[25-28] 128
Inc.) at 37 °C according to the published procedure.[25-28]
129
2.5 Lipid peroxidation inhibitory assay
130
The antioxidant activity of all isolates (compounds 1-5) and cholesterol was
131
determined by the LPO inhibitory assay using fluorescence spectroscopy on a Turner
132
model 450 fluorometer (Barnstead/Thermolyne Corp.) according to the reported
133
procedure.[17-20]
134
2.6 Molecular Docking
135
Docking was performed to investigate the molecular interactions between the pure
136
compounds and cyclooxygenase using the AutoDock Vina open-source program (ver. 137
1.1.2) according to the reported procedure.[29, 30] The crystallographic structures of
138
ovine COX-1 (PDB: 3N8Z) and human COX-2 (PDB: 5F1A) were retrieved from the
139
Protein Data Bank in SDF format. The 3D conformations of compounds 1-5 were
140
provided from PubChem (http://pubchem.ncbi.nlm.nih.gov/). Virtual screening of all
141
compounds was performed into the active site of the COX-1 and COX-2 domain by
142
using AutoDock Vina. The whole protein structures were targeted for compounds
143
docking.[31] Molecular docking coordinates for COX-1 receptor were determined as
144
center_x = -34.054, center_y =56.874, center_z = -11.092, dimensions parameters are
145
size_x = 94, size_y = 96, size_z = 126, spacing parameter is 0.808 Å. Docking
146
parameters used for COX-2 receptor were center_x = 34.099, center_y = 27.4, center_z
147
= 217.829, size_x = 96, size_y = 96, size_z = 126, spacing parameter is 0.789 Å. Cluster
148
analysis was performed on the docked results using an RMS tolerance of 2.0 Å. The 84
2.3 Extraction and isolation Fraction A-1, B-1, J-1, J-
121
2, and K-1 mainly contained fatty acids and triglycerides based on preliminary analyses
122
were kept aside due to prior published work. [25]
123
2.4 Cyclooxygenase enzymes (COX-1 and -2) inhibitory assays
124
The COX-1 and-2 enzyme inhibitory activity of pure isolates (compounds 1-5) 106
purified by HPLC by eluting with acetonitrile: methanol 50:50 v/v, 4 mL/min under
107
isocratic conditions yielded fraction LJ-1(a) (62 mg), compound 3 (39 mg, 42 min, 24-
108
methylenecholesterol, Figure S1 SI File) and compound 4 (58 mg, 51 min, fucosterol,
109
Figure S1 SI File).[22-24] Repeated HPLC purification of the fraction LJ-1(a) (40 mg)
110
eluting with acetonitrile:methanol 95:5 v/v, 3 mL/min under isocratic conditions
111
yielded compound 1 (12 mg, 46 min, 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol,
112
Figure S1 SI File) and compound 2 (22 mg, 51 min, saringosterol Figure S1 SI
113
File).[22] 114
Fraction I (137 mg) was purified by Prep-TLC on silica gel with chloroform-
115
Methanol (40: 1) as the mobile phase, yielded fraction L (33 mg). An aliquot of fraction
116
L (20 mg) was further separated by recrystallization from hexane: acetone to afford
117
compound 5 (17 mg, 24-hydroperoxy-24-vinyl-cholesterol).[22] 118
Pre-TLC with chloroform: methanol (60: 1, v/v) of fraction J (51 mg) produced
119
J-1 (15 mg, 87B) and J-2 (8 mg, 87D). Fraction K (75 mg) fractionated by Pre-TLC
120
with hexanes-acetone (8: 1, v/v) afforded K-1 (43 mg, 69B). Fraction A-1, B-1, J-1, J-
121
2, and K-1 mainly contained fatty acids and triglycerides based on preliminary analyses
122
were kept aside due to prior published work. [25] 123
2.4 Cyclooxygenase enzymes (COX-1 and -2) inhibitory assays 124
The COX-1 and-2 enzyme inhibitory activity of pure isolates (compounds 1-5)
125
from L. japonica and cholesterol were measured by monitoring the initial rate of O2
126
uptake using an Instech micro oxygen chamber and electrode (Instech Laboratories)
127
attached to a YSI model 5300 biological oxygen monitor (Yellow Springs Instrument, 124
The COX-1 and-2 enzyme inhibitory activity of pure isolates (compounds 1-5)
125
from L. japonica and cholesterol were measured by monitoring the initial rate of O2
126
uptake using an Instech micro oxygen chamber and electrode (Instech Laboratories)
127
attached to a YSI model 5300 biological oxygen monitor (Yellow Springs Instrument, 7 7 . CC-BY 4.0 International license
perpetuity. 152
2.7 Statistical Analysis 153
The data was presented as mean ± SD (n = 3) and evaluated by one-way analysis
154
of variance (ANOVA) followed by the Duncan’s test. All statistical analyses were
155
carried out using R statistical package and SPSS for Windows, Version 17.0 (SPSS
156
Inc., Chicago, IL, USA). 129
2.5 Lipid peroxidation inhibitory assay 130
The antioxidant activity of all isolates (compounds 1-5) and cholesterol was
131
determined by the LPO inhibitory assay using fluorescence spectroscopy on a Turner
132
model 450 fluorometer (Barnstead/Thermolyne Corp.) according to the reported
133
procedure.[17-20] 135
Docking was performed to investigate the molecular interactions between the pure
136
compounds and cyclooxygenase using the AutoDock Vina open-source program (ver. 137
1.1.2) according to the reported procedure.[29, 30] The crystallographic structures of
138
ovine COX-1 (PDB: 3N8Z) and human COX-2 (PDB: 5F1A) were retrieved from the
139
Protein Data Bank in SDF format. The 3D conformations of compounds 1-5 were
140
provided from PubChem (http://pubchem.ncbi.nlm.nih.gov/). Virtual screening of all
141
compounds was performed into the active site of the COX-1 and COX-2 domain by
142
using AutoDock Vina. The whole protein structures were targeted for compounds
143
docking.[31] Molecular docking coordinates for COX-1 receptor were determined as
144
center_x = -34.054, center_y =56.874, center_z = -11.092, dimensions parameters are
145
size_x = 94, size_y = 96, size_z = 126, spacing parameter is 0.808 Å. Docking
146
parameters used for COX-2 receptor were center_x = 34.099, center_y = 27.4, center_z
147
= 217.829, size_x = 96, size_y = 96, size_z = 126, spacing parameter is 0.789 Å. Cluster
148
analysis was performed on the docked results using an RMS tolerance of 2.0 Å. The
149
docked formation with the lowest energy value was obtained, and enzyme-ligand 8 . CC-BY 4.0 International license
perpetuity. It is made available under a
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The copyright holder for this
this version posted October 11, 2021. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
this version posted October 11, 2021. ;
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doi:
bioRxiv preprint 150
interactions were visualized using open-source PyMOL created by Warren Lyford 150
interactions were visualized using open-source PyMOL created by Warren Lyford 151
DeLano (https://github.com/schrodinger/pymol-open-source). 151
DeLano (https://github.com/schrodinger/pymol-open-source). 158
3.1 Structure elucidation 159
Compound 1 was isolated as a white powder and exhibited a molecular ion peak
160
at m/z 427.3563 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the
161
molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in
162
compound 1 (figure S2 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
163
two olefin methine proton signal at δH 5.33 (1H, brd, J=4.9 Hz, H-6) and δH 4.53 (1H,
164
d, J=6.8 Hz, H-28), one oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and
165
five methyl proton signals at δH 0.99 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.95
166
(3H, d, J=6.8 Hz, H-27), 0.89 (3H, d, J=6.7 Hz, H-26), and 0.65 (3H, s, H-18) (figure
167
S3 SI File). The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals
168
including 2 olefin quaternary carbon signals at δC 155.3 (C-24) and 140.7 (C-5), two
169
olefin methine carbon signals at δC121.7 (C-6) and δC114.8 (C-28), one oxygenated
170
methine carbon signal at δC 71.8 (C-3), one oxygenated methylene carbon signal at δC
171
73.5 (C-29) and 5 methyl carbon signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 159
Compound 1 was isolated as a white powder and exhibited a molecular ion peak
160
at m/z 427.3563 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the
161
molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in
162
compound 1 (figure S2 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
163
two olefin methine proton signal at δH 5.33 (1H, brd, J=4.9 Hz, H-6) and δH 4.53 (1H,
164
d, J=6.8 Hz, H-28), one oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and
165
five methyl proton signals at δH 0.99 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.95
166
(3H, d, J=6.8 Hz, H-27), 0.89 (3H, d, J=6.7 Hz, H-26), and 0.65 (3H, s, H-18) (figure
167
S3 SI File). The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals
168
including 2 olefin quaternary carbon signals at δC 155.3 (C-24) and 140.7 (C-5), two
169
olefin methine carbon signals at δC121.7 (C-6) and δC114.8 (C-28), one oxygenated
170
methine carbon signal at δC 71.8 (C-3), one oxygenated methylene carbon signal at δC
171
73.5 (C-29) and 5 methyl carbon signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 9 9 . 158
3.1 Structure elucidation CC-BY 4.0 International license
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bioRxiv preprint 172
18.7 (C-21), and 11.8 (C-18) (figure S4 SI File). According to the spectral data,
173
compound 1 was identified as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol (Figure
174
1).[22]
175
Compound 2 was isolated as a white powder and exhibited a molecular ion peak
176
at m/z 427.3562 [M+H-2H]+ in its positive-ion HRESITOFMS consistent with the
177
molecular formula C29H48O2, This confirmed six indices of hydrogen deficiency in
178
compound 2 (figure S5 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
179
two olefin methine proton signals at δH 5.34 (1H, brd, J=4.9 Hz, H-6) and 5.75 (1H, m,
180
H-28), one olefin methylene signal at δH 5.11 - 5.27 (2H, m, H-29a and H-29b), one
181
oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl proton
182
signals at δH 0.98 (3H, s, H-19), 0.90 (3H, d, J=6.8 Hz, H-21), 0.89 (3H, d, J=6.8 Hz,
183
H-27), 0.85 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S6 SI File). The
184
13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including one olefin
185
quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at
186
δC121.7 (C-29), two olefinic methines carbon signals at δC137.1 (C-5) and δC116.3
187
(C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 89.1 (C-24),
188
and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C-
189
21), and 11.8 (C-18) (figure S7 SI File). Therefore, according to the spectral data, 174
1).[22]
175
Compound 2 was isolated as a white powder and exhibited a molecular ion peak
176
at m/z 427.3562 [M+H-2H]+ in its positive-ion HRESITOFMS consistent with the
177
molecular formula C29H48O2, This confirmed six indices of hydrogen deficiency in
178
compound 2 (figure S5 SI File). 158
3.1 Structure elucidation ;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint 194
molecular formula C28H46O, This confirmed six indices of hydrogen deficiency in
195
compound 3 (figure S8 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
196
one olefin methine proton signal at δH 5.33 (1H, brd, J=4.9 Hz, H-6), one olefinic
197
methylene proton signals at δH 4.69 (1H, d, J=1.0, H-28a) and 4.64 (1H, d, J=1.0, H-
198
28b), one oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl
199
proton signals at δH 0.98 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.96 (3H, d, J=6.8
200
Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S9 SI File). 201
The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 28 carbon signals including 2
202
olefin quaternary carbon signals at δC 156.9 (C-24) and 140.7 (C-5), one olefin methine
203
carbon signals at δC121.7 (C-6), one olefin methylene carbon signals at δC105.9 (C-
204
28), one oxygenated methine carbon signal at δC 71.8 (C-3), and 5 methyl carbon
205
signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18)
206
(figure S10 SI File). According to the spectral data, compound 3 was identified as 24-
207
methylenecholesterol (Figure 1).[22, 23] 194
molecular formula C28H46O, This confirmed six indices of hydrogen deficiency in
195
compound 3 (figure S8 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
196
one olefin methine proton signal at δH 5.33 (1H, brd, J=4.9 Hz, H-6), one olefinic
197
methylene proton signals at δH 4.69 (1H, d, J=1.0, H-28a) and 4.64 (1H, d, J=1.0, H-
198
28b), one oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl
199
proton signals at δH 0.98 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.96 (3H, d, J=6.8
200
Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S9 SI File). 201
The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 28 carbon signals including 2
202
olefin quaternary carbon signals at δC 156.9 (C-24) and 140.7 (C-5), one olefin methine
203
carbon signals at δC121.7 (C-6), one olefin methylene carbon signals at δC105.9 (C-
204
28), one oxygenated methine carbon signal at δC 71.8 (C-3), and 5 methyl carbon
205
signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18)
206
(figure S10 SI File). 158
3.1 Structure elucidation The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
179
two olefin methine proton signals at δH 5.34 (1H, brd, J=4.9 Hz, H-6) and 5.75 (1H, m,
180
H-28), one olefin methylene signal at δH 5.11 - 5.27 (2H, m, H-29a and H-29b), one
181
oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl proton
182
signals at δH 0.98 (3H, s, H-19), 0.90 (3H, d, J=6.8 Hz, H-21), 0.89 (3H, d, J=6.8 Hz,
183
H-27), 0.85 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S6 SI File). The
184
13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including one olefin
185
quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at
186
δC121.7 (C-29), two olefinic methines carbon signals at δC137.1 (C-5) and δC116.3
187
(C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 89.1 (C-24),
188
and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C-
189
21), and 11.8 (C-18) (figure S7 SI File). Therefore, according to the spectral data,
190
compound 2 was identified as saringosterol (24-vinyl-cholest-5-ene-3β,24-diol)
191
(Figure 1). [22]
192
Compound 3 was isolated as a white powder and exhibited a molecular ion peak 192
Compound 3 was isolated as a white powder and exhibited a molecular ion peak
193
at m/z 381.3507 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the 192
Compound 3 was isolated as a white powder and exhibited a molecular ion peak
193
at m/z 381.3507 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the 192
Compound 3 was isolated as a white powder and exhibited a molecular ion peak
193
at m/z 381.3507 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the 10 . CC-BY 4.0 International license
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The copyright holder for this
this version posted October 11, 2021. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
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this version posted October 11, 2021. 158
3.1 Structure elucidation ;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S12 SI File) 216
Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S12 SI File). 217
The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including 2
218
olefin quaternary carbon signals at δC 146.9 (C-24) and 140.7 (C-5), two olefin methine
219
carbon signals at δC121.7 (C-6) and 115.5 (C-28), one oxygenated methine carbon
220
signal at δC 71.7 (C-3), and six methyl carbon signals at δC 13.2 (C-29), 22.2 (C-26),
221
22.1 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18) (figure S13 SI File). Therefore,
222
according to the spectral data, compound 4 was identified as fucosterol (stigmasta-5,24-
223
diene-3β-ol) (Figure 1).[22, 26] 216
Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S12 SI File). 217
The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including 2
218
olefin quaternary carbon signals at δC 146.9 (C-24) and 140.7 (C-5), two olefin methine
219
carbon signals at δC121.7 (C-6) and 115.5 (C-28), one oxygenated methine carbon
220
signal at δC 71.7 (C-3), and six methyl carbon signals at δC 13.2 (C-29), 22.2 (C-26),
221
22.1 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18) (figure S13 SI File). Therefore,
222
according to the spectral data, compound 4 was identified as fucosterol (stigmasta-5,24-
223
diene-3β-ol) (Figure 1).[22, 26] 216
Hz, H-27), 0.94 (3H, d, J=6.7 Hz, H-26), and 0.66 (3H, s, H-18) (figure S12 SI File). 217
The 13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including 2
218
olefin quaternary carbon signals at δC 146.9 (C-24) and 140.7 (C-5), two olefin methine
219
carbon signals at δC121.7 (C-6) and 115.5 (C-28), one oxygenated methine carbon
220
signal at δC 71.7 (C-3), and six methyl carbon signals at δC 13.2 (C-29), 22.2 (C-26),
221
22.1 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18) (figure S13 SI File). Therefore,
222
according to the spectral data, compound 4 was identified as fucosterol (stigmasta-5,24-
223
diene-3β-ol) (Figure 1).[22, 26] 224
Compound 5 was also isolated as a white powder and exhibited a molecular ion
225
peak at m/z 427.3562 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with
226
the molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in
227
compound 5 (figure S14 SI File). 158
3.1 Structure elucidation According to the spectral data, compound 3 was identified as 24-
207
methylenecholesterol (Figure 1).[22, 23] 208
Compound 4 was also isolated as a white powder and exhibited a molecular ion
209
peak at m/z 395.3669 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with
210
the molecular formula C29H48O, This confirmed six indices of hydrogen deficiency in
211
compound 4 (figure S11 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
212
two olefin methine proton signals at δH 5.33 (1H, brd, J=4.9 Hz, H-6) and 5.16 (1H,
213
dd, J=6.8 and 6.8 Hz, H-28), one oxygenated methine proton signal at δH 3.49 (1H, m,
214
H-3), one allyl methyl proton signal at δH 1.55 (3H, d, J=6.8 Hz, H-29), and five methyl
215
proton signals at δH 0.98 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.96 (3H, d, J=6.8 208
Compound 4 was also isolated as a white powder and exhibited a molecular ion
209
peak at m/z 395.3669 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with
210
the molecular formula C29H48O, This confirmed six indices of hydrogen deficiency in
211
compound 4 (figure S11 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
212
two olefin methine proton signals at δH 5.33 (1H, brd, J=4.9 Hz, H-6) and 5.16 (1H,
213
dd, J=6.8 and 6.8 Hz, H-28), one oxygenated methine proton signal at δH 3.49 (1H, m,
214
H-3), one allyl methyl proton signal at δH 1.55 (3H, d, J=6.8 Hz, H-29), and five methyl
215
proton signals at δH 0.98 (3H, s, H-19), 0.97 (3H, d, J=6.8 Hz, H-21), 0.96 (3H, d, J=6.8 11 11 . CC-BY 4.0 International license
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3.1 Structure elucidation It is made available under a
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21), and 11.8 (C-18) (figure S16 SI File). Therefore, according to the spectral data,
239
compound 5 was identified as 24-Hydroperoxy-24-vinyl-cholesterol (Figure 1).[22] 240
3.2 Cyclooxygenase enzymes (COX-1 and -2) inhibitory assay 241
Prostaglandins, the inflammation-causing hormones, were converted from
242
arachidonic acid by the catalysis of COX enzymes. Therefore, inhibition of COX
243
enzymes could prevent the production of prostaglandins and reduce inflammation.[26,
244
32] The anti-inflammatory activity of the pure isolates from L. japonica was revealed
245
by their COX-1 and -2 enzyme inhibitions. In this study, compounds (1-5) at 25 μg/mL
246
inhibited COX-1 enzyme by 32, 19, 50, 59, and 26% and COX-2 enzyme by 23, 14,
247
33, 47, and 9%, respectively (Figure 2). Commercial NSAIDs aspirin, Celebrex®,
248
naproxen and ibuprofen were used as positive control at 108, 1, 12, and 15 μg/mL,
249
respectively (Figure 2). Apparently, the COX enzyme inhibitory activity of 24-
250
methylenecholesterol (compound 3) and fucosterol (compound 4) was comparable to
251
the activity of the over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs)
252
aspirin and ibuprofen. Compound 3 and 4 inhibited the COX-1 enzyme at a higher rate
253
than the COX-2 enzyme, which is similar to aspirin and ibuprofen. 241
Prostaglandins, the inflammation-causing hormones, were converted from
242
arachidonic acid by the catalysis of COX enzymes. Therefore, inhibition of COX
243
enzymes could prevent the production of prostaglandins and reduce inflammation.[26,
244
32] The anti-inflammatory activity of the pure isolates from L. japonica was revealed
245
by their COX-1 and -2 enzyme inhibitions. In this study, compounds (1-5) at 25 μg/mL
246
inhibited COX-1 enzyme by 32, 19, 50, 59, and 26% and COX-2 enzyme by 23, 14,
247
33, 47, and 9%, respectively (Figure 2). 158
3.1 Structure elucidation The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
228
two olefin methine proton signals at δH 5.34 (1H, brd, J=4.9 Hz, H-6) and 5.75 (1H, m,
229
H-28), one olefin methylene signal at δH 5.09 - 5.19 (2H, m, H-29a and H-29b), one
230
oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl proton
231
signals at δH 0.99 (3H, s, H-19), 0.91 (3H, d, J=6.8 Hz, H-21), 0.89 (3H, d, J=6.8 Hz,
232
H-27), 0.86 (3H, d, J=6.7 Hz, H-26), and 0.64 (3H, s, H-18) (figure S15 SI File). The
233
13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including one olefin
234
quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at
235
δC121.6 (C-29), two olefinic methines carbon signals at δC 112.8 (C-5) and δC 142.5
236
(C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 77.7 (C-24),
237
and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C- 224
Compound 5 was also isolated as a white powder and exhibited a molecular ion
225
peak at m/z 427.3562 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with
226
the molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in
227
compound 5 (figure S14 SI File). The 1H-NMR (500 MHz, CDCl3) spectrum exhibited
228
two olefin methine proton signals at δH 5.34 (1H, brd, J=4.9 Hz, H-6) and 5.75 (1H, m,
229
H-28), one olefin methylene signal at δH 5.09 - 5.19 (2H, m, H-29a and H-29b), one
230
oxygenated methine proton signal at δH 3.49 (1H, m, H-3), and five methyl proton
231
signals at δH 0.99 (3H, s, H-19), 0.91 (3H, d, J=6.8 Hz, H-21), 0.89 (3H, d, J=6.8 Hz,
232
H-27), 0.86 (3H, d, J=6.7 Hz, H-26), and 0.64 (3H, s, H-18) (figure S15 SI File). The
233
13C-NMR (125 MHz, CDCl3) spectrum exhibited 29 carbon signals including one olefin
234
quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at
235
δC121.6 (C-29), two olefinic methines carbon signals at δC 112.8 (C-5) and δC 142.5
236
(C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 77.7 (C-24),
237
and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C- 12 12 . CC-BY 4.0 International license
perpetuity. 158
3.1 Structure elucidation Commercial NSAIDs aspirin, Celebrex®,
248
naproxen and ibuprofen were used as positive control at 108, 1, 12, and 15 μg/mL,
249
respectively (Figure 2). Apparently, the COX enzyme inhibitory activity of 24-
250
methylenecholesterol (compound 3) and fucosterol (compound 4) was comparable to
251
the activity of the over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs)
252
aspirin and ibuprofen. Compound 3 and 4 inhibited the COX-1 enzyme at a higher rate
253
than the COX-2 enzyme, which is similar to aspirin and ibuprofen. 254
3.3 Lipid peroxidation inhibitory assay 255
Potential antioxidant activity of the pure compounds 1-5 was determined by the
256
LPO assays using the large unilamellar vesicles (LUVs) model system, the results of
257
which are shown in Fig. 3. The LPO assay detects compounds that can scavenge free
258
radicals. At 25 μg/mL, the LPO inhibitory activity of sterols 1-5 and cholesterol was
259
21, 51, 58, 56, 25 and 20%, respectively, as compared to the commercial antioxidants 255
Potential antioxidant activity of the pure compounds 1-5 was determined by the
256
LPO assays using the large unilamellar vesicles (LUVs) model system, the results of
257
which are shown in Fig. 3. The LPO assay detects compounds that can scavenge free
258
radicals. At 25 μg/mL, the LPO inhibitory activity of sterols 1-5 and cholesterol was
259
21, 51, 58, 56, 25 and 20%, respectively, as compared to the commercial antioxidants 13 13 . CC-BY 4.0 International license
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BHA, BHT and TBHQ at 85, 85 and 82%, respectively. The concentrations used to test
261
LPO inhibitory activity for compounds were at 1.8, 2.2 and 1.6 μg/mL, respectively
262
(Figure 3). Compared to compound 1 and 5, saringosterol (compound 2), 24-
263
methylenecholesterol (compound 3) and fucosterol (compound 4) showed the higher
264
LPO inhibitory activity at >50%, at 25 μg/mL concentration. 260
BHA, BHT and TBHQ at 85, 85 and 82%, respectively. The concentrations used to test
261
LPO inhibitory activity for compounds were at 1.8, 2.2 and 1.6 μg/mL, respectively
262
(Figure 3). Compared to compound 1 and 5, saringosterol (compound 2), 24-
263
methylenecholesterol (compound 3) and fucosterol (compound 4) showed the higher
264
LPO inhibitory activity at >50%, at 25 μg/mL concentration. 265
3.4 Molecular docking It is made available under a
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COX-2 (Fig. 6). Compounds 1 and 4 located in the same active pocket of COX-1, which
283
was surrounded by active site at ILE-46, CYS-47, CYS-36, ASN-34, ARG-49, THR-
284
322, GLY-324, GLU-326, GLN-327, TYR-136, ASP-135 and PRO-153. The docking
285
results also revealed that compound 1 typically bind within the COX-1 channel via H-
286
bonding interactions with the side chain of ASP-135 and ASP-158, while compound 4
287
was embedded in the active pocket of COX-1 primarily through hydrophobic
288
interactions (Fig.6, Table 1). 282
COX-2 (Fig. 6). Compounds 1 and 4 located in the same active pocket of COX-1, which
283
was surrounded by active site at ILE-46, CYS-47, CYS-36, ASN-34, ARG-49, THR-
284
322, GLY-324, GLU-326, GLN-327, TYR-136, ASP-135 and PRO-153. The docking
285
results also revealed that compound 1 typically bind within the COX-1 channel via H-
286
bonding interactions with the side chain of ASP-135 and ASP-158, while compound 4
287
was embedded in the active pocket of COX-1 primarily through hydrophobic
288
interactions (Fig.6, Table 1). 289
Furthermore, the binding sites and interactions between compounds 1-5 and COX-
290
2 are represented in Fig 5 and 6. Compounds 1-4 were located in the hydrophobic
291
pocket A of COX-2 (surrounded by active site at GLY-225, ASN-375, ARG-376, PHE-
292
142, VAL-538, ASN-537, GLY-227, GLY-536, LEU-224, HIS-226, GLN-374, TYR-
293
373), while compound 5 was located in the pocket B (surrounded by active site at PRO-
294
84, LYS-83, LEU-80, LYS-473, LEU-472, PHE-470, GLU-524, ARG-120, TYR-115,
295
THR-85, MET-471, PRO-86, LEU-123, HIS-122, SER-119). All of the five
296
compounds had interactions with surrounding residues in the active site of COX-2. 297
Compounds 1, 3 and 4 showed possible hydrogen bond with GLY-225, predicting
298
which might be favorable binding site of COX-2 for sterols (Fig.6). Differ from
299
compounds 1-4, compound 5 bound with COX-2 at another active pocket and showed
300
possible hydrogen bonds with ARG-120 and GLU-524. Moreover, there was a high
301
binding energy between compound 4 and COX-2 (-9.02 kcal/mol), which was
302
comparable to that of the compound 5 (-6.97 kcal/mol). 265
3.4 Molecular docking 266
Molecular docking was used to predict the interactions between the 5 sterols and
267
the COX-1 and -2 enzyme at the molecular level. Root-Mean-Squared-Deviation
268
(RMSD) is a similarity metric between molecular conformations, the lower the RMSD
269
value is, the more similar the molecular conformations of ligands (before and after the
270
docking) is. As shown in Table 1, the RMSD values of all the detected ligands were
271
below the maximum allowed value of 2 Å, indicating ligands underwent minimal
272
deformation during docking and the docking results were credible.[33] Docking results
273
revealed that the binding energy of compounds 1-5 were -7.64, -6.36, -7.79, -7.85 and
274
-6.44 kcal/mol for COX-1, and -8.49, -8.33, -8.72, -9.02 and -6.97 kcal/mol for COX-
275
2, which have the similar trends with their COX-1 and -2 enzyme inhibitory activities
276
(Table 1). 277
The possible mechanisms beneath the inhibition of compounds 1-5 against COX-
278
1 and -2 was presented through molecular docking (Fig. 4-6, Table 1). As shown in Fig. 4
d 1 5
di
d
b l
d i diff
k
f COX 1 (
k 266
Molecular docking was used to predict the interactions between the 5 sterols and
267
the COX-1 and -2 enzyme at the molecular level. Root-Mean-Squared-Deviation
268
(RMSD) is a similarity metric between molecular conformations, the lower the RMSD
269
value is, the more similar the molecular conformations of ligands (before and after the
270
docking) is. As shown in Table 1, the RMSD values of all the detected ligands were
271
below the maximum allowed value of 2 Å, indicating ligands underwent minimal
272
deformation during docking and the docking results were credible.[33] Docking results
273
revealed that the binding energy of compounds 1-5 were -7.64, -6.36, -7.79, -7.85 and
274
-6.44 kcal/mol for COX-1, and -8.49, -8.33, -8.72, -9.02 and -6.97 kcal/mol for COX-
275
2, which have the similar trends with their COX-1 and -2 enzyme inhibitory activities
276
(Table 1). 14 . CC-BY 4.0 International license
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COX-1 and -2 are bifunctional enzymes that convert arachidonic acid (AA) to 265
3.4 Molecular docking 289
Furthermore, the binding sites and interactions between compounds 1-5 and COX-
290
2 are represented in Fig 5 and 6. Compounds 1-4 were located in the hydrophobic
291
pocket A of COX-2 (surrounded by active site at GLY-225, ASN-375, ARG-376, PHE-
292
142, VAL-538, ASN-537, GLY-227, GLY-536, LEU-224, HIS-226, GLN-374, TYR-
293
373), while compound 5 was located in the pocket B (surrounded by active site at PRO-
294
84, LYS-83, LEU-80, LYS-473, LEU-472, PHE-470, GLU-524, ARG-120, TYR-115,
295
THR-85, MET-471, PRO-86, LEU-123, HIS-122, SER-119). All of the five
296
compounds had interactions with surrounding residues in the active site of COX-2. 297
Compounds 1, 3 and 4 showed possible hydrogen bond with GLY-225, predicting
298
which might be favorable binding site of COX-2 for sterols (Fig.6). Differ from
299
compounds 1-4, compound 5 bound with COX-2 at another active pocket and showed
300
possible hydrogen bonds with ARG-120 and GLU-524. Moreover, there was a high
301
binding energy between compound 4 and COX-2 (-9.02 kcal/mol), which was
302
comparable to that of the compound 5 (-6.97 kcal/mol). 303
COX-1 and -2 are bifunctional enzymes that convert arachidonic acid (AA) to 15 15 . CC-BY 4.0 International license
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prostaglandin G2 (PGG2) in their cyclooxygenase active site.[35] Our results suggested
305
that fucosterol (compound 4) was located in the hydrophobic pocket of COX-1 and -2
306
enzyme with the highest binding energy (-7.85 and -9.02 kcal/mol) in molecular
307
docking, compared to the other sterols. The possible mechanism underlying the highest
308
COX-1 and -2 enzyme inhibitory activities of fucosterol might be attributed to the
309
distinct olefin methine presented in its molecular structure, which could occupy the
310
active site of the enzyme and prevent the enzyme from contacting with the substrate. 265
3.4 Molecular docking 311
This is in agreement with previous molecular docking results that tetracyclic skeletons
312
and incorporation of an aliphatic chain in sterols were the key structural requirements
313
theoretically for their good COX-1 and COX-2 binding activity.[36] Thus, these
314
findings confirmed that fucosterol with tetracyclic skeletons and olefin methine
315
achieved the good COX-1 and -2 enzyme inhibitory activities through hydrophobic
316
interactions and hydrogen bond. 317
4. Conclusions For the LPO assays, saringosterol
330
(compound 2), 24-methylenecholesterol (compound 3) and fucosterol (compound 4)
331
showed higher LPO inhibitory activity at >50% than the other compounds. In addition,
332
the results of molecular docking predicted that 5 sterols were located in different pocket
333
of COX-1 and -2 and confirmed that fucosterol with tetracyclic skeletons and olefin
334
methine achieved the best COX-1 and -2 enzyme inhibitory activities through
335
hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5
336
sterols in L. japonica and its significant anti-inflammatory and antioxidant activity. 337
Therefore, it appears that L. japonica have the potential to be developed as a dietary
338
supplement. 317
4. Conclusions 318
Purification of hexane extracts from L. japonica afforded 5 sterols, and their
319
structures were identified using spectroscopic and chemical evidence. Spectroscopic
320
methods characterized these compounds as 29-Hydroperoxy-stigmasta-5,24(28)-dien-
321
3β-ol (compound 1), saringosterol (24-vinyl-cholest-5-ene-3β,24-diol) (compound 2),
322
24-methylenecholesterol (compound 3), fucosterol (stigmasta-5,24-diene-3β-ol)
323
(compound 4), and 24-Hydroperoxy-24-vinyl-cholesterol (compound 5). These pure
324
isolates were tested for antioxidant and anti-inflammatory activities using LPO and
325
COX-1 and -2 enzyme inhibitory assays. Compared to the other compounds, fucosterol 16 16 . CC-BY 4.0 International license
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(compound 4) exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59
327
and
47%,
respectively. The
COX
enzyme
inhibitory
activity
of
24-
328
methylenecholesterol (compound 3) and fucosterol (compound 4) was comparable to
329
the activity of the NSAIDs aspirin and ibuprofen. For the LPO assays, saringosterol
330
(compound 2), 24-methylenecholesterol (compound 3) and fucosterol (compound 4)
331
showed higher LPO inhibitory activity at >50% than the other compounds. In addition,
332
the results of molecular docking predicted that 5 sterols were located in different pocket
333
of COX-1 and -2 and confirmed that fucosterol with tetracyclic skeletons and olefin
334
methine achieved the best COX-1 and -2 enzyme inhibitory activities through
335
hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5
336
sterols in L. japonica and its significant anti-inflammatory and antioxidant activity. 337
Therefore, it appears that L. japonica have the potential to be developed as a dietary
338
supplement. 326
(compound 4) exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59
327
and
47%,
respectively. The
COX
enzyme
inhibitory
activity
of
24-
328
methylenecholesterol (compound 3) and fucosterol (compound 4) was comparable to
329
the activity of the NSAIDs aspirin and ibuprofen. 339
Abbreviations 340
HRESITOFMS: high-resolution electrospray ionization mass spectrometry; NMR:
341
nuclear magnetic resonance; LPO: lipid peroxidation; COX: cyclooxygenase; MPLC:
342
medium-pressure liquid chromatography; VLC: vacuum liquid chromatography; Prep-
343
TLC: preparative thin-layer chromatography; HPLC: high pressure liquid
344
chromatography; TBHQ: t-butylhydroquinone; BHA: butylated hydroxyanisole; BHT:
345
butylated hydroxytoluene; NSAIDs: nonsteroidal anti-inflammatory drugs; SLPC: 1-
346
stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine; LUVs: large unilamellar vesicles;
347
AA: arachidonic acid. 340
HRESITOFMS: high-resolution electrospray ionization mass spectrometry; NMR:
341
nuclear magnetic resonance; LPO: lipid peroxidation; COX: cyclooxygenase; MPLC:
342
medium-pressure liquid chromatography; VLC: vacuum liquid chromatography; Prep-
343
TLC: preparative thin-layer chromatography; HPLC: high pressure liquid
344
chromatography; TBHQ: t-butylhydroquinone; BHA: butylated hydroxyanisole; BHT:
345
butylated hydroxytoluene; NSAIDs: nonsteroidal anti-inflammatory drugs; SLPC: 1-
346
stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine; LUVs: large unilamellar vesicles;
347
AA: arachidonic acid. 17 17 . CC-BY 4.0 International license
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bioRxiv preprint 348
Acknowledgement 349
Jie Gao gratefully acknowledge the financial support from China Scholarship Council
350
(CSC) and National Natural Science Foundation of China (Project No. 32001696). This
351
research is also a contribution from Michigan State University AgBioResearch
352
(MICL01680). The nuclear magnetic resonance data were obtained on instrumentation
353
that was purchased, in part, with the funds from NIH grant no. 1-S10-RR04750, NSF
354
grant no. CHE-88-00770, and NSF grant no. CHE-92-13241. HRMS data were
355
obtained at the Michigan State University Mass Spectrometry Facility, which is
356
supported, in part, by a grant (DRR-00480) from the Biotechnology Research
357
Technology Program, National Centre for Research Resources, National Institutes of
358
Health. 369
2.
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aggregation-related eicosanoid production by dietary F-fucoidan from brown alga Laminaria japonica in
371
human subjects. British journal of nutrition. 2013;110(5):880-90. 359
Author Contributions It is made available under a
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The copyright holder for this
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bioRxiv preprint 375
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Author Contributions 360
Muraleedharan G. Nair and Mouming Zhao conceived the idea. Xingyu Lu and Jie Gao
361
conducted the experiment and write manuscript. Amila Dissanayake analyzed the data. 360
Muraleedharan G. Nair and Mouming Zhao conceived the idea. Xingyu Lu and Jie Gao 360
Muraleedharan G. Nair and Mouming Zhao conceived the idea. Xingyu Lu and Jie Gao
361
conducted the experiment and write manuscript. Amila Dissanayake analyzed the data. 361
conducted the experiment and write manuscript. Amila Dissanayake analyzed the data. 362
Chuqiao Xiao conducted the molecular docking. All authors contributed to the 363
interpretation and discussion of the results. 366
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371
human subjects. British journal of nutrition. 2013;110(5):880-90. 369
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Author Contributions CC-BY 4.0 International license
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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bioRxiv preprint Table 1. Docking results of all the 5 sterols against COX-1 and -2 receptors. Table 1. Docking results of all the 5 sterols against COX-1 and -2 receptors. 466 Receptor
with PDB
IDs
Ligand
Binding
affinity
(kcal/mol)
RMSD(Å)
No. of H-
bonds
Amino acid
residues forming
H-bonds
Comp. 1
-7.64
1.28
3
ASP-135,
ASP-158
Comp. 2
-6.36
1.43
1
GLU-326
Comp. 3
-7.79
1.09
1
ASP-158
Comp. 4
-7.85
1.08
0
None
COX-1
(3N8Z)
Comp. 5
-6.44
1.69
1
LEU-115
Comp. 1
-8.49
0.88
2
GLY-225,
TYR-373
Comp. 2
-8.33
1.82
2
ASN-375
Comp. 3
-8.72
1.06
1
GLY-225
Comp. 4
-9.02
1.43
1
GLY-225
COX-2
(5F1A)
Comp. 5
-6.97
1.83
2
ARG-120,
GLU-524 467 22 . CC-BY 4.0 International license
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Figure captions 469
Figure 1: Chemical structures of the biologically active sterols (compounds 1-5)
470
isolated from the hexane extract of L. japonica. 469
Figure 1: Chemical structures of the biologically active sterols (compounds 1-5)
470
isolated from the hexane extract of L. japonica. 471
Figure 2: Cyclooxygenase enzymes (COX-1 and -2) enzyme inhibitory activities
472
sterols (1-5) from the hexane extract of L. japonica and cholesterol were tested at 25
473
μg/mL concentration. Commercial NSAIDs aspirin, ibuprofen, Celebrex® and
474
naproxen used as positive control and tested at 108, 12, 1 and 12 µg/mL, respectively. 475
The varying concentrations of positive controls used were to yield a comparable activity
476
profile between 50-100% by test compounds and positive controls alike. Vertical bars
477
represent the standard deviation of each data point (n=2). 471
Figure 2: Cyclooxygenase enzymes (COX-1 and -2) enzyme inhibitory activities
472
sterols (1-5) from the hexane extract of L. japonica and cholesterol were tested at 25
473
μg/mL concentration. Commercial NSAIDs aspirin, ibuprofen, Celebrex® and
474
naproxen used as positive control and tested at 108, 12, 1 and 12 µg/mL, respectively. 475
The varying concentrations of positive controls used were to yield a comparable activity
476
profile between 50-100% by test compounds and positive controls alike. Vertical bars
477
represent the standard deviation of each data point (n=2). 478
Figure 3: Lipid peroxidation (LPO) inhibitory activities of sterols (1-5) isolated from
479
the hexane extract of L. japonica and commercial cholesterol tested at 25 μg/mL. 480
Commercial antioxidants BHA, BHT and TBHQ used as positive controls at 1.8, 2.2
481
and 1.6 µg/mL. The oxidation of lipid was initiated by the addition of Fe2+ ions. The
482
varying concentrations of positive controls used were to yield a comparable activity
483
profiles between 50-100% by test compounds and positive controls alike. Vertical bars
484
represent the standard deviation of each data point (n=2). 478
Figure 3: Lipid peroxidation (LPO) inhibitory activities of sterols (1-5) isolated from
479
the hexane extract of L. japonica and commercial cholesterol tested at 25 μg/mL. 480
Commercial antioxidants BHA, BHT and TBHQ used as positive controls at 1.8, 2.2
481
and 1.6 µg/mL. The oxidation of lipid was initiated by the addition of Fe2+ ions. The
482
varying concentrations of positive controls used were to yield a comparable activity
483
profiles between 50-100% by test compounds and positive controls alike. 468
Figure captions Vertical bars
484
represent the standard deviation of each data point (n=2). 485
Figure 4: Molecular docking of COX-1 with compounds 1-5. Different compounds
486
were located in different active pockets. Compounds 1 and 4 were located in the
487
hydrophobic pocket A of COX-1 (Comp. 1 shown in green, Comp.4 shown in
488
yellow), compounds 2 and 3 were located in the pocket B of COX-1 (Comp.2 shown
489
in cyan, Comp.3 shown in magenta), while compound 5 was located in the pocket C
490
(Comp.5 shown in salmon). 485
Figure 4: Molecular docking of COX-1 with compounds 1-5. Different compounds
486
were located in different active pockets. Compounds 1 and 4 were located in the
487
hydrophobic pocket A of COX-1 (Comp. 1 shown in green, Comp.4 shown in
488
yellow), compounds 2 and 3 were located in the pocket B of COX-1 (Comp.2 shown
489
in cyan, Comp.3 shown in magenta), while compound 5 was located in the pocket C
490
(Comp.5 shown in salmon). 491
Figure 5: Molecular docking of COX-2 with compounds 1-5. Different compounds
492
were located in different active pockets. Compounds 1, 2, 3 and 4 were located in the
493
hydrophobic pocket A of COX-2 (Comp. 1 shown in green, Comp.2 shown in cyan,
494
Comp.3 shown in magenta, Comp.4 shown in yellow), while compound 5 was located
495
in the pocket B (Comp.5 shown in salmon). 496
497
Figure 6: 3-D docked poses of COX-1 and -2 with five sterols (compounds 1-5). The
498
first column represents COX-1 and the second column represents COX-2. The rows
499
present: A) compound 1, B) compound 2, C) compound 3, D) compound 4, E)
500
compound 5. 501
502
503 497
Figure 6: 3-D docked poses of COX-1 and -2 with five sterols (compounds 1-5). The
498
first column represents COX-1 and the second column represents COX-2. The rows
499
present: A) compound 1, B) compound 2, C) compound 3, D) compound 4, E)
500
compound 5. 23 . CC-BY 4.0 International license
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Figure captions It is made available under a
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505
Figure 1: Chemical structures of the biologically active sterols (compounds 1-5)
506
isolated from the hexane extract of L. japonica. 504
505
Figure 1: Chemical structures of the biologically active sterols (compounds 1-5)
506
isolated from the hexane extract of L. japonica. 504
505
Figure 1: Chemical structures of the biologically active sterols (compounds 1-5)
506
isolated from the hexane extract of L. japonica. 507 24 . CC-BY 4.0 International license
perpetuity. It is made available under a
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bioRxiv preprint 508
509
Figure 2: Cyclooxygenase enzymes (COX-1 and -2) enzyme inhibitory activities of
510
sterols (1-5) from the hexane extract of L. japonica and cholesterol were tested at 25
511
μg/mL concentration. Commercial NSAIDs aspirin, ibuprofen, Celebrex® and
512
naproxen used as positive control and tested at 108, 12, 1 and 12 µg/mL, respectively. 513
The varying concentrations of positive controls used were to yield a comparable
514
activity profiles between 50-100% by test compounds and positive controls alike. 515
Vertical bars represent the standard deviation of each data point (n=2). 508
509
Figure 2: Cyclooxygenase enzymes (COX-1 and -2) enzyme inhibitory activities of
510
sterols (1-5) from the hexane extract of L. japonica and cholesterol were tested at 25
511
μg/mL concentration. Commercial NSAIDs aspirin, ibuprofen, Celebrex® and
512
naproxen used as positive control and tested at 108, 12, 1 and 12 µg/mL, respectively. 513
The varying concentrations of positive controls used were to yield a comparable
514
activity profiles between 50-100% by test compounds and positive controls alike. 515
Vertical bars represent the standard deviation of each data point (n=2). 25 . 468
Figure captions CC-BY 4.0 International license
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bioRxiv preprint 516
517
Figure 3: Lipid peroxidation (LPO) inhibitory activities of sterols (1-5) isolated from
518
the hexane extract of L. japonica and commercial cholesterol tested at 25 μg/mL. 519
Commercial antioxidants BHA, BHT and TBHQ used as positive controls at 1.8, 2.2
520
and 1.6 µg/mL. The oxidation of lipid was initiated by the addition of Fe2+ ions. The
521
varying concentrations of positive controls used were to yield a comparable activity
522
profiles between 50-100% by test compounds and positive controls alike. Vertical bars
523
represent the standard deviation of each data point (n=2). 516 517
Figure 3: Lipid peroxidation (LPO) inhibitory activities of sterols (1-5) isolated from
518
the hexane extract of L. japonica and commercial cholesterol tested at 25 μg/mL. 519
Commercial antioxidants BHA, BHT and TBHQ used as positive controls at 1.8, 2.2
520
and 1.6 µg/mL. The oxidation of lipid was initiated by the addition of Fe2+ ions. The
521
varying concentrations of positive controls used were to yield a comparable activity
522
profiles between 50-100% by test compounds and positive controls alike. Vertical bars
523
represent the standard deviation of each data point (n=2). 524 26 . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
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bioRxiv preprint . CC-BY 4.0 International license
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
this version posted October 11, 2021. 468
Figure captions ;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint 525
526
Figure 4: Molecular docking of COX-1 with compounds 1-5. Different compounds
527
were located in different active pockets. Compounds 1 and 4 were located in the
528
hydrophobic pocket A of COX-1 (Comp. 1 shown in green, Comp.4 shown in
529
yellow), compounds 2 and 3 were located in the pocket B of COX-1 (Comp.2 shown
530
in cyan, Comp.3 shown in magenta), while compound 5 was located in the pocket C
531
(Comp.5 shown in salmon). 526
Figure 4: Molecular docking of COX-1 with compounds 1-5. Different compounds
527
were located in different active pockets. Compounds 1 and 4 were located in the
528
hydrophobic pocket A of COX-1 (Comp. 1 shown in green, Comp.4 shown in
529
yellow), compounds 2 and 3 were located in the pocket B of COX-1 (Comp.2 shown
530
in cyan, Comp.3 shown in magenta), while compound 5 was located in the pocket C
531
(Comp.5 shown in salmon). 27 . CC-BY 4.0 International license
perpetuity. It is made available under a
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this version posted October 11, 2021. ;
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doi:
bioRxiv preprint 532
533
Figure 5: Molecular docking of COX-2 with compounds 1-5. Different compounds
534
were located in different active pockets. Compounds 1, 2, 3 and 4 were located in the
535
hydrophobic pocket A of COX-2 (Comp. 1 shown in green, Comp.2 shown in cyan,
536
Comp.3 shown in magenta, Comp.4 shown in yellow), while compound 5 was located
537
in the pocket B (Comp.5 shown in salmon). 532
533
Figure 5: Molecular docking of COX-2 with compounds 1-5. Different compounds
534
were located in different active pockets. Compounds 1, 2, 3 and 4 were located in the
535
hydrophobic pocket A of COX-2 (Comp. 1 shown in green, Comp.2 shown in cyan,
536
Comp.3 shown in magenta, Comp.4 shown in yellow), while compound 5 was located
537
in the pocket B (Comp.5 shown in salmon). 468
Figure captions 28 . CC-BY 4.0 International license
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this version posted October 11, 2021. ;
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bioRxiv preprint Figure 6: 3-D docked poses of COX-1 and -2 with five sterols (compounds 1-5). The
first column represents COX-1 and the second column represents COX-2. The rows
present: A) compound 1, B) compound 2, C) compound 3, D) compound 4, E)
compound 5. 539
Figure 6: 3-D docked poses of COX-1 and -2 with five sterols (compounds 1-5). The
540
first column represents COX-1 and the second column represents COX-2. The rows
541
present: A) compound 1, B) compound 2, C) compound 3, D) compound 4, E)
542
compound 5. 29 . CC-BY 4.0 International license
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bioRxiv preprint 543
Graphical abstract 543
Graphical abstract 544 544
545 545
546 30 . CC-BY 4.0 International license
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
this version posted October 11, 2021.
;
https://doi.org/10.1101/2021.10.11.463984
doi:
bioRxiv preprint 547
Highlights 548
1. Sterols 29-hydroperoxy-stigmasta-5,24(28)-dien-3β-ol and 24-hydroperoxy-24-
549
vinyl-cholesterol are identified for the first time in L. japonica. 550
2. Saringosterol, 24-methylenecholesterol and fucosterol showed strong LPO
551
inhibitory activity. 552
3. Fucosterol showed highest binding affinity for COX-1 and -2 enzymes through
553
hydrophobic interactions. 554 548
1. Sterols 29-hydroperoxy-stigmasta-5,24(28)-dien-3β-ol and 24-hydroperoxy-24- 549
vinyl-cholesterol are identified for the first time in L. japonica. 31
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https://openalex.org/W2767975443
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https://europepmc.org/articles/pmc5766906?pdf=render
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English
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Pregnane X receptor promotes ethanol-induced hepatosteatosis in mice
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Journal of biological chemistry/The Journal of biological chemistry
| 2,018
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cc-by
| 14,436
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Pregnane X receptor promotes ethanol-induced
hepatosteatosis in mice Despite intense research efforts in the past decade to under-
stand the pathogenesis of EtOH–induced liver damage, there
are no Food and Drug Administration-approved treatments for
ALD. The development of an ALD treatment is hindered both
by a limited understanding of the molecular mechanisms
involved in EtOH–induced liver damage and the lack of animal
models that recapitulate advanced ALD. Although cumulative
alcohol intake correlates with liver disease, only 35% of alcohol
abusers develop more severe forms of ALD, such as advanced
fibrosis and cirrhosis, suggesting the contribution of genetic
factors (1, 3). To date, only genes encoding the principal alco-
hol-metabolizing enzymes alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (ALDH) have been linked to alcohol-
ism and ALD (4–7). Interestingly, a recent report indicates that
the nuclear receptors (NRs) peroxisome proliferator–activated
receptor (PPAR) and are linked with alcohol consumption
in mice and withdrawal and dependence in humans (8). consume ethanol (EtOH) develop steatosis (fatty liver), the first
hit, which can advance to inflammation, fibrosis, cirrhosis, and
even liver cancer (1). Thus, it was suggested that the prevention
or suppression of EtOH–induced steatosis may block or delay
the progression of fatty liver to hepatitis and fibrosis (2). Despite intense research efforts in the past decade to under-
stand the pathogenesis of EtOH–induced liver damage, there
are no Food and Drug Administration-approved treatments for
ALD. The development of an ALD treatment is hindered both
by a limited understanding of the molecular mechanisms
involved in EtOH–induced liver damage and the lack of animal
models that recapitulate advanced ALD. Although cumulative
alcohol intake correlates with liver disease, only 35% of alcohol
abusers develop more severe forms of ALD, such as advanced
fibrosis and cirrhosis, suggesting the contribution of genetic
factors (1, 3). To date, only genes encoding the principal alco-
hol-metabolizing enzymes alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (ALDH) have been linked to alcohol-
ism and ALD (4–7). Interestingly, a recent report indicates that
the nuclear receptors (NRs) peroxisome proliferator–activated
receptor (PPAR) and are linked with alcohol consumption
in mice and withdrawal and dependence in humans (8). The involvement of NRs in the pathogenesis of ALD has been
demonstrated (9–11). Specifically, knock-out mice lacking ret-
inoid X receptor (RXR) in hepatocytes, PPAR, constitutive
androstane receptor (CAR), and the farnesoid X receptor (FXR)
are all highly susceptible to liver injury induced by EtOH (9, 10,
12–14). This work was supported by National Institutes of Health Grants U54
AA019765, 1U54MD012392, P20 MD000175, and U01-021898 and in part
by the NCI Intramural Research Program of the National Institutes of
Health. The authors declare that they have no conflicts of interest with
the contents of this article. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the
National Institutes of Health. Pregnane X receptor promotes ethanol-induced
hepatosteatosis in mice Received for publication,August 29, 2017, and in revised form, November 2, 2017 Published, Papers in Press, November 9, 2017, DOI 10.1074/jbc.M117.815217
Sora Choi‡, Prince Neequaye‡, Samuel W. French§, Frank J. Gonzalez¶, and Maxwell A. Gyamfi‡1
From the ‡Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, North
Carolina 27707, the §Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90509, and the ¶Laboratory of
Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 Edited by Jeffrey E. Pessin The pregnane X receptor (PXR, NR1I2) is a xenobiotic-sens-
ing nuclear receptor that modulates the metabolic response to
drugs and toxic agents. Both PXR activation and deficiency pro-
mote hepatic triglyceride accumulation, a hallmark feature of
alcoholic liver disease. However, the molecular mechanism of
PXR-mediated activation of ethanol (EtOH)-induced steatosis
is unclear. Here, using male wildtype (WT) and Pxr-null mice,
we examined PXR-mediated regulation of chronic EtOH–
induced hepatic lipid accumulation and hepatotoxicity. EtOH
ingestion for 8 weeks significantly (1.8-fold) up-regulated Pxr
mRNA levels in WT mice. The EtOH exposure also increased
mRNAs encoding hepatic constitutive androstane receptor
(3-fold) and its target, Cyp2b10 (220-fold), in a PXR-dependent
manner. Furthermore, WT mice had higher serum EtOH levels
and developed hepatic steatosis characterized by micro- and mac-
rovesicular lipid accumulation. Consistent with the development
of steatosis, lipogenic gene induction was significantly increased in
WT mice, including sterol regulatory element–binding protein 1c
target gene fatty-acid synthase (3.0-fold), early growth response-1
(3.2-fold), and TNF (3.0-fold), whereas the expression of peroxi-
some proliferator–activated receptor target genes was sup-
pressed. Of note, PXR deficiency suppressed these changes and
steatosis. Protein levels, but not mRNAs levels, of EtOH-metabo-
lizingenzymes,includingalcoholdehydrogenase1,aldehydedehy-
drogenase 1A1, and catalase, as well as the microsomal triglyceride
transfer protein, involved in regulating lipid output were higher in
Pxr-null than in WT mice. These findings establish that PXR sig-
naling contributes to ALD development and suggest that PXR
antagonists may provide a new approach for ALD therapy. consume ethanol (EtOH) develop steatosis (fatty liver), the first
hit, which can advance to inflammation, fibrosis, cirrhosis, and
even liver cancer (1). Thus, it was suggested that the prevention
or suppression of EtOH–induced steatosis may block or delay
the progression of fatty liver to hepatitis and fibrosis (2). gy
2 The abbreviations used are: ALD, alcoholic liver disease; PXR, pregnane X
receptor; NR, nuclear receptor; CAR, constitutive androstane receptor; FXR,
farnesoid X receptor; SHP, small heterodimer partner; EtOH, ethanol; ALT,
alanine aminotransferase; PPAR, peroxisome proliferator–activated recep- tor; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; NEFA,
non-esterified fatty acid; BEC, blood ethanol concentration; MTP, micro-
somal triglyceride transfer protein; CAT, catalase; IBD, inflammatory bowel
disease; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; hPXR,
PXR-humanized; NAFLD, non-alcoholic fatty liver disease; ER, endoplasmic
reticulum; HFD, high-fat diet. 1 Towhomcorrespondenceshouldbeaddressed:Cardio-metabolicResearch
Program, JLC-Biomedical/Biotechnology Research Institute, North Caro-
lina Central University, 700 George St., Durham, NC 27707. Tel.: 919-530-
7832; Fax: 919-530-6815; E-mail: mgyamfi@nccu.edu. ARTICLE ARTICLE Pregnane X receptor promotes ethanol-induced
hepatosteatosis in mice Furthermore, ligands of the NRs, including PPAR,
PPAR, PPAR/, and hepatocyte nuclear factor-4 (HNF4)
were reported to attenuate EtOH–induced hepatic steatosis,
insulin resistance, and hepatotoxicity (16–19). Unexpectedly,
previous reports indicate that pharmacological activation of
mouse CAR by its potent activator, 1,4-bis[2-(3,5-dichloropyri-
dyloxy)] benzene (TCPOBOP), enhances both acute and
chronic EtOH–induced lipid accumulation, oxidative stress,
and liver injury (12). Furthermore, CAR activation also increased
both methionine- and choline-deficient diets and acetamino-
phen-induced hepatotoxicities (20, 21). Interestingly, although
pregnane X receptor (PXR) and CAR are considered to be sister Alcoholic liver disease (ALD)2 is a common pathology of
excessive alcohol use. About 90% of individuals who chronically gy
2 The abbreviations used are: ALD, alcoholic liver disease; PXR, pregnane X
receptor; NR, nuclear receptor; CAR, constitutive androstane receptor; FXR,
farnesoid X receptor; SHP, small heterodimer partner; EtOH, ethanol; ALT,
alanine aminotransferase; PPAR, peroxisome proliferator–activated recep- J. Biol. Chem. (2018) 293(1) 1–17 1 cro cro Table 1 The major
target of genes of PXR are those involved in xenobiotic metab-
olism and transport, including the cytochrome P450 (CYP)
enzymes and the ATP-binding cassette drug transporters (24). Recent reports indicate that activation of PXR inhibits nuclear
factor B, inflammation, and fibrosis (25–28). In addition to
these beneficial effects, PXR activation has also been implicated
in gut dysbiosis and metabolic abnormalities, tumor promo-
tion, chemoresistance, and response of cancer cells to chemo-
therapeutic agents (29–31). Furthermore, PXR activation can
also result in hepatic triglyceride accumulation and reactive
metabolite formation leading to drug-induced liver injury (32,
33). Finally, a recent report indicates that human PXR gene
variants are associated with liver injury in non-alcoholic fatty
liver disease (NAFLD), in which PXR polymorphisms increase
the risk of susceptibility to a more severe form of NAFLD (34). Data represent mean S.D. (n 6–7). Parameter
Wildtype (C57BL/6)
PXR-KO
Control
(n 7)
EtOH
(n 7)
Control
(n 6)
EtOH
(n 7)
Initial body weight (g)
23.2 1.6
24.2 1.7
25.4 1.3
24.5 2.5
Week 8 body weight (g)
34.1 4.6
28.2 1.8a
34.3 1.4
28.2 1.6a
Absolute liver weight (g) 1.6 0.3
1.4 0.2
1.7 0.2
1.4 0.2
Liver/body weight (%)
4.6 0.5
4.9 0.4
4.9 0.5
4.9 0.3
Macrovesicular steatosis
1.0 0.8
2.4 1.1a
0.3 0.5
0.4 0.5b
Microvesicular steatosis
0.0 0.0
2.1 1.1a
0.3 0.5
0.4 0.5b
Necrosis score
0.1 0.4
0.7 1.1
0.0 0.0
0.0 0.0
a p 0.05 was between mice fed control diet and EtOH. b p 0.05 was between mice fed EtOH. which were higher in WT mice but absent in Pxr-null mice
(Table 1 and Fig. 1, A–C). Histologic scoring via H&E staining
of lipid accumulation showed that both microvesicular (multi-
ple smaller vacuoles in each hepatic cell without significant
nuclear displacement) and macrovesicular steatosis (observed
as a single vacuole displacing the nucleus in each hepatic cell) as
well as mild necrosis were present in EtOH-fed WT mice (Table
1 and Fig. 1, A–C). In contrast, lipid droplets and necrosis were
absent in the livers of EtOH-fed Pxr-null mice (Fig. 1A). Table 1 Table 1
Effect of chronic EtOH ingestion on body weight, liver weight, and
pathology score deficiency also promotes EtOH–induced hepatotoxicity (22). The PXR, a xenobiotic-sensing nuclear receptor expressed
primarily in the liver and intestine, is involved in the detoxifi-
cation of drugs and other harmful substances (23). The major
target of genes of PXR are those involved in xenobiotic metab-
olism and transport, including the cytochrome P450 (CYP)
enzymes and the ATP-binding cassette drug transporters (24). Recent reports indicate that activation of PXR inhibits nuclear
factor B, inflammation, and fibrosis (25–28). In addition to
these beneficial effects, PXR activation has also been implicated
in gut dysbiosis and metabolic abnormalities, tumor promo-
tion, chemoresistance, and response of cancer cells to chemo-
therapeutic agents (29–31). Furthermore, PXR activation can
also result in hepatic triglyceride accumulation and reactive
metabolite formation leading to drug-induced liver injury (32,
33). Finally, a recent report indicates that human PXR gene
variants are associated with liver injury in non-alcoholic fatty
liver disease (NAFLD), in which PXR polymorphisms increase
the risk of susceptibility to a more severe form of NAFLD (34). Valid animal models are a prerequisite for developing new
drug treatment for ALD. Recently, two independent groups
have generated mice that lack PXR to study the physiological or
functional significance of PXR in xenobiotic regulation (35, 36). Although none of the two lines of Pxr-null mice has been eval-
uated for EtOH–induced hepatotoxicity, recent reports indi-
cate that PXR deficiency protects mice against high-fat diet
(HFD)-induced obesity (37–39). In addition, HFD-fed male
obese wildtype (WT) mice had more severe liver injury than
male obese Pxr-null mice (38). Thus, PXR may potentiate the
transition from fatty liver to steatohepatitis. Accumulating evi-
dence indicates that PXR may have roles in lipid metabolism
(33, 40, 41). Interestingly, loss of PXR and PXR activation leads
to hepatic lipid accumulation, an important feature of ALD (41,
42). Furthermore, reports indicate that EtOH can induce both
PXR and its target Cyp3a11/CYP3A gene in rodents, human
primary hepatocytes, and human liver samples of alcoholics
(43–45). Currently, little is known regarding the molecular
mechanism of PXR-mediated activation of EtOH–induced
steatosis. In this study, male WT and Pxr-null mice were fed control The PXR, a xenobiotic-sensing nuclear receptor expressed
primarily in the liver and intestine, is involved in the detoxifi-
cation of drugs and other harmful substances (23). Table 1 In
agreement with the morphology data, after EtOH treatment,
hepatic triglyceride and NEFA levels were significantly higher
only in WT mice and not in EtOH-fed Pxr-null mice (Fig. 1, B
and C). However, inflammation and fibrosis were absent in
both EtOH-fed genotypes (data not shown). These data indi-
cate that the PXR promotes chronic EtOH–induced steatosis
and hepatic triglyceride accumulation. p
y
(
)
Valid animal models are a prerequisite for developing new
drug treatment for ALD. Recently, two independent groups
have generated mice that lack PXR to study the physiological or
functional significance of PXR in xenobiotic regulation (35, 36). Although none of the two lines of Pxr-null mice has been eval-
uated for EtOH–induced hepatotoxicity, recent reports indi-
cate that PXR deficiency protects mice against high-fat diet
(HFD)-induced obesity (37–39). In addition, HFD-fed male
obese wildtype (WT) mice had more severe liver injury than
male obese Pxr-null mice (38). Thus, PXR may potentiate the
transition from fatty liver to steatohepatitis. Accumulating evi-
dence indicates that PXR may have roles in lipid metabolism
(33, 40, 41). Interestingly, loss of PXR and PXR activation leads
to hepatic lipid accumulation, an important feature of ALD (41,
42). Furthermore, reports indicate that EtOH can induce both
PXR and its target Cyp3a11/CYP3A gene in rodents, human
primary hepatocytes, and human liver samples of alcoholics
(43–45). Currently, little is known regarding the molecular
mechanism of PXR-mediated activation of EtOH–induced
steatosis. Published in the U.S.A. Published in the U.S.A. Chronic EtOH ingestion significantly up-regulated mRNA
levels of PXR and CAR and CAR target gene Cyp2b10 in WT
mice Chronic EtOH ingestion significantly up-regulated Pxr
mRNA expression (1.8-fold) in WT mice, but not in Pxr-null
mice, confirming the absence of Pxr mRNA in the Pxr-null mice
(Fig. 2A). The basal hepatic Car mRNA levels did not vary
between WT and Pxr-null mice suggesting that Car gene
expression does not appear to be dependent on PXR but that
EtOH–induced up-regulation of Car mRNA might be. Chronic
EtOH exposure induced the hepatic Car mRNA 2.9-fold only in
WT mice (Fig. 2B). The Fxr and small heterodimer partner
(Shp) basal mRNA levels did not differ between the two geno-
types (Fig. 2, C and D). However, chronic EtOH significantly
reduced both Fxr and Shp mRNA levels, each by 61% in WT
mice compared with their respective control-fed mice. In con-
trast, EtOH decreased Shp mRNA by 40% but not Fxr mRNA
levels in Pxr-null mice (Fig. 2, C and D). The basal hepatic gene
expression of the PXR target gene, Cyp3a11, was significantly
increased in Pxr-null mice (3.0-fold) compared with WT con-
trols (Fig. 2E), as reported previously (35). Chronic EtOH inges-
tion non-significantly induced the Cyp3a11 gene (1.6-fold) in
WT mice and had no effect in Pxr-null mice (Fig. 2E). Further-
more, the basal hepatic mRNA levels of the specific PXR target
genes Slco1a4 (Oatp2) and Cyp7a1 (47) were lower, 39 and 57%,
respectively, compared with their respective WT controls (Fig. 2, F and G). EtOH had no effect on Cyp7a1 and Sclo1a4 mRNA In this study, male WT and Pxr-null mice were fed control
(pair-fed) or the standard Lieber-Decarli liquid diet containing
5% EtOH for 8 weeks and were evaluated for changes in hepatic
gene expression, histology, and hepatotoxicity. 2
J. Biol. Chem. (2018) 293(1) 1–17 PXR and chronic ethanol-induced steatosis NRs with overlapping functions, it is not known whether PXR
deficiency also promotes EtOH–induced hepatotoxicity (22). Table 1
Effect of chronic EtOH ingestion on body weight, liver weight, and
pathology score EtOH induces lipid accumulation in WT but not Pxr-null mice During the 8-week feeding period, whereas food intake was
similar in all groups, a significant decrease in body weight was
observed in both EtOH-fed WT and Pxr-null mice, compared
with their respective controls (Table 1) and consistent with
what was observed by others (46). However, liver weight and
liver-to-body weight ratios were not significantly different
between the control and EtOH-fed groups of both genotypes
(Table 1). Hematoxylin and eosin (H&E) staining revealed that
the genotype influenced EtOH–induced hepatic lipid droplet
accumulation, histology score, triglycerides, and NEFA levels, PXR and chronic ethanol-induced steatosis Figure 1. Characterization of hepatic histology and hepatic lipids in male WT and Pxr-null mice pair-fed control diets or EtOH-containing diets. Male
WT and Pxr-null mice were pair-fed control diets (control) or standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric
intake) for 8 weeks. H&E staining (original magnification 400) (A), hepatic triglyceride levels (B), and non-esterified fatty acid (NEFA) levels (C) from different
treatment groups were performed as described under “Experimental procedures.” Chronic EtOH feeding produced both macrovesicular (indicated by arrows)
and microvesicular steatosis (indicated by arrowheads) only in the WT mice. Data represent mean S.D. (n 5–6). #, p 0.05 between mice fed control diet
and EtOH. †, p 0.05 between mice fed EtOH. Figure 1. Characterization of hepatic histology and hepatic lipids in male WT and Pxr-null mice pair-fed control diets or EtOH-containing diets. Male
WT and Pxr-null mice were pair-fed control diets (control) or standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric
intake) for 8 weeks. H&E staining (original magnification 400) (A), hepatic triglyceride levels (B), and non-esterified fatty acid (NEFA) levels (C) from different
treatment groups were performed as described under “Experimental procedures.” Chronic EtOH feeding produced both macrovesicular (indicated by arrows)
and microvesicular steatosis (indicated by arrowheads) only in the WT mice. Data represent mean S.D. (n 5–6). #, p 0.05 between mice fed control diet
and EtOH. †, p 0.05 between mice fed EtOH. levels in Pxr-null mice but decreased Cyp7a1 mRNA levels by
69% in WT mice without any effect on Sclo1a4 mRNA levels
(Fig. 2, F and G). The basal Cyp2b10 mRNA levels did not differ
between the two genotypes (Fig. 2H). EtOH induces lipid accumulation in WT but not Pxr-null mice Even though EtOH
induced Car mRNA expression by about 3-fold in WT mice, the
mRNA of its target gene Cyp2b10 was increased dramatically
(about 220-fold) in WT mice but not in EtOH-fed Pxr-null mice
(Fig. 2, B and H). In agreement with the increased Cyp2b10 gene
expression by EtOH, CYP2B10 protein levels were significantly
higher in EtOH-fed WT mice (27-fold) compared with WT
controls (Fig. 2I). null mice (Fig. 3A). The basal hepatic mRNA levels of the
SREBP-1C target genes fatty acid synthase (Fas), acetyl-CoA
carboxylase 1 (Acc-1), and stearoyl-CoA desaturase 1 (Scd1)
were not different between the two genotypes, and EtOH did
not have any significant effects on Acc-1 and Scd1 mRNAs in
both genotypes (Fig. 3, B–D). In contrast, EtOH significantly
increased Fas mRNA (2.9-fold) only in WT mice (Fig. 3B). Con-
stitutive Srebp2 mRNA levels did not vary between the two
genotypes, and their mRNA levels were not different after
EtOH treatment (Fig. 3E). Similarly, basal Egr-1 mRNA and
protein levels did not vary between the two genotypes. How-
ever, EtOH treatment significantly increased Egr-1 mRNA (3.2-
fold) and protein (12.3-fold) levels only in WT mice (Fig. 3, F
and G). PXR deficiency suppresses chronic EtOH–induced lipogenic
gene induction The steatosis observed in EtOH-fed WT mice prompted us
to examine the expression of Srebp-1c and early growth
response-1 (Egr-1), both of which have been implicated in
EtOH–induced steatosis (48, 49). Basal hepatic Srebp-1c
mRNA levels were higher (1.9-fold) in control-fed Pxr-null
mice compared with their WT counterparts (Fig. 3A). EtOH
non-significantly increased Srebp-1c mRNA levels in WT mice
(1.6-fold) compared with control-fed WT mice (Fig. 3A). In
contrast, EtOH decreased Srebp-1c mRNA levels by 48% in Pxr- PXR deficiency protects against chronic EtOH–induced
suppression of hepatic fatty acid -oxidation genes Ppar mRNA and its target genes carnitine palmitoyltrans-
ferase 1 (Cpt1), acyl-CoA oxidase 1 (Acox-1), liver fatty
acid-binding protein 1 (Lfabp-1), and microsomal triglyceride
transfer protein (Mtp) mRNA levels were comparable between
control WT and Pxr-null mice (Fig. 4, A–E). EtOH did not
significantly affect Ppar and Mtp mRNA levels in both WT 4
J. Biol. Chem. (2018) 293(1) 1–17 PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis and Pxr-null mice (Fig. 4A). However, chronic EtOH ingestion
significantly decreased Cpt-1, Acox-1, and Lfabp-1 mRNA lev-
els by 38, 64, and 56%, respectively, only in WT mice (Fig. 4,
B–D). Even though the basal hepatic Mtp mRNA levels were
not different between the two genotypes, the constitutive MTP
protein levels were significantly higher in Pxr-null mice (2.4-
fold) (Fig. 4, E and F). Furthermore, after EtOH ingestion,
hepatic MTP protein levels were significantly higher in Pxr-null
mice than in WT mice (Fig. 4F). both control-fed WT and Pxr-null mice, whereas EtOH signif-
icantly increased Ucp2 mRNA levels only in WT mice (3.3-fold)
(Fig. 7C). Expression of hepatic ER stress marker GRP78 pro-
tein levels was significantly up-regulated 1.7-fold in EtOH-fed
WT mice but not in Pxr-null mice (Fig. 7D). Interestingly, after
EtOH treatment, hepatic GRP78 protein levels were signifi-
cantly higher in WT mice than in Pxr-null mice (Fig. 7D). Basal
expression of hepatic apoptotic protein Bax was elevated in
Pxr-null mice (4.2-fold) compared with control-fed WT mice
(Fig. 7E). Although EtOH significantly decreased Bax protein
levels in Pxr-null mice, it up-regulated the Bax protein in WT
mice (2.9-fold), although the increase was not statistically sig-
nificant (Fig. 7E). Discussion This study investigated the effect of chronic EtOH adminis-
tration on steatosis, hepatotoxicity, hepatic lipid profiles, lipid
and alcohol metabolism pathways, PXR target genes, and
inflammatory and cellular stress gene and protein expression in
Pxr-null mice. The results demonstrated that when exposed to
chronic EtOH, mice that lacked PXR showed dramatically
reduced levels of hepatic steatosis; the reduced levels of histo-
logic score for steatosis, hepatic triglycerides, and NEFA
and serum ALT were consistent with this. Also, dramatically
reduced were serum EtOH levels in Pxr-null mice, suggesting
that compensatory mechanisms exist in the absence of PXR
that counteract EtOH–induced steatosis and reduce blood
EtOH concentration (BEC). Among 27 genes and 11 associated
proteins known to function in hepatotoxicity, lipogenesis, Protein but not gene expression of EtOH-metabolizing
enzymes is altered by EtOH in mice with differential PXR
expression The basal hepatic alcohol dehydrogenase (Adh1), aldehyde
dehydrogenase 2 (Aldh2), Aldh1b1 and Aldh1a1, and catalase
(Cat) mRNA levels did not vary significantly between the two
genotypes, and EtOH did not significantly affect their mRNA
levels (Fig. 5, A–E). However, after EtOH treatment, Aldh1a1
mRNA levels in Pxr-null mouse livers were significantly higher
than in WT mice (Fig. 5D). CYP2E1 protein expression in the
liver did not differ between control WT and Pxr-null mice (Fig. 6A). However, CYP2E1 protein expression was up-regulated by
EtOH treatment in WT mice (2.4-fold) and Pxr-null mice (2.6-
fold) (Fig. 6A). Similar to their gene expression, the basal
hepatic ALDH2 and ALDH1B1 protein levels did not differ
between the two genotypes; however, the basal hepatic ADH1,
ALDH1A1, and CAT protein levels were significantly higher,
20-, 4.6-, and 2.8-fold in Pxr-null than in WT mice (Fig. 6, B, E,
and F). Although EtOH significantly increased hepatic ALDH2
protein levels in WT mice (1.4-fold), it had no significant effect
on the levels in Pxr-null mice (Fig. 6C). In contrast, EtOH main-
tained the significantly higher hepatic ADH1, ALDH1A1, and
CAT protein levels in Pxr-null mice, and both ADH1 and
ALDH1A1 protein levels were significantly higher in EtOH-fed
Pxr-null mice than in EtOH-fed WT mice (Fig. 6, B, E, and F). Figure 2. Hepatic gene expression and immunoblot analysis of mouse type II nuclear receptors and their target genes important for xenobiotic
metabolism and lipid homeostasis. Male WT and Pxr-null mice were pair-fed control diets (control) or standard Lieber-Decarli liquid diet containing 5% EtOH
(representing 27.5% of the total caloric intake) for 8 weeks. Pregnane X receptor (Pxr) (A), constitutive androstane receptor (Car) (B), farnesoid X receptor (Fxr)
(C), small heterodimer partner (Shp) (D), Cyp3a11 (E), solute carrier organic anion transporter family, member 1a4 (Slco1a4) (F), Cyp7a1 (G), Cyp2b10 (H), and
Gapdh mRNAs were quantified as described under “Experimental procedures.” Data represent mean S.D. (n 4–6). Furthermore, Western blottings of liver
homogenate (40 g/lane) were probed with antibodies to CYP2B10 (I). The induction of the CYP2B10 protein expression by EtOH only in WT mice was
unexpected, so we decided to probe for different target proteins using the same blot. Following initial probing with CYP2B10 antibody, this blot was stripped
and reprobed with primary antibodies against ALDH2 and ALDH1A1 in Fig. 6, C and E. Finally, the blot was stripped and reprobed with -tubulin antibody.
Bands were quantified and normalized to -tubulin. Data represent mean S.D. (n 3–4). *, p 0.05 between WT and Pxr-null mice fed control. #, p 0.05
between mice fed control diet and EtOH. †, p 0.05 between mice fed EtOH. Residual EtOH concentrations are significantly higher in WT
mice compared with their Pxr-null counterparts The basal serum triglyceride concentration tended to be
higher in Pxr-null mice than in WT mice (Fig. 8A). However,
EtOH did not have any significant effect on the serum triglyc-
eride levels in both genotypes (Fig. 8A). Serum ALT activity was
measured as an index of hepatocyte injury. Serum ALT activity
was not induced in EtOH-fed Pxr-null mice, but the levels were
increased 2.3-fold in EtOH-fed WT mice, although not statis-
tically significantly (Fig. 8B). However, after EtOH treatment,
serum ALT levels were significantly higher in WT mice than in
Pxr-null mice (Fig. 8B). To determine the impact on EtOH
metabolism of elevated levels of ADH1, ALDH1A1, and CAT
protein expression observed in Pxr-null mice, residual concen-
trations of EtOH were measured after the 8 weeks of chronic
EtOH ingestion. The residual EtOH concentration was signifi-
cantly higher in WT mice compared with their Pxr-null coun-
terparts (Fig. 8C). 3 J. Biol. Chem. (2018) 293(1) 1–17 3 PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis 4
J. Biol. Chem. (2018) 293(1) 1–17 Pro-inflammatory cytokines and cellular stress markers are
up-regulated in EtOH-fed WT mice but not in Pxr-null mice To determine the effect of PXR deficiency on the expression
of inflammatory and endoplasmic reticulum (ER) stress medi-
ators during EtOH exposure, mRNA levels of several pro-in-
flammatory cytokines and ER stress markers were measured. Ethanol increased mRNAs encoding tumor necrosis factor
(Tnf, 3.0-fold) and toll-like receptor 7 (Tlr7, 3.0-fold) in WT
mice but not in Pxr-null mice (Fig. 7, A and B). After EtOH
treatment, both hepatic Tnf and Tlr7 mRNA levels were sig-
nificantly higher in WT mice than in Pxr-null mice (Fig. 7, A
and B). The basal hepatic levels of the oxidative stress marker
uncoupling protein 2 (Ucp2) mRNA were not different between 5 J. Biol. Chem. (2018) 293(1) 1–17 PXR and chronic ethanol-induced steatosis Previous reports indicate that both Cyp2b10 and its human
ortholog, CYP2B6, as well as murine CAR, can be directly reg-
ulated by PXR (51–53). It was hypothesized that PXR-mediated
activation of CAR could be involved in EtOH–induced hepato-
toxicity observed in this study. Indeed, Car mRNA expression
was up-regulated in WT mice exposed to chronic EtOH but not
in Pxr-null mice. Besides, this study also demonstrates a robust
Pxr-dependent induction of the mRNA and protein of the CAR
target gene Cyp2b10 in EtOH-fed WT mice. Expression of
Cyp2b10 is thought to be mediated by CAR activation (54). The
PXR-dependent effect of EtOH in WT mice may have clinical
relevance to ALD. Interestingly, previous reports have impli-
cated pharmacological activation of Car/Cyp2b10 and EtOH
infusion in suppression of EtOH metabolism, enhanced steato-
sis, oxidative stress, and hepatotoxicity (12). Furthermore, in a
recent study, while activation of CAR by its potent agonist,
TCPOBOP reversed EtOH–induced hyperbilirubinemia, it was
associated with marked hepatotoxicity (55). In contrast, Car-
null mice with dampened Cyp2b10 expression completely
inhibited jet-lag–induced steatosis and inflammatory gene
expression (56), suggesting that the Cyp2b10 induction by
EtOH in WT mice seen in this study as a result of PXR/CAR
cross-talk could be a major pathway for EtOH–induced
hepatotoxicity. Figure 3. Gene expression and immunoblot analysis of mouse hepatic
enzymes that promote lipogenesis Male WT and Pxr-null mice were pair- Figure 3. Gene expression and immunoblot analysis of mouse hepatic
enzymes that promote lipogenesis. Male WT and Pxr-null mice were pair-
fedcontroldiets(control)orstandardLieber-Decarliliquiddietcontaining5%
EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Sterol regu-
latory element–binding protein 1c (Srebp-1c) (A), fatty-acid synthase (Fas) (B),
acetyl-CoA carboxylase 1 (Acc-1) (C), stearoyl-CoA desaturase 1 (Scd1) (D),
Srebp2 (E), early growth response-1 (Egr-1) (F), and Gapdh mRNAs were quan-
tified as described under “Experimental procedures.” Data represent mean
S.D. (n 4–6). Western blottings of liver homogenate (40 g/lane) were
probed with antibodies to EGR-1 (G). Bands were quantified and normalized
to -tubulin. Data represent mean S.D. (n 3–4). *, p 0.05 between WT
and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and
EtOH. †, p 0.05 between mice fed EtOH. PXR and chronic ethanol-induced steatosis Figure 3. Gene expression and immunoblot analysis of mouse hepatic
enzymes that promote lipogenesis. Male WT and Pxr-null mice were pair-
fedcontroldiets(control)orstandardLieber-Decarliliquiddietcontaining5%
EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Sterol regu-
latory element–binding protein 1c (Srebp-1c) (A), fatty-acid synthase (Fas) (B),
acetyl-CoA carboxylase 1 (Acc-1) (C), stearoyl-CoA desaturase 1 (Scd1) (D),
Srebp2 (E), early growth response-1 (Egr-1) (F), and Gapdh mRNAs were quan-
tified as described under “Experimental procedures.” Data represent mean
S.D. (n 4–6). Western blottings of liver homogenate (40 g/lane) were
probed with antibodies to EGR-1 (G). Bands were quantified and normalized
to -tubulin. Data represent mean S.D. (n 3–4). *, p 0.05 between WT
and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and
EtOH †
0 05 b t
i
f d EtOH EtOH metabolism, and inflammation, five were strongly up-
regulated in response to chronic EtOH exposure in a PXR-
dependent manner; these included the Car and its target
Cyp2b10, Egr-1, Tnf, and Tlr7. Furthermore, the PPAR tar-
get genes Cpt-1, Acox-1, and Lfabp-1 as well as Fxr were sup-
pressed only in WT mice. The protein (but not the gene)
expression of ADH1, ALDH1A1, and CAT, three enzymes
involved in alcohol metabolism, as well as the lipid transfer
protein MTP, was significantly higher in Pxr-null mice, sug-
gesting post-transcriptional suppression of these enzyme
levels by a PXR-dependent mechanism. These results estab-
lish for the first time that PXR signaling during chronic
EtOH ingestion promotes steatosis, triglyceride accumula-
tion, and inflammation, while inhibiting EtOH oxidation,
suggesting that PXR deficiency is protective against chronic
EtOH–induced hepatotoxicity. Reports indicate that PXR and the closely related CAR cross-
talk by sharing response elements and overlapping affinities for
some ligands resulting in co-regulation of their target genes (22,
47). We first determined the effect of chronic EtOH on several
nuclear receptors and their target genes, including PXR. In this
study, no Pxr expression was in Pxr-null mice. Surprisingly,
although the Pxr gene was up-regulated by EtOH in WT mice,
the specific PXR target genes Slco1a4 (Oatp2) and Cyp7a1 were
not increased. Furthermore, the prototypical PXR target gene
Cyp3a11 (50), which can also be up-regulated by CAR, was
mildly induced (1.6-fold) but not significantly by EtOH (47). PXR and chronic ethanol-induced steatosis 6
J. Biol. Chem. (2018) 293(1) 1–17 PXR and chronic ethanol-induced steatosis Although the Pxr-null mice used in this study constitutively
express CAR, an intriguing and more unexpected finding was
the PXR-dependent robust increases in hepatic expression of
Cyp2b10 (220-fold) in WT mice but not in Pxr-null mice by
EtOH. The current results are consistent with an in vitro study
showing Cyp2b10 up-regulation is observed when the ratio
of PXR to CAR is high (57). However, Cyp2b10 repression 6 J. Biol. Chem. (2018) 293(1) 1–17 PXR and chronic ethanol-induced steatosis igure 4. Hepatic gene expression and immunoblot analysis of mouse Ppar and its target genes. Male WT and Pxr-null mice were pair-fed control diets
r standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Ppar (A), carnitine palmitoyltransferase
(Cpt1) (B), acyl-CoA oxidase-1 (Acox-1) (C), liver fatty acid-binding protein 1 (Lfabp-1) (D), microsomal triglyceride transport protein (Mtp) (E), and Gapdh
mRNAs were quantified as described under “Experimental procedures.” Data represent mean S.D. (n 5–6). Western blottings of liver homogenate (40
g/lane) were probed with antibodies to MTP (F). Following initial probing with the MTP antibody and the results showing elevation of basal hepatic MTP
roteininPxr-nullmice,thisblotwasstrippedandreprobedwithprimaryantibodyagainstCYP2E1,knowntobeinducedbyEtOHinFig.6A.Followingprobing,
he blot was finally stripped and reprobed with -tubulin antibody. Bands were quantified and normalized to -tubulin. Data represent mean S.D. (n 3–4). , p 0.05 between WT and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05 between mice fed EtOH. PXR and chronic ethanol induced steatosis Figure 4. Hepatic gene expression and immunoblot analysis of mouse Ppar and its target genes. Male WT and Pxr-null mice were pair-fed control diets
or standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Ppar (A), carnitine palmitoyltransferase
1 (Cpt1) (B), acyl-CoA oxidase-1 (Acox-1) (C), liver fatty acid-binding protein 1 (Lfabp-1) (D), microsomal triglyceride transport protein (Mtp) (E), and Gapdh
mRNAs were quantified as described under “Experimental procedures.” Data represent mean S.D. (n 5–6). Western blottings of liver homogenate (40
g/lane) were probed with antibodies to MTP (F). Following initial probing with the MTP antibody and the results showing elevation of basal hepatic MTP
proteininPxr-nullmice,thisblotwasstrippedandreprobedwithprimaryantibodyagainstCYP2E1,knowntobeinducedbyEtOHinFig.6A.Followingprobing,
the blot was finally stripped and reprobed with -tubulin antibody. Bands were quantified and normalized to -tubulin. Data represent mean S.D. (n 3–4). PXR and chronic ethanol-induced steatosis *, p 0.05 between WT and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05 between mice fed EtOH. play a pivotal role in the pathogenesis of ALD (62). Because
Cyp2b10 is a prototypical PXR/CAR target gene, it is important
to clarify whether PXR and/or CAR regulates EtOH–induced
Cyp2b10 activation. Thus, further studies are warranted using
Car-null mice together with Pxr/Car double-null mice to pro-
vide further insight into the precise roles of both PXR and CAR
in ALD pathogenesis. occurred when the PXR-to-CAR ratio was low, as found in Pxr-
null mice (57). Furthermore, other factors, including PXR, were
found to be required for optimal CYP2B activation (47). Similar
to the current findings, up-regulation of Cyp2b10 expression by
EtOH and by other toxicants has been reported (58, 59). Fur-
thermore, not only CYP2E1 but other CYPs, including CYP2B,
can be induced by alcohol hepatitis (60). It is also possible that
the marked induction of Cyp2b10 by EtOH in WT mice, may be
a source of reactive oxygen species and increased oxidative
stress as reported by others (61). Oxidative stress is known to Chronic alcohol ingestion contributes to the development of
fatty liver through multiple mechanisms. Furthermore, ligand-
dependent activation and knockdown of PXR both resulted in 8
J. Biol. Chem. (2018) 293(1) 1–17 J. Biol. Chem. (2018) 293(1) 1–17 7 gure 5. Gene expression of mouse hepatic enzymes involved in EtOH metabolism. Male WT and Pxr-null mice were pair-fed control diets or standard
eber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Alcohol dehydrogenase 1 (Adh1) (A), aldehyde
hydrogenase2(Aldh2)(B),Aldh1b1(C),Aldh1a1(D),catalase(Cat)(E),andGapdhmRNAswerequantifiedasdescribedunder“Experimentalprocedures.”Data
present mean S.D. (n 4–6). †, p 0.05 between mice fed EtOH. XR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis ronic ethanol-induced steatosis Figure 5. Gene expression of mouse hepatic enzymes involved in EtOH metabolism. Male WT and Pxr-null mice were pair-fed control diets or standard
Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Alcohol dehydrogenase 1 (Adh1) (A), aldehyde
dehydrogenase2(Aldh2)(B),Aldh1b1(C),Aldh1a1(D),catalase(Cat)(E),andGapdhmRNAswerequantifiedasdescribedunder“Experimentalprocedures.”Data
represent mean S.D. (n 4–6). †, p 0.05 between mice fed EtOH. enhanced steatosis of HepG2 cells (42). Although numerous
reports have shown that clinically relevant PXR ligands can
increase plasma lipid levels in patients (40), it was unclear
whether PXR signaling specifically contributes to EtOH–
induced lipid accumulation. Furthermore, the effect of EtOH
on livers of Pxr-null mice has not been reported. Our results
show that the expression of hepatic PXR was significantly
enhanced in EtOH–induced steatotic livers of WT mice fed
EtOH. Importantly, expression of fatty acid oxidation genes,
including Ppar, Cpt1, Acox-1, and Lfabp-1, were suppressed in
EtOH-fed WT mice but not in Pxr-null mice. These results are
consistent with a previous report in which PXR activation by its
ligand, pregnenolone 16-carbonitrile, was found to suppress
PPAR target genes by binding directly to forkhead box A2 (FoxA2) while up-regulating SCD1, a key enzyme in lipogenesis
(41). Although the effect of chronic EtOH ingestion on lipid
accumulation has not been examined in PXR-humanized
(hPXR) mice, previous reports revealed that hepatic lipid com-
position and pathology are differentially affected by mouse and
human PXR by obesity induction and human PXR ligand treat-
ment (33, 38, 63). For example, whereas HFD feeding resulted
in mainly macrovesicular steatosis in WT mice, in contrast,
microvesicular steatosis was most prominent in the livers of
hPXR mice. Furthermore, long-term treatment of rifaximin,
a potent gut-specific human PXR agonist, caused hepatic
triglyceride accumulation in hPXR mice but not in WT mice
(63). Moreover, hepatic lipid accumulations have also been
reported in hPXR mice treated with the human PXR activa- J. Biol. Chem. (2018) 293(1) 1–17 8 PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis Figure 6. Immunoblot analysis of mouse hepatic enzymes involved in EtOH metabolism. Male WT and Pxr-null mice were pair-fed control diets o
standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Western blottings of liver homoge-
nate (40 g/lane) were probed with antibodies to CYP2E1 (A), ADH1 (B), ALDH2 (C), ALDH1B1 (D), ALDH1A1 (E), and catalase (F). The blot used to probe
for the CYP2E1 protein was initially used to also probe for the MTP protein in Fig. 4F. The membrane used to probe for CYP2B10 protein expression in
Fig. 2I was stripped and reprobed with primary antibodies against ALDH2 and ALDH1A1. Bands were quantified and normalized to -tubulin. Data
represent mean S.D. (n 3–4). *, p 0.05 between WT and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05
between mice fed EtOH. J. Biol. Chem. (2018) 293(1) 1–17 9 Figure 6. Immunoblot analysis of mouse hepatic enzymes involved in EtOH metabolism. Male WT and Pxr-null mice were pair-fed control diets or
standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake) for 8 weeks. Western blottings of liver homoge-
nate (40 g/lane) were probed with antibodies to CYP2E1 (A), ADH1 (B), ALDH2 (C), ALDH1B1 (D), ALDH1A1 (E), and catalase (F). The blot used to probe
for the CYP2E1 protein was initially used to also probe for the MTP protein in Fig. 4F. The membrane used to probe for CYP2B10 protein expression in
Fig. 2I was stripped and reprobed with primary antibodies against ALDH2 and ALDH1A1. Bands were quantified and normalized to -tubulin. Data
represent mean S.D. (n 3–4). *, p 0.05 between WT and Pxr-null mice fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05
between mice fed EtOH. J. Biol. Chem. (2018) 293(1) 1–17 9 PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis Figure 7. Hepatic gene expression and immunoblot analysis of inflammatory, oxidative stress, and endoplasmic reticulum stress markers. Male WT
and Pxr-null mice were pair-fed control diets (control) or standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake)
for 8 weeks. Tumor necrosis factor (Tnf) (A), toll-like receptor 7 (Tlr7) (B), uncoupling protein 2 (Ucp2) (C), and Gapdh mRNAs were quantified as described
under “Experimental procedures.” Data represent mean S.D. (n 4–6). Western blottings of liver homogenate (40 g/lane) were probed with antibodies to
anti-glucose-regulated protein 78 (GRP78) antibody (D) and anti-Bax antibody (E). Bands were quantified and normalized to -tubulin. Data represent mean
S.D. (n 3–4). *, p 0.05 between WT and Pxr-null mice treated fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05 between mice fed
EtOH. PXR and chronic ethanol-induced steatosis matory, oxidative stress, and endoplasmic reticulum stress markers. Male WT Figure 7. Hepatic gene expression and immunoblot analysis of inflammatory, oxidative stress, and endoplasmic reticulum stress markers. Male WT
and Pxr-null mice were pair-fed control diets (control) or standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5% of the total caloric intake)
for 8 weeks. Tumor necrosis factor (Tnf) (A), toll-like receptor 7 (Tlr7) (B), uncoupling protein 2 (Ucp2) (C), and Gapdh mRNAs were quantified as described
under “Experimental procedures.” Data represent mean S.D. (n 4–6). Western blottings of liver homogenate (40 g/lane) were probed with antibodies to
anti-glucose-regulated protein 78 (GRP78) antibody (D) and anti-Bax antibody (E). Bands were quantified and normalized to -tubulin. Data represent mean
S.D. (n 3–4). *, p 0.05 between WT and Pxr-null mice treated fed control. #, p 0.05 between mice fed control diet and EtOH. †, p 0.05 between mice fed
EtOH tor, rifampicin (33). Because PXR activation is species-spe-
cific in terms of ligand binding, there may be important dif-
ferences in PXR actions between mice and humans. Thus,
whether human PXR activation is involved in steatosis and
liver injury in human alcoholics is important and remains to
be investigated. increased cytokine gene expression; however, contrary to the
notion that PXR activation promotes hepatotoxicity, a signifi-
cant down-regulation of PXR has been identified in the patho-
genesis of inflammatory bowel disease (IBD) in humans (70–
72). 10
J. Biol. Chem. (2018) 293(1) 1–17 9 PXR and chronic ethanol-induced steatosis suggesting the involvement of increased MTP activity in the
protection against EtOH–induced steatosis in Pxr-null mice. Taken together, the present results revealed increased
hepatic expression of SREBP-1C target gene Fas in associa-
tion with decreased mRNA levels of PPAR target genes,
Cpt-1, Acox-1, and Lfabp-1, Fxr and Shp in EtOH-fed WT
mice. Furthermore, these data suggest that increased de novo
synthesis together with inhibition of the transport of fatty
acids into mitochondria for fatty acid -oxidation contribute
to steatosis in WT mice. Figure 8. Serum triglycerides, alanine aminotransferase, and EtOH con
centration. Male WT and Pxr-null mice were pair-fed control diets (control) o
standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5%
of the total caloric intake) for 8 weeks. Serum was assayed for triglycerides (A
alanine aminotransferase (B), and EtOH concentration (C) as described unde Accumulating evidence indicates that EGR-1, a zinc finger
transcription factor, contributes to the development of EtOH–
induced steatosis (48, 78). Interestingly, recent reports indicate
that EGR-1 is an essential factor required for CAR to activate
the CYP2B6 gene, suggesting that the up-regulation of Egr-1
expression by EtOH in WT mice may play a role in the Cyp2b10
activation seen in this study (79, 80). Furthermore, Egr-1 has
also been implicated in the regulation of inflammatory gene
expression, including TNF (48). Egr-1-null mice are pro-
tected against chronic EtOH–induced steatosis, indicating
that Egr-1 is required for the induction of alcoholic fatty liver
and inflammatory gene expression (48, 81). EtOH induced
both Egr-1 mRNA and protein as well as mRNA levels of
Tnf, Tlr7, and oxidative stress marker Ucp2 and the endo-
plasmic reticulum stress marker GRP78 only in WT mice,
suggesting that PXR deficiency is protective against EtOH–
induced Egr-1 activation and its associated Cyp2b10 up-reg-
ulation, steatosis, oxidative stress, and inflammatory cyto-
kine activation. EtOH is metabolized primarily via oxidation to acetaldehyde
through the enzymatic activity of ADH, CAT, and CYP2E1 (7). Among the ADH isozymes, ADH1 is known to play a major role
in hepatic EtOH clearance due to its low Km value for EtOH (7). Acetaldehyde generated from EtOH oxidation is then con-
verted to acetate by the ALDHs, in which three isoforms
ALDH2, ALDH1B1, and ALDH1A1 are responsible for metab-
olizing acetaldehyde (82–84). In the human liver, mitochon-
drial (low Km) ALDH2 oxidizes most of the EtOH-derived acet-
aldehyde (83). PXR and chronic ethanol-induced steatosis IBD refers to either ulcerative colitis or Crohn’s disease
with chronic inflammation of the intestinal tract. Besides the
liver, PXR is highly expressed in the intestinal tract (73, 74). Interestingly, PXR activation in the intestinal tract has been
found to ameliorate experimentally-induced IBD in rodents,
suggesting that tissue-specific activation of PXR may have dif-
ferent outcomes (74, 75). g
Previous reports also linked other nuclear receptors FXR,
CAR, and SHP to lipid homeostasis (64). Specifically, EtOH
ingestion suppresses FXR activity leading to hepatic steatosis
and liver injury, whereas pharmacological activation of FXR
ameliorates EtOH–induced hepatic steatosis (65). Further-
more, FXR inhibits hepatic lipogenesis through up-regulation
of SHP (66). In this study, both Fxr and Shp mRNAs were inhib-
ited by EtOH in WT mice, but only Shp mRNA in Pxr-null mice. These findings are in agreement with a recent report indicating
that ablation of mouse PXR suppresses hepatic lipid accumula-
tion (37, 39, 67), whereas PXR activation increases triglyceride
formation and steatosis (33, 68, 69). Expression of an activated
PXR in the livers of transgenic mice resulted in hepatic triglyc-
eride accumulation and steatosis (33). In this study, PXR acti-
vation in the liver by EtOH also resulted in hepatic steatosis and Recent studies have shown that preventing or reducing
EtOH–induced fatty liver may inhibit or delay the transition
from fatty liver to steatohepatitis and then fibrosis (76). In this
study, massive steatosis in EtOH-fed WT mice but not in Pxr-
null mice was observed. Meanwhile, EtOH inhibition of MTP, a
key enzyme regulating lipid export and very low density lipo-
protein (VLDL) synthesis, contributes to alcoholic fatty liver; in
contrast, MTP up-regulation reverses alcoholic steatosis (77). In this study EtOH up-regulated the MTP protein in livers of
EtOH-fed Pxr-null mice but not in similarly treated WT mice, J. Biol. Chem. (2018) 293(1) 1–17 10 “Experimentalprocedures.”DatarepresentmeanS.D.(n4–6).#,p0.05
between mice fed control diet and EtOH. †, p 0.05 between mice fed EtOH. PXR and chronic ethanol-induced steatosis However, in rodents both mitochondrial and
cytosolic ALDH isozymes are important in acetaldehyde oxida-
tion (83). Previous reports have shown that mouse cytosolic
ALDH1A1 has a high affinity for acetaldehyde, and it is an
important mediator of alcohol and acetaldehyde metabolism
(83, 85). Furthermore, cytosolic ALDH1A1 with micromolar
Km values also contributes to oxidation of acetaldehyde, partic-
ularly in individuals who lack active ALDH2 (86). Another
novel finding observed in this study was that protein expression
but not the mRNAs of major genes regulating EtOH metabo-
lism were up-regulated in the livers of Pxr-null mice compared
with their WT counterparts. Specifically, both the basal and
EtOH-treated hepatic ADH1, CAT, and ALDH1A1 protein lev-
els were significantly higher in Pxr-null mice than in EtOH-fed
WT mice. These findings are consistent with previous reports
showing that hepatic ADH regulation is post-transcriptional
and that ADH1 protein expression increases without a corre- Figure 8. Serum triglycerides, alanine aminotransferase, and EtOH con-
centration. Male WT and Pxr-null mice were pair-fed control diets (control) or
standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5%
of the total caloric intake) for 8 weeks. Serum was assayed for triglycerides (A), Figure 8. Serum triglycerides, alanine aminotransferase, and EtOH con-
centration. Male WT and Pxr-null mice were pair-fed control diets (control) or
standard Lieber-Decarli liquid diet containing 5% EtOH (representing 27.5%
of the total caloric intake) for 8 weeks. Serum was assayed for triglycerides (A),
alanine aminotransferase (B), and EtOH concentration (C) as described under 11 J. Biol. Chem. (2018) 293(1) 1–17 11 Figure 9. Proposed mechanism of PXR-dependent regulation of EtOH-
induced steatosis. Chronic EtOH exposure significantly up-regulated the
mRNAlevelsofPxr,constitutiveandrostanereceptor(Car),anditstargetgene
Cyp2b10inWTmice,butnotinPxr-nullmice.TheincreaseinPxrexpressionby
EtOH in WT mice was also associated with PXR-dependent increase in gene
and protein expression of early growth response-1 (Egr-1). EGR-1 is a key
signal necessary for CAR to activate the CYP2B gene (79, 80). EGR-1 can also
up-regulate the sterol regulatory element binding protein-1c (Srebp-1c) tar-
get gene fatty acid synthase (Fas), and tumor necrosis factor (TNF), leading
to EtOH–induced steatosis (78). Both histochemical staining of liver sections
with H&E and biochemical assay revealed development of steatosis and accu-
mulationofneutrallipidsonlyinWTmice.Steatosis,“thefirsthit,”canserveas
a potent stimulus for the “second hit,” including increased generation of oxi-
dative, endoplasmic reticulum stress ,and inflammation markers, seen in the
livers of the EtOH-fed WT mice. Unexpectedly, PXR deficiency not only inhib-
ited EtOH–induced increases in lipogenic gene expression but also up-regu-
lated MTP, a lipid-transfer protein and suppressed lipid accumulation and
steatosis in Pxr-null mice. PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis PXR and chronic ethanol-induced steatosis ADH1 protein expression may protect Pxr-null mice against
EtOH–induced steatosis, because ADH1 deficiency is a deter-
mining factor for increased EtOH–induced lipid accumulation
and hepatotoxicity (88). Recent evidence indicates that PXR and its agonists, includ-
ing those used in the clinic such as rifampicin, rifaximin clo-
trimazole, and lovastatin, play a role in drug-induced liver
injury, a leading cause of acute liver failure (32, 89, 90). Com-
pared with PXR agonists, relatively few PXR antagonists have
been reported (91, 92). Similar to our current results in which
Pxr-null mice are resistant to EtOH–induced steatosis, both
cholic acid and HFD-induced hepatotoxicity are more severe in
WT mice than in their Pxr-null counterparts (38, 93), suggest-
ing that PXR deficiency protects against drug-induced hepato-
toxicity and that inhibition of PXR signaling could be a means
to prevent or treat ALD. Indeed, a recent report indicates
that a novel human PXR antagonist, 1-(4-(4-(((2R,4S)-2-
(2,4-difluorophenyl)-2-methyl-1,3-dioxolan-4-yl) methoxy)
phenyl)piperazin-1-yl)ethanone (FLB-12) that specifically
disrupts the function of activated PXR decreased acetamino-
phen-induced hepatotoxicity in mice (92, 94). Figure 9. Proposed mechanism of PXR-dependent regulation of EtOH-
induced steatosis. Chronic EtOH exposure significantly up-regulated the
mRNAlevelsofPxr,constitutiveandrostanereceptor(Car),anditstargetgene
Cyp2b10inWTmice,butnotinPxr-nullmice.TheincreaseinPxrexpressionby
EtOH in WT mice was also associated with PXR-dependent increase in gene
and protein expression of early growth response-1 (Egr-1). EGR-1 is a key
signal necessary for CAR to activate the CYP2B gene (79, 80). EGR-1 can also
up-regulate the sterol regulatory element binding protein-1c (Srebp-1c) tar-
get gene fatty acid synthase (Fas), and tumor necrosis factor (TNF), leading
to EtOH–induced steatosis (78). Both histochemical staining of liver sections
with H&E and biochemical assay revealed development of steatosis and accu-
mulationofneutrallipidsonlyinWTmice.Steatosis,“thefirsthit,”canserveas
a potent stimulus for the “second hit,” including increased generation of oxi-
dative, endoplasmic reticulum stress ,and inflammation markers, seen in the
livers of the EtOH-fed WT mice. Unexpectedly, PXR deficiency not only inhib-
ited EtOH–induced increases in lipogenic gene expression but also up-regu-
lated MTP, a lipid-transfer protein and suppressed lipid accumulation and
steatosis in Pxr-null mice. In summary, these findings suggest that PXR ablation pre-
vented EtOH induced up-regulation of the CAR target gene,
Cyp2b10, implicated in EtOH hepatotoxicity. The results of this
study clearly revealed the protective effects of PXR deficiency
on EtOH–induced steatosis, lipid accumulation, inflammation,
and ER stress as evidenced by inhibition of lipogenic transcrip-
tion factor induction, increased MTP protein expression, and
induction of major EtOH catabolism enzymes leading to
increased EtOH clearance and low BEC. Thus, these results
demonstrate that PXR aggravates chronic EtOH–induced
steatosis, inflammation, and endoplasmic reticulum stress as
depicted in Fig. 9. To our knowledge, this is the first report
to explore the effects of PXR deficiency on chronic EtOH–
induced hepatotoxicity. This work suggests the possibility that
inhibition of PXR signaling could be a means to prevent or treat
ALD. sponding increase in Adh1 mRNA levels (10, 87). A previous
study found that leptin can increase ADH1 protein expression
but not the mRNA (87). We have shown that serum leptin levels
are higher in male Pxr-null mice (38). Therefore, it is plausible
to hypothesize that the observed increased ADH1 protein
expression in Pxr-null mice might involve increased ADH1
protein synthesis (38). It is not clear why basal CAT and
ALDH1A1 protein levels are higher in Pxr-null mice. However,
previous reports indicate that Aldh1a1 is a target gene of both
PXR and CAR (47). Furthermore, the high ALDH1A1 protein
levels observed in Pxr-null mice may be related to the high
ADH1 protein levels in these mice, because previous reports
indicate that there is a delicate balance between net EtOH oxi-
dation rate via ADH1 and the acetaldehyde oxidation rate via
ALDH in the liver, so that each enzyme contributes to the over-
all control of the rate of EtOH oxidation (4). The BEC, the EtOH
concentration in the blood after metabolism of consumed
EtOH in WT and Pxr-null mice, was measured to determine
whether the metabolic pathway converting EtOH to acetate via
acetaldehyde is different between the two genotypes. In agree-
ment with increased ADH1, CAT, and ALDH1A1 protein
expression in EtOH-fed Pxr-null mice, the residual EtOH con-
centration was significant higher in WT mice compared with
their Pxr-null counterparts. Thus, the current results indicate
that PXR deficiency increases protein expression of EtOH-me-
tabolizing enzymes, thereby increasing EtOH catabolism and
EtOH elimination in Pxr-null mice. Furthermore, the high 12
J. Biol. Chem. (2018) 293(1) 1–17 Preparation of liver extracts for Western blot analyses Frozen livers were homogenized at 4 °C, and Western blot
analysis was performed on extracts as described previously (99). Protein contents in liver homogenates were determined by the
BCATM protein assay kit (Thermo Fisher Scientific, Rockford,
IL). Liver homogenates (40 g/lane) were diluted in Laemmli
loading buffer containing -mercaptoethanol, boiled for 5 min,
separated on 10 or 15% SDS-polyacrylamide gels, and trans-
ferred to polyvinylidene difluoride (PVDF) membranes. The
membranes were probed overnight with rabbit polyclonal pri-
mary antibodies from Sigma at a concentration of 1:5000 for
anti-CYP2B10 (AB9916, lot no. 2794496). Rabbit polyclonal
primary antibodies from Abcam (Cambridge, MA) were used at
a concentration of 1:1000 for anti-CYP2E1 (ab28146, lot no. GR182611-20) and anti-catalase (ab16731, lot no. GR1693791). Primary antibodies from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), were used as follows: concentration of 1:200 for
mouse monoclonal anti-ADH1 (Sc-137078 lot no. L3015); goat
polyclonal antibody at a concentration of 1:1000 for anti-
ALDH2 (Sc-48837, lot no. D1709); mouse monoclonal antibody
at a concentration of 1:500 for anti-ALDH1B1 (Sc-393583, lot
no. K2613); mouse monoclonal antibody at a concentration of
1:200 for anti-MTP (Sc-135994, lot no. G2115); or rabbit poly-
clonal antibody at a concentration of 1:200 for anti-GRP78 (Sc-
13968, lot no. H1809). Rabbit monoclonal primary antibodies
from Novus Biologicals (Littleton, CO) were used at a concen-
tration of 1:1000 for anti-ALDH1A1 (NB110-55451, lot no. GR182706-7). Primary antibodies from Cell Signaling Technol-
ogy (Boston, MA) were used at a concentration of 1:1000 for
rabbit polyclonal anti-Bax (no. 2772, lot no.: 10) or were used at
a concentration of 1:1000 for rabbit monoclonal anti-EGR-1
(no. 4154, lot no. 3). Blots were then incubated with the
appropriate peroxidase-conjugated secondary antibodies
from Cell Signaling Technology or Santa Cruz Biotechnol-
ogy, Inc., and diluted in TBST plus 1% milk for 60 min at
room temperature. Following initial probing, blots were
stripped and reprobed with rabbit monoclonal antibody
from Cell Signaling Technology at a concentration of 1:1000
for anti--tubulin (no. 2125, lot no. 9). Proteins were visual-
ized using enhanced chemiluminescence, and band intensity
was quantified using ImageJ software (National Institutes of
Health, Bethesda). PXR and chronic ethanol-induced steatosis caloric intake of mice in this group. Liquid diets were based
upon the Lieber-DeCarli EtOH formulation and provide 1 kcal/
ml, which was purchased from a single source (DYETS Inc.,
Bethlehem, PA). Protein content accounted for 18.9% of total
calories and fat comprised 16.5% of total calories, whereas
64.5% of calories were derived from carbohydrate. The Lieber-
DeCarli liquid diet model with 5% EtOH in the diet used in this
study provides an excellent means to reproduce the early stages
of chronic human ALD, including steatosis, oxidative stress,
and liver injury (95, 96). Control mice were pair-fed diets that
isocalorically substituted cornstarch for EtOH during the entire
study period. After 8 weeks, mice were anesthetized with iso-
flurane and killed. Sections of liver were rapidly dissected,
weighed, snap-frozen in liquid nitrogen, and kept at 80 °C. Blood samples collected by cardiac puncture from anesthetized
mice were centrifuged at 3000 rpm for 15 min to collect serum
and stored at 80 °C to determine EtOH concentration, liver
enzymes, and triglycerides. All procedures were conducted in
accordance with the National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals and approved by
the North Carolina Central University Institutional Animal
Care and Use Committee. triglyceride and NEFA test kits (Wako Pure Chemical Indus-
tries, Richmond, VA). triglyceride and NEFA test kits (Wako Pure Chemical Indus-
tries, Richmond, VA). Serum ALT and triglyceride measurements Serum was processed from blood and stored at 80 °C. Serum ALT activity was determined using a commercially
available kit (Sigma). Serum triglyceride levels were quantified
using assay test kits (Wako Pure Chemical Industries, Rich-
mond, VA). H&E staining of liver sections and morphology analysis Liver slices were fixed in 10% formalin/phosphate-buffered
saline, and then stained with H&E for histological examination. Slides were viewed blindly and scored for steatosis, inflamma-
tion, necrosis, and fibrosis using our previously described
criteria (97). The liver pathology was scored as follows: steatosis
(the percentage of liver cells containing fat), 25% 1,
50% 2, 75% 3, 75% 4; inflammation, necro-
sis, and fibrosis, 1 focus 1, 2 or more foci 2. Quantification of mRNA levels using real-time PCR Total RNA was isolated from frozen liver tissues using the
TRIzol reagent according to the manufacturer’s protocol (Invit-
rogen). Total RNA (5 g) was reverse-transcribed into cDNA
with random hexamer primers using Tetro cDNA synthesis kit
(Bioline, Taunton, MA) as we described previously (38). The
cDNA was then diluted 20-fold with water and subjected to
real-time quantitative PCR by the SensiFast SYBR Hi-ROX Kit
(Bioline, Taunton, MA) to quantify the mRNA levels of Ppar,
Cpt1, Acox-1, Lfabp-1, Mtp, Srebp-1c, Scd1, Acc-1, Fas,
Srebp2, Egr-1, Cyp2b10, Cyp7a1, Tnf, Tlr7, Fxr, Shp, and
GAPDH. The primers for the SYBR Green assay (Table 2) were Determination of serum alcohol concentration Blood samples were collected after 8 weeks of treatment with
either control or EtOH diet and centrifuged at 3000 rpm for 15
min to collect serum for BEC. The EtOH L3K assay kit for
quantitative measurement of EtOH concentration (Sekisui
Diagnostics P.E.I. Inc., Charlottetown, Canada) was used
according to the manufacturer’s instructions as reported
previously (98). The reaction is based on the enzymatic con-
version of EtOH by ADH to acetaldehyde and NADH. EtOH
concentration in the serum was quantified as the rate of
increase in NADH absorbance due to the reduction of NAD
at 380 nm. Experimental procedures
Animal care and treatment Genetically identical male C57BL/6 mice (which served as
WT) and Pxr-null mice on the C57BL/6 genetic background
were used in this study (35). Although the influence of gut
microbiota was not examined in this study, all mice (3–5 mice/
cage) were housed under equivalent housing conditions in a
specific pathogen-free animal facility within a single holding
room in polycarbonate cages on racks directly vented via the
facility’s exhaust system at 22 °C with a 12/12-h light/dark cycle
at the Animal Resources Complex at North Carolina Central
University. After 2 days of feeding a control liquid diet, age-
matched (8–10 weeks of age) male WT and Pxr-null mice were
each randomly separated into two groups (n 6–7), pair-fed
(control) or EtOH groups. The EtOH-fed group was allowed
free access to EtOH-containing diets with increasing concen-
trations of EtOH (1–5%) over a 7-day period as described pre-
viously (9). The EtOH concentration was kept thereafter at 5%
for an additional 7 weeks. EtOH made up 27.5% of the total 12
J. Biol. Chem. (2018) 293(1) 1–17 12 Hepatic triglyceride and NEFA levels Total liver lipids were extracted from 100 mg of liver homo-
genate using methanol and chloroform as described previously
(9). Hepatic triglyceride and NEFA levels were quantified using 13 J. Biol. Chem. (2018) 293(1) 1–17 References 1. Gao, B., and Bataller, R. (2011) Alcoholic liver disease: pathogenesis and
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Sequences of primers used for a SYBR Green real-time quantitative PCR
Name
Sequence
Accession
no. PPAR
Sense
GATTCAGAAGAAGAACCGGAACA
NM011144
Antisense
TGCTTTTTCAGATCTTGGCATTC
CPT-1
Sense
CGATCATCATGACTATGCGCTACT
NM013495
Antisense
GCCGTGCTCTGCAAACATC
ACOX1
Sense
TTTGTTGTCCCTATCCGTGAGA
NM 015729
Antisense
GCCGATATCCCCAACAGTGA
L-FABP
Sense
TGCATGAAGGGAAGAAAATCAAA
NM017399
Antisense
CCCCCAGGGTGAACTCATT
MTP
Sense
CCGCTGTGCTTGCAGAAGA
NM008642
Antisense
TTTGACACTATTTTTCCTGCTATGGT
EGR1
Sense
GCCTCGTGAGCATGACCAAT
NM 007913
Antisense
GCAGAGGAAGACGATGAAGCA
SREBP2
Sense
ATGATCACCCCGACGTTCAG
NM 033218
Antisense
GCTGCGTTCTGGTATATCAAAGG
SREBP-1c
Sense
CATGCCATGGGCAAGTACAC
NM011480
Antisense
TGTTGCCATGGAGATAGCATCT
SCD1
Sense
CGTTCCAGA ATGACGTGTACGA
NM009127
Antisense
AGGGTCGGCGTGTGTTTC
TNF
Sense
ACAAGGCTGCCCCGACTAC
NM 013693
Antisense
TTTCTCCTGGTATGAGATAGCAAATC
ACC-1
Sense
ATGTCCGCACTGACTGTAACCA
NM133360
Antisense
TGCTCCGCACAGATTCTTCA
FAS
Sense
CCCGGAGTCGCTTGAGTATATT
NM007988
Antisense
GGACCGAGTAATGCCATTCAG
CYP7a1
Sense
CCATGATGCAAAACCTCCAAT
NM 007824
Antisense
TGACCCAGACAGCGCTCTTT
CYP2B10
Sense
TTTCTGCCCTTCTCAACAGGAA
NM 009999
Antisense
ATGGACGTGAAGAAAAGGAACAAC
FXR
Sense
CACGAAGATCAGATTGCTTTGC
NM 009108
Antisense
CCGCCGAACGAAGAAACAT
SHP
Sense
CAGGCACCCTTCTGGTAGATCT
NM 011850
Antisense
GTCTTCAAGGAGTTCAGTGATGTCA
GAPDH
Sense
TGTGTCCGTCGTGGATCTGA
NM001001303
Antisense
CCTGCTTCACCACCTTCTTGA TGTGTCCGTCGTGGATCTGA
CCTGCTTCACCACCTTCTTGA designed using Primer Express 2.0 (Applied BioSystems, Foster
City, CA). The primer sequence for TLR7 was previously pub-
lished (15). Furthermore, the following proprietary TaqMan
Gene Expression Assays were purchased from Applied Biosys-
tems/Life Technologies, Inc., and used for real-time quan-
titative PCR: Adh1 (No. Mm00507711_m1); Aldh2 (No. Mm00477463_m1); Aldh1a1 (No. Mm00657317_m1); Aldh1b1
(No. Mm00728303_s1); Pxr (No. Mm01344139_m1); Cyp3a11
(No. Mm00731567_m1);
Car
(No. Mm01283978_m1);
Ucp2 (No. Mm00627599_m1); Slco1a4 (Oatp2, No. Mm01267407_
m1); Cat (No. Mm00437992_m1); and Gapdh (housekeeping
gene, No. 99999915_g1). The amplification reactions were per-
formed in the ABI QuantStudioTM 3 Systems (Applied Biosys-
tems, Foster City, CA). Results were presented as levels of
expression relative to that of controls after normalizing with
Gapdh mRNA using the comparative CT method. designed using Primer Express 2.0 (Applied BioSystems, Foster
City, CA). The primer sequence for TLR7 was previously pub-
lished (15). Furthermore, the following proprietary TaqMan
Gene Expression Assays were purchased from Applied Biosys-
tems/Life Technologies, Inc., and used for real-time quan-
titative PCR: Adh1 (No. Mm00507711_m1); Aldh2 (No. Mm00477463_m1); Aldh1a1 (No. Mm00657317_m1); Aldh1b1
(No. Mm00728303_s1); Pxr (No. Mm01344139_m1); Cyp3a11
(No. Mm00731567_m1);
Car
(No. Mm01283978_m1);
Ucp2 (No. Mm00627599_m1); Slco1a4 (Oatp2, No. Mm01267407_
m1); Cat (No. Mm00437992_m1); and Gapdh (housekeeping
gene, No. 99999915_g1). The amplification reactions were per-
formed in the ABI QuantStudioTM 3 Systems (Applied Biosys-
tems, Foster City, CA). Results were presented as levels of
expression relative to that of controls after normalizing with
Gapdh mRNA using the comparative CT method. Author contributions—M. A. G. conceived and designed experi-
ments. S. C., P. N., and M. A. G. conducted experiments. M. A. G. 14
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English
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Vaticanol C, a resveratrol tetramer, activates PPARα and PPARβ/δ in vitro and in vivo
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Nutrition & metabolism
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cc-by
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BioMed Central
© 2010 Tsukamoto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original work is properly cited. Abstract Background: Appropriate long-term drinking of red wine is associated with a reduced risk of cardiovascular disease. Resveratrol, a well-known SIRT1 activator is considered to be one of the beneficial components contained in red wine,
and also developed as a drug candidate. We previously demonstrated that resveratrol protects brain against ischemic
stroke in mice through a PPARα-dependent mechanism. Here we report the different effects of the oligomers of
resveratrol. Methods: We evaluated the activation of PPARs by ε-viniferin, a resveratrol dimer, and vaticanol C, a resveratrol
tetramer, in cell-based reporter assays using bovine arterial endothelial cells, as well as the activation of SIRT1. Moreover, we tested the metabolic action by administering vaticanol C with the high fat diet to wild-type and PPARα-
knockout male mice for eight weeks. Results: We show that vaticanol C activates PPARα and PPARβ/δ in cell-based reporter assays, but does not activate
SIRT1. ε-Viniferin shows a similar radical scavenging activity as resveratrol, but neither effects on PPARs and SIRT-1. Eight-week intake of vaticanol C with a high fat diet upregulates hepatic expression of PPARα-responsive genes such as
cyp4a10, cyp4a14 and FABP1, and skeletal muscle expression of PPARβ/δ-responsive genes, such as UCP3 and PDK4
(pyruvate dehydrogenase kinase, isoform 4), in wild-type, but not PPARα-knockout mice. Conclusion: Vaticanol C, a resveratrol tetramer, activated PPARα and PPARβ/δ in vitro and in vivo. These findings
indicate that activation of PPARα and PPARβ/δ by vaticanol C may be a novel mechanism, affording beneficial effects
against lifestyle-related diseases. * Correspondence: inoue@cc.nara-wu.ac.jp
1 Department of Food Science and Nutrition, Nara Women's University, Nara,
630-8506, Japan
† Contributed equally
Full list of author information is available at the end of the article RESEARCH Research
Vaticanol C, a resveratrol tetramer, activates PPARα
and PPARβ/δ in vitro and in vivo Tomoko Tsukamoto†1, Rieko Nakata†1, Emi Tamura1, Yukiko Kosuge1, Aya Kariya1, Michiko Katsukawa1,
Satoshi Mishima2, Tetsuro Ito3, Munekazu Iinuma3, Yukihiro Akao4, Yoshinori Nozawa4, Yuji Arai5, Shobu Namura6 and
Hiroyasu Inoue*1 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46
Open Access
RESEARCH
Research
Vaticanol C, a resveratrol tetramer, activates PPARα
and PPARβ/δ in vitro and in vivo
Tomoko Tsukamoto†1, Rieko Nakata†1, Emi Tamura1, Yukiko Kosuge1, Aya Kariya1, Michiko Katsukawa1,
Satoshi Mishima2, Tetsuro Ito3, Munekazu Iinuma3, Yukihiro Akao4, Yoshinori Nozawa4, Yuji Arai5, Shobu Namura6 and
Hiroyasu Inoue*1 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46
Open Access
RESEARCH
Research
Vaticanol C, a resveratrol tetramer, activates PPARα
and PPARβ/δ in vitro and in vivo
Tomoko Tsukamoto†1, Rieko Nakata†1, Emi Tamura1, Yukiko Kosuge1, Aya Kariya1, Michiko Katsukawa1,
Satoshi Mishima2, Tetsuro Ito3, Munekazu Iinuma3, Yukihiro Akao4, Yoshinori Nozawa4, Yuji Arai5, Shobu Namura6 and
Hiroyasu Inoue*1 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46 http://www.nutritionandmetabolism.com/content/7/1/46 Background tive compounds, and experimental data on resveratrol
has been accumulating [1,2]. The polymers of resveratrol
are also known phytoalexins and antioxidants. The res-
veratrol dimer, ε-viniferin, is a major phytoalexin in
response to UV-C irradiation [3] and has been reported
to be an antioxidant [4]. The resveratrol tetramer, vati-
canol C, exhibited a more potent cytotoxic effect against
human cultured cancer cells than resveratrol [5-7]. By
screening 20 derivatives of resveratrol, vaticanol C was
found to have the strongest suppressive activity of cell
growth in colon cancer cell lines [6]. However, the molec-
ular mechanisms for these actions remain to be deter-
mined. Prevention of lifestyle-related diseases, such as cardiovas-
cular disease, diabetes, and stroke, has become of world-
wide interest. In such lifestyle-related matters, people pay
attention not only to drugs, but also to the functionality
of natural chemicals contained in food and drink, such as
polyphenols and their polymers. In this context, resvera-
trol, a phytoalexin and antioxidant polyphenol included
in red wine and various plants, is one of the most attrac- * Correspondence: inoue@cc.nara-wu.ac.jp
1 Department of Food Science and Nutrition, Nara Women's University, Nara,
630-8506, Japan
† Contributed equally
Full list of author information is available at the end of the article Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 Page 2 of 8 We demonstrated that resveratrol activates peroxisome
proliferator-activated receptor (PPAR) α and γ in cell-
based reporter assays and protects the brain against isch-
emic stroke in mice through a PPARα-dependent mecha-
nism [8]. PPARs are members of nuclear receptor family
of ligand-dependent transcription factors [9]. The PPAR
subfamily consists of three isotypes, PPARα, β/δ, and γ,
which play various roles in lipid and carbohydrate metab-
olism, cell proliferation and differentiation, and inflam-
mation and are considered to be molecular targets
against lifestyle-related diseases [10,11]. For example,
PPARα and γ agonists, such as fibrates and thiazolidine
derivatives, are used to treat dyslipidemia and diabetes,
respectively. Moreover, eicasapentaenoic acid, a natural
ligand for PPARα, has been used as a hypolipidemic drug
and has been reported to lower plasma and liver choles-
terol levels in a PPARα-dependent manner [12]. Thus, we
have focused on PPARs as possible molecular targets of
resveratrol in preventing lifestyle-related diseases, while
others have studied SIRT1, a NAD+-dependent protein
deacetylase, and PGC-1α [13-17]. Cell Culture Bovine arterial endothelial cells (BAEC) [8,18] were
grown in DMEM supplemented with 10% fetal calf serum
and 50 μM 2-mercaptoethanol. DPPH Radical Scavenging and SIRT1 Activation Assays DPPH Radical Scavenging and SIRT1 Activation Assays
Resveratrol and its derivatives were dissolved in DMSO
to prepare samples at 500 μM. Twenty microliters of each
sample, 80 μL of 0.1 M Tris HCl buffer (pH 7.4), and 100
μL of 500 μM 1,1-diphenyl-2-picryl-hydrazyl (DPPH)
solution were mixed well and incubated at room temper-
ature for 20 min, then the absorbance was recorded at
540 nm with a plate reader (Immuno mini NJ2300, Nal-
gene) as described [20]. Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) was the reference
antioxidant, used as a positive control, and each result is
expressed as the equivalent per mole of trolox from the
percentage decrease with respect to the negative control
value. Data are presented as mean ± SD (n = 3). Background via real-time quantitative RT-PCR (Mx3005, Stratagene)
as described previously [18]. The primer pairs for genes
in this study and their cycling conditions are shown in
Additional file 1. Expression levels of each mRNA were
normalized to those of 36B4 mRNA. Methods
Reagents ε-Viniferin, a resveratrol dimmer, and vaticanol C, a res-
veratrol tetramer, were isolated from the genus belonging
to Vatica, Shorea, and Vateria (Dipterocarpaceae) as
described previously [5,6]. A stock solution of these com-
pounds was prepared in 10 mM dimethyl sulfoxide
(DMSO), and was further diluted to the working concen-
tration before use. The Fluor de Lys fluorescence assay for in vitro SIRT1
activity was performed by the SIRT1 activity assay kit
(BIOMOL, catalog number AK-555), according to the
manufacturer's instructions. Fluorescent intensity was
measured using an Infinite F200 microplate fluorometer
(TECAN). DMSO was used as a negative control and also
as a solvent for resveratrol and its derivatives to a final
concentration of 100 μM in the assay buffer. Transcription Assays In the case of activation assay for PPARα, BAEC was
transfected with 0.15 μg of tk-PPREx3-Luc reporter plas-
mid, 0.15 μg of human PPARα expression vector pGS-
hPPARα (Invitrogen, GeneStorm™ clone L02932) and
0.02 μg of pSV-βgal as described previously using Trans
IT-LT-1 (Mirus) [19]. In the activation assay for PPARβ/δ
and γ, their expression vectors pCMX-hNUCI (PPAR β/
δ) and pCMX-hPPARγ1 were used for the transfection,
instead of the human PPARα expression vector [18]. The
transfected BAEC were harvested, lysed, and assayed for
both luciferase and β-galactosidase activities. The results
are represented as relative luciferase activities, which
were normalized against the β-galactosidase standard. Transfection efficiency of BAEC is over 20% estimated by
cotransfection of pEGFP-N1, an expression vector for
Green Fluorescent Protein. pCMX-hNUCI and pCMX-
hPPARγ1 were human PPARγ1 and PPARβ/δ expression
vectors under control of a cytomegalovirus promoter
[18]. In the positive control experiments, selective activa-
tors of PPARs, such as Wy-14643 (α), GW501516 (β/δ),
and pioglitazone (γ), had PPARα, β/δ, and γ agonistic
activities, respectively [18]. In the present study, we evaluated the activation of
PPARs, antioxidant, and SIRT1-activator properties of
resveratrol, ε-viniferin, and vaticanol C and found that
vaticanol C activates PPARα and PPARβ/δ in cell-based
reporter assays, but does not have antioxidant or SIRT1-
activator properties. Moreover, 8-week intake of vati-
canol C with a high fat (HF) diet upregulates hepatic
expression of PPARα-responsive genes and skeletal mus-
cle expression of PPARβ/δ-responsive genes in wild-type,
but not PPARα-knockout mice. These findings indicate
that activation of PPARα and PPARβ/δ by vaticanol C
may provide beneficial effects against lifestyle-related dis-
eases. Activation of PPARs In Vitro by Resveratrol and Vaticanol C
but not by ε-Viniferin y
Resveratrol activated PPARα and γ in our cell-based assay
using bovine arterial endothelial cells (BAEC) [8]. In this
assay system, we also confirmed that selective activators
of PPARs, such as Wy-14643 (α), GW501516 (β/δ), and
pioglitazone (γ), had PPARα, β/δ, and γ agonistic activi-
ties, respectively [18]. Using this assay system, we evalu-
ated the activation of PPARs by resveratrol, ε-viniferin,
and vaticanol C (Figure 1A). Activation of PPARα and β/δ
were observed by resveratrol and vaticanol C, whereas
only resveratrol activated PPARγ (Figure 1B). These effi-
cacies of resveratrol and vaticanol C on PPARα and β/δ
were roughly similar to those of selective activators such
as Wy-14643 and GW501516, respectively [18]. Remark-
ably, at lower concentrations, 1.25-5 μM, vaticanol C was
a more potent activator for PPARα than resveratrol. On
the other hand, ε-viniferin showed no agonistic activity
for PPARs. At a concentration of 10 μM, resveratrol
showed triple agonistic activities for α, β/δ and γ, whereas
vaticanol C showed dual agonistic activities for α and β/δ
Figure 2A). These properties of resveratrol and vaticanol
C will provide beneficial effects against lifestyle-related
disease since several drugs were reported to have similar
properties. We evaluated the effects of vaticanol C administration
on the expression of PPAR-dependent genes in wild-type
and PPARα-knockout mice. In the liver, expression of
cyp4a10, cyp4a14 and fatty acid binding protein 1
(FABP1) was induced by 0.04% vaticanol C in the wild-
type, but not in the knockout mice (Figure 3A). In the
positive control, expression of these genes was also
induced by 0.02% fenofibrate, a PPARα agonist, in the
wild-type mice (Figure 3B). On the other hand, expres-
sion of FGF21, a PPARα-responsive protein, was not
induced by 0.04% vaticanol C, and by 0.02% fenofibrate
(Figure 3A, B), which may be due to feedback control of
the PPARα-FGF21 endocrine pathway [21,22]. Expres-
sion of other PPARα-dependent genes, such as acyl-CoA
oxidase (Acox1) and long-chain type acyl-CoA dehydro-
genase (LCAD) was induced by 0.02% fenofibrate (Figure
3B) but not 0.04% vaticanol C (Figure 3A) in the wild-
type mice, and was reduced in the knockout mice (Figure
3A). We also tested fenofibrate in PPARα-knockout mice
on high fat diet (n = 2) or on normal fat diet (n = 4); how-
ever, no induction of PPARα-responsive genes was
observed (data not shown), which is similar to the results
of vaticanol C with a HF diet. Statistical analyses All results are expressed as means ± SD. Comparisons
between groups were performed with the unpaired t test. Values were deemed to be statistically significantly differ-
ent at p < 0.05. Activation of PPARs In Vitro by Resveratrol and Vaticanol C
but not by ε-Viniferin The discrepancies between
fenofibrate and vaticanol C were unknown, but may be
involved in other regulating factors for gene expression. In this point, we found that 0.04% resveratrol treatment
with a normal diet, but not with a HF diet, showed RNA Extraction and Analysis Total RNA was isolated using the acid guanidinium thio-
cyanate procedure, and was analyzed for gene expression Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 Page 3 of 8 No Activation of SIRT1 by Vaticanol C or ε-Viniferin Because resveratrol has been reported to be an activator
for SIRT1 [13], we evaluated the activation of SIRT1 by ε-
viniferin and vaticanol C. As a positive control, resvera-
trol activated SIRT1; however, ε-viniferin and vaticanol C
did not (Figure 2C). Rather, vaticanol C seemed to inhibit
SIRT1 activity in this assay. Taken together, vaticanol C, a
resveratrol tetramer, is a dual agonist for PPARα and β/δ,
but not a radical scavenger and not a SIRT1 activator. Vaticanol C Administration Induces Expression of PPAR-
dependent Genes The metabolic actions of vaticanol C were then evaluated
by administering the compound by food admixture (0
and 0.04%) to wild-type and PPARα-knockout male mice
challenged with the HF diet for 8 weeks. During this
experiment, there was no statistically significant differ-
ence in body weight between the eight groups. Animal Experiments iferin showed similar radical-scavenging activities,
comparable to the positive control radical scavenger,
trolox, a water-soluble derivative of vitamin E. On the
other hand, vaticanol C showed no radical scavenging
activity (Figure 2B), indicating that there was no correla-
tion between agonistic activities for PPARs and radical-
scavenging activity. Male 12-week-old 129SV-strain (wild-type) and PPARα-
knockout (129SV background) mice were obtained by
Jackson laboratory (USA). These mice were housed in a
room at 24 ± 2°C with a 12/12 h light-dark cycle, and fed a
HF diet containing 60% energy as fat, supplemented with
vaticanol C (0 and 0.04%) for 8 weeks. Diet and water
were available ad libitum. After 8 weeks of feeding, the
mice were sacrificed under diethyl ether anesthesia to
obtain tissues. Liver and skeletal muscle were stored in
RNAlater™ solution (Ambion, USA) at -30°C until gene
expression analysis. These experimental procedures were
approved by the Animal Care Committee of Nara
Women's University. No Correlation with Resveratrol or its Derivatives between
Activation of PPARs and Radical-Scavenging Activities Next, we evaluated resveratrol and its derivatives for their
radical-scavenging activity using DPPH, a stable free rad-
ical [20]. As shown in Figure 2B, resveratrol and ε-vin- Tsukamoto et al. Nutrition & Metabolism 2010, 7:46 Page 4 of 8 http://www.nutritionandmetabolism.com/content/7/1/46 Figure 1 Activation of PPARs by Resveratrol, ε-Viniferin and Vaticanol C. (A) Structures of resveratrol (monomer), ε-viniferin (dimer) and vaticanol
C (tetramer). (B) Activation of PPARα, β/δ, and γ was evaluated by transfection assays using BAEC with PPRE-luc together with GS-hPPARα, pCMX-NUC1
or pCMX-hPPARγ1, respectively. Results are presented as relative luciferase activities obtained by dividing the normalized luciferase activity from the
reporter vector PPRE-luc. *, P < 0.05, **, P < 0.01 compared with the value of cells treated with ethanol (control) by unpaired t-test (n = 3). Figure 1 Activation of PPARs by Resveratrol, ε-Viniferin and Vaticanol C. (A) Structures of resveratrol (monomer), ε-viniferin (dimer) and vaticanol
C (tetramer). (B) Activation of PPARα, β/δ, and γ was evaluated by transfection assays using BAEC with PPRE-luc together with GS-hPPARα, pCMX-NUC1
or pCMX-hPPARγ1, respectively. Results are presented as relative luciferase activities obtained by dividing the normalized luciferase activity from the
reporter vector PPRE-luc. *, P < 0.05, **, P < 0.01 compared with the value of cells treated with ethanol (control) by unpaired t-test (n = 3). induced expression of Acox1 and LCAD (manuscript in
preparation), which are not induced by vaticanol C treat-
ment with a HF diet (Figure 3A). In skeletal muscles,
expression of PPARβ/δ-dependent genes, such as UCP3
and PDK4 [23,24], was induced by vaticanol C in wild-
type, but not in knockout mice (Figure 3A). an inducible key enzyme for prostaglandin synthesis, was
regulated by 15d-PGJ2, a natural ligand for PPARγ [19]. Interestingly, vaticanol C as well as resveratrol [25] sup-
pressed the expression of COX-2 in several kinds of
tumor cell lines (data not shown) whereas vaticanol C is
not an activator for PPARγ. Vaticanol C did not activate SIRT1 whereas resveratrol
activated SIRT1 by the assay utilizing a fluorophore-con-
taining peptide (Figure 2C). Recent report showed that
this assay was not suitable to direct measurement of the
SIRT1 activity and that resveratrol did not activate SIRT1
in the assay without the fluorophore-containing peptide
[26]. Therefore, by the different assay system, we may
confirm that vaticanol C is not activator for SIRT1. No Correlation with Resveratrol or its Derivatives between
Activation of PPARs and Radical-Scavenging Activities Taken
together, vaticanol C is a dual activator for PPARα and β/
δ, whereas resveratrol is a triple activator for PPARα, β/δ
and γ, indicating that vaticanol C is thought to be differ-
ently effective on lifestyle-related diseases, compared
with resveratrol. Discussion Resveratrol is known to be a SIRT1-activator. However,
various effects of resveratrol are not explained by activa-
tion of SIRT1 alone. We found that resveratrol is an acti-
vator not only for PPARα and γ [8], but also for PPARβ/δ
(Figure 1B) in this report. Moreover, vaticanol C, a res-
veratrol tetramer, is an activator for PPARα and β/δ in
cell-based reporter assays (Figure 1B), which is confirmed
by induced expression of PPAR-responsive genes in wild-
type, but not PPARα-knockout, mice (Figure 3A). At
lower concentrations of 1.25-5 μM, vaticanol C showed
higher PPARα-agonistic activity than resveratrol. How-
ever, at 10 μM, resveratrol showed higher activity than
vaticanol C (Figure 1A), and this activity of resveratrol
was dose-dependent, at less than 40 μM (data not
shown). Previously, we found that expression of COX-2, A resveratrol dimer, ε-viniferin has radical-scavenging
activity, but does not have agonistic activity for PPARs or
SIRT1, which differs from resveratrol and vaticanol C
(Figure 2). These differences indicate the importance of Tsukamoto et al. Nutrition & Metabolism 2010, 7:46 Page 5 of 8 http://www.nutritionandmetabolism.com/content/7/1/46 Figure 2 Distinct Effects of Resveratrol, ε-Viniferin and Vaticanol C on Activation of PPARs, Radical Scavenging Activity and SIRT1 Activa-
tion. (A) Activation of PPARα, β/δ, and γ was evaluated by cell-based transfection assays at a dose of 10 μM. Trolox, a standard antioxidant, was used
as control. (B) Radical scavenging activity was evaluated by the DPPH method. Each result is expressed as the equivalent per mole of trolox from the
percentage decrease with respect to the negative control values. (C) SIRT1 activation in vitro was evaluated by the Fluor de Lys fluorescence assay. DMSO was used as a negative control and also as a solvent for resveratrol and its derivatives to a final concentration of 100 μM in the assay buffer. Figure 2 Distinct Effects of Resveratrol, ε-Viniferin and Vaticanol C on Activation of PPARs, Radical Scavenging Activity and SIRT1 Activa-
tion. (A) Activation of PPARα, β/δ, and γ was evaluated by cell-based transfection assays at a dose of 10 μM. Trolox, a standard antioxidant, was used
as control. (B) Radical scavenging activity was evaluated by the DPPH method. Each result is expressed as the equivalent per mole of trolox from the
percentage decrease with respect to the negative control values. (C) SIRT1 activation in vitro was evaluated by the Fluor de Lys fluorescence assay. Discussion DMSO was used as a negative control and also as a solvent for resveratrol and its derivatives to a final concentration of 100 μM in the assay buffer. PPARα is also involved in the expression of PPARβ/δ-
responsive genes. the structure of these chemicals. Concerning the binding
pocket of PPARα, the molecular sizes of resveratrol and
vaticanol C are smaller and larger than those of synthetic
PPAR agonists such as fenofibrate, respectively. Resvera-
trol but not vaticanol C activates PPAR γ, indicating the
importance of the structure of these molecules in the
binding pocket of PPARs. It was reported that a single
amino acid, which was Tyr in PPARα and His in PPARγ,
imparted subtype selectivity for both thiazolidinedione
and nonthiazolidinedione ligands [27]. We need more
experimental data to discuss about these points including
the possibility of indirect activation of PPARs by resvera-
trol and vaticanol C. Resveratrol has been reported to prevent body weight
gain with a HF diet [15]. No such effect on body weight
was observed after vaticanol C treatment. We found that
129SV mice used in our study showed milder body
weight gain with a HF diet, versus the C57/black strain
mice used in several reports. This difference on body
weight gain will be due to the different strains between
129SV and C57/black. These genetic differences are also
usual common in human genomes. To address these
questions, we are studying on the molecular mechanism
involved in the physiological differences between 129SV
and C57/black mice. There are distinct expression patterns of PPAR-respon-
sive genes in the liver and skeletal muscle, indicating that
PPAR-responsive genes are controlled in a tissue-specific
manner (Figure 3A). In this context, especially in skeletal
muscles, vaticanol C upregulates expression of PPARβ/δ-
responsive genes, such as UCP3 and PDK4, in the wild-
type, but not PPARα-knockout mice, indicating that Conclusion Vaticanol C, a resveratrol tetramer, activates PPARα and
PPARβ/δ in cell-based reporter assays, but does not acti-
vates SIRT1. Eight-week intake of vaticanol C with a HF
diet upregulates hepatic expression of PPARα-responsive Tsukamoto et al. Nutrition & Metabolism 2010, 7:46 Page 6 of 8 ,
http://www.nutritionandmetabolism.com/content/7/1/46 Figure 3 Induction of PPAR-dependent Genes by Vaticanol C and Fenofibrate. (A) mRNA levels of the indicated genes were measured by RT-
qPCR in liver and skeletal muscle from WT (open column) and PPARα-knockout (closed column) mice fed a HF diet containing 60% energy as fat, sup-
plemented with vaticanol C (0 and 0.04%) for 8 weeks. Results are presented as means ± S.D. (n = 4~7). * compared with the value of 0% vaticanol C
(*, P < 0.05, **, P < 0.01). # compared with the value of the corresponding wild-type mice (#, p < 0.05, ##, p < 0.01).(B) mRNA levels of the indicated
genes were measured by RT-qPCR in liver from WT fed a HF diet containing 60% energy as fat, supplemented with fenofibrate (0 and 0.02%) for 4
weeks. Results are presented as means ± S.D. (n = 4). * compared with the value of 0% fenofibrate (*, P < 0.05, **, P < 0.01). A
B A A Figure 3 Induction of PPAR-dependent Genes by Vaticanol C and Fenofibrate. (A) mRNA levels of the indicated genes were measured by RT-
qPCR in liver and skeletal muscle from WT (open column) and PPARα-knockout (closed column) mice fed a HF diet containing 60% energy as fat, sup-
plemented with vaticanol C (0 and 0.04%) for 8 weeks. Results are presented as means ± S.D. (n = 4~7). * compared with the value of 0% vaticanol C
(*, P < 0.05, **, P < 0.01). # compared with the value of the corresponding wild-type mice (#, p < 0.05, ##, p < 0.01).(B) mRNA levels of the indicated
genes were measured by RT-qPCR in liver from WT fed a HF diet containing 60% energy as fat, supplemented with fenofibrate (0 and 0.02%) for 4
weeks. Results are presented as means ± S.D. (n = 4). * compared with the value of 0% fenofibrate (*, P < 0.05, **, P < 0.01). B B Figure 3 Induction of PPAR-dependent Genes by Vaticanol C and Fenofibrate. Author Details 1Department of Food Science and Nutrition, Nara Women's University, Nara,
630-8506, Japan, 2API research Center, API Co. Ltd., Gifu, 502-0071, Japan,
3Laboratory of Pharmacognosy, Gifu Pharmaceutical University, Gifu 502-8585,
Japan, 4Gifu International Institute of Biotechnology, Gifu 504-0838, Japan,
5Department of Bioscience, National Cardiovascular Center Research Institute,
Suita, Osaka 565-8565, Japan and 6Department of Neurobiology, Morehouse
School of Medicine, 720 Westview Drive, SW, Atlanta, GA30310-1495, USA 17. Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M, Tschöp MH: Sirt1
protects against high-fat diet-induced metabolic damage. Proc Natl
Acad Sci 2008, 105:9793-9798. 18. Hotta M, Nakata R, Katsukawa M, Hori K, Takahashi S, Inoue H: Carvacrol, a
component of thyme oil, activates PPARα and γ, and suppresseses
COX-2 expression. J Lipid Res 2010, 51:132-139. Suita, Osaka 565-8565, Japan and 6Department of Neurobiology, Morehouse
School of Medicine, 720 Westview Drive, SW, Atlanta, GA30310-1495, USA 19. Inoue H, Tanabe T, Umesono K: Feedback control of cyclooxygenase-2
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Chem 2002, 50:1213-1217. 23. Son C, Hosoda K, Matsuda J, Fujikura J, Yonemitsu S, Iwakura H, Masuzaki
H, Ogawa Y, Hayashi T, Itoh H, Nishimura H, Inoue G, Yoshimasa Y, Yamori
Y, Nakao K: Up-regulation of uncoupling protein 3 gene expression by
fatty acid and agonists for PPARs in L6 myotubes. Endocrinology 2001,
142:4189-4194. 5. Ito T, Akao Y, Tanaka T, Iinuma M, Nozawa Y: Vaticanol C, a novel
resveratrol tetramer, inhibits cell growth through induction of
apoptosis in cancer cell lines. Biol Pharm Bull 2002, 25:147-148. 5. Conclusion superfamily: the second decade. Cell 1995, 83:835-839. 10. Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass GK, Gonzalez FJ,
Grimaldi PA, Kadowaki T, Lazar MA, O'Rahilly S, Palmer CN, Plutzky J, Reddy
JK, Spiegelman BM, Staels B, Wahli W: International Union of
Pharmacology. LX1. Peroxisome proliferators- acitivated receptors. Pharmacol Rev 2006, 58:726-741. Acknowledgements 15. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F,
Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P,
Auwerx J: Resveratrol improves mitochondrial function and protects
against metabolic disease by activating SIRT1 and PGC-1alpha. Cell
2006, 127:1109-1122. We thank Dr. Takanobu Matsubara, Haruka Takeuchi, Mariko Hotta, and Kanako
Kojima for technical assistance. This work was supported by Grants-in-Aid for
Scientific Research (Nos. 15310158 to HI and 19300250 to HI and RN) from the
Ministry of Education, Culture, Sports, Science and Technology, Japan. SN was
supported by NS048532 from NIH/NINDS, USA. 16. Feige JN, Lagouge M, Canto C, Strehle A, Houten SM, Milne JC, Lambert
PD, Mataki C, Elliott PJ, Auwerx J: Specific SIRT1 activation mimics lower
energy levels and protects against diet-induced metabolic disorders
by enhancing fat oxidation. Cell Metab 2008, 8:347-358. Authors' contributions 14. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV,
Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O,
Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG,
Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R,
Sinclair DA: Resveratrol improves health and survival of mice on a high-
caloric diet. Nature 2006, 444:337-342. TT, RN, ET, YK, AK, MK and YA carried out the experiments and analyzed the
data. SM, TI, MI, YA and YN prepared ε-viniferin and vaticanol C. RN, SN and HI
designed the experiments and wrote the manuscript. All authors read and
approved the final manuscript. Received: 16 March 2010 Accepted: 27 May 2010
Published: 27 May 2010
Thi
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t iti
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© 2010 T
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2010 7 46 20. Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon
JM: Comparative study of radical scavenger and antioxidant properties
of phenolic components from Vitis vinifera cell cultures using in vitro
tests. Life Sci 1997, 61:2103-2110. Additional material 11. Sonoda J, Pei L, Evans RM: Nuclear receptors: decoding metabolic
disease. FEBS Lett 2008, 9:2-9. 12. Sugiyama E, Ishikawa Y, Li Y, Kagai T, Nobayashi M, Tanaka N, Kamijo Y,
Yokoyama S, Hara A, Aoyama T: Eicosapentaenoic acid lowers plasma
and liver cholesterol levels in the presence of peroxisome proliferators-
activated receptor alpha. Life Sci 2008, 83:19-28. Additional file 1 Supplementary Table 1. Primer pairs for genes and
their cycling conditions are shown. Competing interests Competing interests 13. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG,
Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA: Small
molecule activators of sirtuins extend Saccharomyces cerevisiae. Nature 2003, 425:191-196. Conclusion (A) mRNA levels of the indicated genes were measured by RT-
qPCR in liver and skeletal muscle from WT (open column) and PPARα-knockout (closed column) mice fed a HF diet containing 60% energy as fat, sup-
plemented with vaticanol C (0 and 0.04%) for 8 weeks. Results are presented as means ± S.D. (n = 4~7). * compared with the value of 0% vaticanol C
(*, P < 0.05, **, P < 0.01). # compared with the value of the corresponding wild-type mice (#, p < 0.05, ##, p < 0.01).(B) mRNA levels of the indicated
genes were measured by RT-qPCR in liver from WT fed a HF diet containing 60% energy as fat, supplemented with fenofibrate (0 and 0.02%) for 4
weeks. Results are presented as means ± S.D. (n = 4). * compared with the value of 0% fenofibrate (*, P < 0.05, **, P < 0.01). B B B Figure 3 Induction of PPAR-dependent Genes by Vaticanol C and Fenofibrate. (A) mRNA levels of the indicated genes were measured by RT-
qPCR in liver and skeletal muscle from WT (open column) and PPARα-knockout (closed column) mice fed a HF diet containing 60% energy as fat, sup-
plemented with vaticanol C (0 and 0.04%) for 8 weeks. Results are presented as means ± S.D. (n = 4~7). * compared with the value of 0% vaticanol C
(*, P < 0.05, **, P < 0.01). # compared with the value of the corresponding wild-type mice (#, p < 0.05, ##, p < 0.01).(B) mRNA levels of the indicated
genes were measured by RT-qPCR in liver from WT fed a HF diet containing 60% energy as fat, supplemented with fenofibrate (0 and 0.02%) for 4
weeks. Results are presented as means ± S.D. (n = 4). * compared with the value of 0% fenofibrate (*, P < 0.05, **, P < 0.01). Page 7 of 8 Page 7 of 8 Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 9. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,
Blumberg B, Kastner P, Mark M, Chambon P: The nuclear receptor
superfamily: the second decade. Cell 1995, 83:835-839. genes and skeletal muscle expression of PPARβ/δ-respon-
sive genes in wild-type but not PPARα-knockout mice. These findings indicate that activation of PPARα and
PPARβ/δ by vaticanol C is a novel mechanism that may
afford beneficial effects against cardiovascular disease. References Ito T, Akao Y, Tanaka T, Iinuma M, Nozawa Y: Vaticanol C, a novel
resveratrol tetramer, inhibits cell growth through induction of
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RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qui X, Stockman B, Thanabal V,
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vates PPAR? and PPAR?/? in vitro and in vivo Nutrition & Metabolism 2010, 7:46 26. Pacholec M, Bleasdale JM, CHrunyk B, Chunningham D, Flynn D, Garofalo
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Structural determinants of ligand binding selectivity between the
peroxisome proliferator-activated receptors. Proc Natl Acad Sci 2001,
98:13919-13924. doi: 10.1186/1743-7075-7-46
Cite this article as: Tsukamoto et al., Vaticanol C, a resveratrol tetramer, acti-
vates PPAR? and PPAR?/? in vitro and in vivo Nutrition & Metabolism 2010, 7:46 26. Pacholec M, Bleasdale JM, CHrunyk B, Chunningham D, Flynn D, Garofalo
RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qui X, Stockman B, Thanabal V,
Varghese A, Ward J, Withka J, Ahn K: SRT1720, SRT2183, SRT1460, and
resveratrol are not direct activators of SIRT1. J Biol Chem 2010,
285:8340-8351. 26. Pacholec M, Bleasdale JM, CHrunyk B, Chunningham D, Flynn D, Garofalo
RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qui X, Stockman B, Thanabal V,
Varghese A, Ward J, Withka J, Ahn K: SRT1720, SRT2183, SRT1460, and
resveratrol are not direct activators of SIRT1. J Biol Chem 2010,
285:8340-8351. 27. Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL,
Oplinger JA, Kliewer SA, Gample RT Jr, Mckee DD, Moore JT, Willson TM:
Structural determinants of ligand binding selectivity between the
peroxisome proliferator-activated receptors. Proc Natl Acad Sci 2001,
98:13919-13924. 27. Tsukamoto et al. Nutrition & Metabolism 2010, 7:46
http://www.nutritionandmetabolism.com/content/7/1/46 Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL,
Oplinger JA, Kliewer SA, Gample RT Jr, Mckee DD, Moore JT, Willson TM:
Structural determinants of ligand binding selectivity between the
peroxisome proliferator-activated receptors. Proc Natl Acad Sci 2001,
98:13919-13924. doi: 10.1186/1743-7075-7-46
Cite this article as: Tsukamoto et al., Vaticanol C, a resveratrol tetramer, acti-
vates PPAR? and PPAR?/? in vitro and in vivo Nutrition & Metabolism 2010, 7:46 doi: 10.1186/1743-7075-7-46
Cite this article as: Tsukamoto et al., Vaticanol C, a resveratrol tetramer, acti-
vates PPAR? and PPAR?/? in vitro and in vivo Nutrition & Metabolism 2010, 7:46
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English
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Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity
|
Frontiers in immunology
| 2,019
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cc-by
| 9,051
|
Junin Virus Triggers Macrophage
Activation and Modulates
Polarization According to Viral Strain
Pathogenicity María F. Ferrer 1, Pablo Thomas 1, Aída O. López Ortiz 1,2, Andrea E. Errasti 3, Nancy Charo 2,
Victor Romanowski 1,4, Juan Gorgojo 5, María E. Rodriguez 5, Eugenio A. Carrera Silva 2*†
and Ricardo M. Gómez 1,4*† 1 Laboratorio de Virus Animales, Instituto de Biotecnología y Biología Molecular, CONICET-Universidad Nacional de La Plata,
La Plata, Argentina, 2 Laboratorio de Trombosis Experimental, Instituto de Medicina Experimental, CONICET-Academia
Nacional de Medicina, Buenos Aires, Argentina, 3 Facultad de Medicina, Instituto de Farmacologia, University of Buenos
Aries, Buenos Aires, Argentina, 4 Global Viral Network, Baltimore, MD, United States, 5 Centro de Investigación y Desarrollo
en Fermentaciones Industriales, CONICET-Universidad Nacional de La Plata, La Plata, Argentina ORIGINAL RESEARCH
published: 22 October 2019
doi: 10.3389/fimmu.2019.02499 Keywords: junin virus, human macrophages, TAM receptors, macrophage activation, macrophage polarization,
IFN-I Carrera Silva
carrerasilva@yahoo.com.ar
Ricardo M. Gómez
rmg1426@gmail.com †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Viral Immunology,
a section of the journal
Frontiers in Immunology
Received: 29 May 2019
Accepted: 07 October 2019
Published: 22 October 2019
Citation:
Ferrer MF, Thomas P, López Ortiz AO,
Errasti AE, Charo N, Romanowski V,
Gorgojo J, Rodriguez ME,
Carrera Silva EA and Gómez RM
(2019) Junin Virus Triggers
Macrophage Activation and
Modulates Polarization According to
Viral Strain Pathogenicity. Front. Immunol. 10:2499. doi: 10.3389/fimmu.2019.02499 Specialty section:
This article was submitted to
Viral Immunology,
a section of the journal
Frontiers in Immunology Received: 29 May 2019
Accepted: 07 October 2019
Published: 22 October 2019 The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic
fever (AHF). Previous studies of human macrophage infection by the Old-World
arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus
replicates and activates human macrophages, the pathogenic Lassa virus replicates but
fails to activate human macrophages. Less is known in regard to the impact of New
World arenavirus infection on the human macrophage immune response. Macrophage
activation is critical for controlling infections but could also be usurped favoring immune
evasion. Therefore, it is crucial to understand how the JUNV infection modulates
macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we
compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the
JUNV virus in human macrophage cultures. The results showed that both JUNV strains
similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell
entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively
modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected
with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that
C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted
the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively
regulates the immune response, was down-regulated by C#1 strain and up-regulated
by P strain infection. Similarly, the target genes of MERTK activation, the cytokine
suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition
to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P
strain infection. Together, this differential activation/polarization pattern of macrophages
elicited by P strain suggests a more evasive immune response and may have important
implications in the pathogenesis of AHF and underpinning the development of new
potential therapeutic strategies. Edited by:
Luis F. Garcia,
University of Antioquia, Colombia
Reviewed by:
Jack Nunberg,
University of Montana, United States
Carlos Fernando Narváez,
South Colombian University, Colombia
*Correspondence:
Eugenio A. Carrera Silva
carrerasilva@yahoo.com.ar
Ricardo M. Gómez
rmg1426@gmail.com
†These authors have contributed Edited by:
Luis F. Garcia,
University of Antioquia, Colombia Reviewed by:
Jack Nunberg,
University of Montana, United States
Carlos Fernando Narváez,
South Colombian University, Colombia *Correspondence:
Eugenio A. Carrera Silva
carrerasilva@yahoo.com.ar
Ricardo M. Gómez
rmg1426@gmail.com *Correspondence:
Eugenio A. INTRODUCTION peptide SSP that together will constitute the spikes that decorate
the virus surface (5). Junin virus (JUNV) is the etiological agent of Argentine
hemorrhagic fever (AHF), an endemoepidemic disease mainly
affecting agricultural workers in Argentina. The infection is
usually acquired through small abrasions in the skin or through
aspiration of particles contaminated with urine, saliva, or blood
from carrier rodents. The AHF incubation period ranges from 6
to 12 days, ending with the onset of fever. During first 7 days,
patients are commonly associated with a flu-like syndrome and
the fever persists until the second week, when hemorrhagic or
neurological signs of varied severity may be present. The 80% of
patients improve after the second week. AHF diagnosis is based
on clinical and laboratory data, the latter mainly based on platelet
counts below 100,000/mm3 in combination with white blood cell
counts under 2,500/mm3. Early diagnosis is important, because
the early use of immune plasma from convalescent patients
reduces mortality rates from 20 to 1%. Candid #1 (C#1) is a live
attenuated vaccine against AHF, which is licensed in Argentina
and has been administered to several hundred thousand persons
in endemic areas for more than 20 years, with a major impact on
the incidence of the disease. However, since the first description
of the disease in the 1950s, uninterrupted annual outbreaks
have been observed in a progressively expanding region in
north-central Argentina, to the point that more than 5 million
individuals are today considered to be at risk of AHF (1). Macrophages are the most functionally diverse (plastic) cells
of the hematopoietic system. Macrophages are found in all tissues
and their main function is to respond to pathogens and modulate
the adaptive immune response through antigen processing and
presentation (6, 7). Macrophage activation has emerged as a
key area of study in immunology, tissue homeostasis, disease
pathogenesis and inflammation resolution (8). To accomplish
such diverse functions, they mature under the influence of
signals from the local microenvironment into either classical
M1 or alternatively M2 activated macrophages. M1 macrophages
are characterized by the production of high levels of pro-
inflammatory cytokines, an ability to mediate resistance to
pathogens, strong microbicidal properties, high production of
reactive nitrogen and oxygen intermediates and promotion
of Th1 responses. In contrast, M2 activated macrophages are
characterized by their involvement in parasite control, resolution
of inflammation, tissue remodeling, immune regulation, and Th2
promotion responses (6, 9). INTRODUCTION In this study, we aimed to characterize the infection of
macrophages using two strains of the same arenavirus with
different pathogenic properties. For this purpose, we studied the
infection of human macrophages by the attenuated C#1 and the
pathogenic P strains of JUNV, using an in vitro model represented
by human macrophage cell cultures. y
JUNV belongs to the clade B New World (NW) of genus
mammarenavirus that together with genus reptarenavirus
form the Arenaviridae family (2). Most mammarenavirus
are associated with rodent infections. The Old World (OW)
choriomeningitis
lymphocytic
virus
(LCMV)
infects
Mus
musculus, and this explains its global distribution. In contrast,
other
strains
of
mammarenavirus
infect
rodents
with
a
circumscribed geographical distribution that explains their
endemicity (3). In their natural rodent host, mammarenavirus
usually produce a persistent asymptomatic infection that may
occasionally be transmitted to humans where it can cause severe
hemorrhagic fever (HF). In addition to JUNV, other strains of
mammarenavirus associated with HF are the NW Machupo
(MACV) and Chapare (CHPV) viruses in Bolivia, Sabiá (SABV)
in Brazil and the OW Lassa virus (LASV) in Africa. In contrast,
other members such as the NW Tacaribe (TCRV) and Pichindé
(PICV) or the OW Mopeia (MOPV) viruses do not cause disease
(4). The mammarenavirus are etiological agents of emergent
diseases because human activity facilitates contact with wild
rodents in new ecological niches and, therefore, new isolates
should be expected in the future (3). Cells Cells
BHK-21 and Vero-76 cells (ATCC, USA) were maintained
as monolayers, as previously described (10). Peripheral blood
mononuclear cells (PBMCs) were obtained from healthy
volunteer donors who had not taken any non-steroidal anti-
inflammatory drugs for 10 days prior to sampling as previously
described (11). This study was approved by the Institutional
Ethics Committee, National Academy of Medicine, Argentina. Written consent was obtained from all subjects. Human
monocyte-derived macrophages (HMDM) were obtained as
previously reported (12). Briefly, PBMCs from healthy donors
were isolated by Ficoll-Hypaque (GE, Chicago, IL, USA)
density gradient centrifugation, and positive selection of CD14+
monocytes was performed using an EasySep
TM Human CD14
Positive Selection Kit (StemCell Tech, Vancouver, Canada). Macrophage differentiation was carried out by plating 2.5
× 105 CD14+ monocytes in 48-well plates containing 500
µL of RPMI 1640 plus 10% Fetal Bovine Serum (FBS) and
1% penicillin/streptomycin (PS) in the presence of rM-CSF
(40 ng/ml) and cultured for 7 days. In selected experiments, 24-
well plates were used with a double quantity of cells and medium. Like other members of the same family, JUNVs are enveloped
virions, ∼120 nm in diameter, with a capsid of helicoidal
symmetry that includes a variable number of ribosomes. The
virions contain a bi-segmented single-stranded RNA genome,
with both segments employing an ambisense coding strategy. The
L segment contains genes encoding the RNA-dependent RNA
polymerase (L) and the matrix protein (Z). However, the smaller
S segment encodes the nucleoprotein (N) and the glycoprotein
precursor (GPC) which, after post-translational cleavage, yields
mature virion glycoproteins (G) G1, G2 and the stable signal Citation: Ferrer MF, Thomas P, López Ortiz AO,
Errasti AE, Charo N, Romanowski V,
Gorgojo J, Rodriguez ME,
Carrera Silva EA and Gómez RM
(2019) Junin Virus Triggers
Macrophage Activation and
Modulates Polarization According to
Viral Strain Pathogenicity. Front. Immunol. 10:2499. doi: 10.3389/fimmu.2019.02499 October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org Arenavirus Modulates Macrophage Polarization Ferrer et al. Frontiers in Immunology | www.frontiersin.org Flow Cytometry Analysis MEM, RPMI, and FBS were purchased from Invitrogen (Buenos
Aires, Argentina). rM-CSF was acquired from R&D Systems
(Minneapolis, MN, USA). Anti JUNV antibodies were obtained
from BEI resources, USA. Anti CD71, CD14, CD86, CD80,
HLA-DR, CD11b, CD11c, CD64, CD163, CD206 were obtained
from BioLegend (San Diego, CA, USA). Anti-human APC-
MERTK (mouse IgG1), Biotin-AXL (goat IgG) and isotype
controls were obtained from R&D Systems (Minneapolis, MN,
USA). Anti-TYRO3 (rabbit IgG) was obtained from Novus
Biological (Littleton, CO, USA). DAPI was purchased from
Invitrogen (Buenos Aires, Argentina). ELISA kits (Ready-SET-
Go kits) for TNF-α, IL-1β, IL-6, IL-10, and IL-12p70 were
obtained from eBioscience, Fisher scientific, Pittsburgh, PA, USA. Cytofix/Cytoperm kit was purchased from BD Bioscience (San
Diego, CA, USA). y
y
y
The viability assay on macrophages culture was performed after
72 h of JUNV infection using Annexin V (AnnV) (Immunotools,
Gladiolenweg, Friesoythe, Gemany) together with Fixable
viability dye (eBioscience, USA). Briefly, cell harvesting was
performed by a 20-min incubation with PBS plus 2% FBS (PBSF)
and 1 mM EDTA on ice, followed by up and down pipetting. The
harvested cells were washed once with PBSF and then stained
with fixable viability dye efluor 780 diluted in PBS for 30 min. After washing cells, they were stained with AnnV following
manufacturer’s instruction. After final washing, the cells were
fixed with a Cytofix/Cytoperm Kit (BD Biosciences, USA). y
y
p
The surface staining for CD11b, CD64, CD206, CD14
(phenotypic characterization of macrophages) or CD11b, HLA-
DR, CD86, and CD80 (activation status) was performed
following a standard protocol. Briefly, the harvested cells were
washed with PBS and blocked in PBSF on ice for 30 min. The cells were washed with PBS and the respective antibody
cocktails (prepared in PBSF) were added to the cell pellet and
incubated for 30 min on ice. A fixable viability dye was used
according to the manufacturer’s instructions to gate on live cells. After washing, the cells were fixed with a Cytofix/Cytoperm
Kit, washed again and analyzed in a FACS Canto I (Becton
Dickinson, Franklin Lakes, NJ, USA) or Partec-Sysmex CyFlow
flow cytometer (Görlitz, Germany). All analysis was carried
out with FlowJo software (Tree Star). Intracellular staining was
performed following manufacturer’s recommendation for the
Cytofix/Cytoperm Kit. The preparation of blockage and cocktail
antibodies was performed with PBSF. We have used fluorescent
minus one (FMO) to set the threshold for each marker. Virus A virulent strain of JUNV, originally isolated from an AHF
patient (P3441), as well as the attenuated Candid 1 (C#1) have
been already described (13). The preparation of viral stocks in
BHK-21 cells and infectivity titration using the Vero-76 cell line October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org 2 Arenavirus Modulates Macrophage Polarization Ferrer et al. refrigerated camera under the control of IS listen software and
further processed using Photoshop 6.0 software. has been previously described (13). All work with the infective P
strain was performed in a BSL/3 facility by vaccinated personal. Plaque Formation Assay Ten-fold dilutions of the macrophage-JUNV infected culture
supernatants were added to 24-well plates with a 40–50%
confluence monolayer of Vero E6 cells. The plate was then
incubated at 37◦C for 1 h with gentle rocking. Following
adsorption, the inoculum was removed and overlaid with 2 ml of
MEM containing 0.8% methylcellulose and 2% FBS and further
incubated at 37◦C in a humid atmosphere with 5% CO2. Plaques
were allowed to develop for either 4–6 days before being fixed
(4% w/v paraformaldehyde) and stained with a 1% Crystal Violet
in 20% ethanol and dH2O. Cell Infection For viral infection, cells were washed with PBS twice before
incubating with the virus at a multiplicity of infection (MOI)
of 1 in serum free medium. After 1 h of incubation at 37◦C,
cells were washed with PBS twice again and supplemented with
a complete culture medium. Mock infection was performed by
adding the same volume of BHK-21 cell culture supernatant,
instead of JUNV, to the cell monolayer. Cells were observed daily
using an inverted microscope with an Olympus SP-320 camera
and images were further processed with Photoshop 6.0 software. Enzyme Linked Immunosorbent Assay
(ELISA) IL-6, IL-12p70, TNF-α, and IL-10 levels were assessed in culture
supernatants with ELISA Ready-SET-Go kits (e-Bioscience)
according to the manufacturer’s protocol. Frontiers in Immunology | www.frontiersin.org JUNV Strains Differentially Enhances the
Expression of CD71 Viruses exploit fundamental cellular processes to gain entry into
cells and deliver their genetic cargo. Virus entry pathways are
largely defined by the interactions between virus particles and
their receptors at the cell surface. These interactions determine
the mechanisms of virus attachment and, ultimately, penetration
into the cytosol. In contrast to LASV and other OW arenaviruses,
which use α-dystroglycan to infect cells, the NW arenaviruses,
including JUNV, use human transferrin receptor 1 (hTFR1 or
CD71) (14). We have previously shown that JUNV infection
enhances the expression of hTFR1 in the precursor CD34+ cells,
suggesting that JUNV infection promotes its own dissemination
(15). Compared with other cell types, mature macrophages may
be atypical regarding the requirements for hTFR1 expression
levels (16), and for that reason, we explored the expression
pattern in HMDM infected cells. In this sense, our results showed
that JUNV infection enhances CD71 expression in human
macrophages, but with the highest value associated with P strain
infection (Figures 2A–C). JUNV Selectively Skews Macrophage
Polarization Taking into account the fact that JUNV modulates macrophage
activation depending on which strain was used, we next evaluated
different surface markers to distinguish M1/M2 polarization
in HMDM after JUNV infection. The percentage of CD64+
(M1), CD206+, and CD163+ (M2) cells expressed in CD11b+
cells were analyzed by flow cytometry. CD11b+CD64+CD206−
cells were increased when cells were infected with C#1 strain
as compared to Mock and the P strain with an average
of 18 vs. 8% and 3.2%, respectively. However, the M2
phenotype CD11b+CD206+CD64−was significantly higher
after P infection as compared C#1 strain or to Mock with
an average of 34 vs. 10.1%, and 17.5%, respectively. This
indicates that JUNV modulates polarization according to viral
strain pathogenicity (Figures 4A,B). Additionally, another M2
phenotype analyzed as a double positive, CD206+/CD163+
cells, showed a clear tendency toward an increased percentage
after infection with P strain as compared to Mock and C#1
strain infection (44.2 vs. 29.9% and 33.9%, respectively, see
Supplementary Information S2). JUNV Strains Replicate Similarly in Human
Macrophages p
g
Human monocyte-derived macrophages (HMDM) cells were
infected at a multiplicity of infection (MOI) = 1 with the
attenuated C#1 or the pathogenic P strains of JUNV. HMDM
cells showed clear morphological changes, such as becoming
more flattened and extended, as early as 24 h post-infection (hpi)
with both JUNV strains (Figure 1A). Infectious virus released to
the cell culture supernatants were measured over 6 days by plaque
formation assay. Infection with both viral strains led to similar
levels of infectious viruses, peaking at 3 days post-infection
(dpi) and declining until the end of the study (Figure 1B). Viral
antigen was studied at 3 dpi by immunofluorescence (IF) and
flow cytometry (FC) analysis. Viral protein staining was similarly
positive with both strains (Figure 1C). As expected, FC analysis
confirmed these results showing that 58% of HMDM cells were
infected with C#1 strain meanwhile 57.6% were positive for the P
strain (Figure 1D). PCR Cells were cultured on 12 mm diameter glass inserts before viral
infection. At the indicated time-point after infection, the inserts
were fixed with 4% paraformaldehyde (PFA) for 20 min and
permeabilized with 0.1% Tween for 10 min. The slides were
incubated overnight at 4◦C with a pool of specific monoclonal
antibodies against JUNV (13). FITC-conjugated anti mouse Igs
were then applied to the PBS-washed slides for 30 min at room
temperature (RT). Antibodies were diluted with PBS containing
5% FBS and 5% goat serum as blocking reagents. The slides
were counterstained with DAPI and examined under a Nikon
E200 microscope equipped with fluorescence filters and a 100-
W mercury lamp. Images were acquired with a Tucsen TCC 5.0 For gene expression analysis, cultured cells were washed and
then harvested with Trizol (Life Technologies, Carlsbad, CA,
USA) and total RNA was obtained following the manufacturer’s
instructions. Reverse transcription was performed using 100 ng
of RNA by employing an iScript cDNA synthesis kit (Bio-Rad,
Hercules, CA, USA). qPCR reactions were assessed using 1 µl
of cDNA and using Sso Advanced Universal mix with Sybr
Green and CFX-Connect equipment (Bio-Rad). Primers used in
this study are listed in Table S1. The reaction was normalized
to housekeeping gene expression levels and the specificity
of the amplified products was checked through analysis of
dissociation curves. October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org 3 Arenavirus Modulates Macrophage Polarization Ferrer et al. Statistical Analysis surface marker (HLA-DR). Our results indicate a differential
expression when infected with one or other viral strain. A
significantly higher percentage of CD14+CD86+ cells were
observed after C#1 strain infection, while CD80 did not show
significant differences between infected cells. On the other hand,
P strain-infected macrophages showed the highest percentage
of CD14+ HLA-DR++ cells revealing a differential expression
pattern after infection with C#1 or P strain (Figures 3A,B). Each experiment was performed with 3–7 different donors. All
results are graphed as the median (min-max, horizontal line
indicates the median) and non-parametric one-way analysis
of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s
multiple
comparison
test
was
used
to
detect
significant
differences between groups. In all cases, P-values lower than 0.05
were considered statistically significant. All statistical analyses
were performed using Prism 6 software (GraphPad). p
g
Considering the observed macrophage activation induced by
JUNV infection, we next analyzed the level of several cytokines in
the supernatants of HMDM at 3 dpi. We found a clear distinctive
profile, since higher levels of TNF-α, IL-10, and IL-12 were
detected in the supernatants of C#1 strain-infected macrophages,
but only IL-6 was significantly increased using the P strain. Interestingly, no difference in IL-1 production was observed
compared with mock conditions, suggesting no activation of
the inflammasome pathway (Figures 3C–G). The percentage of
viable cells does not showed significant differences comparing
Mock, C#1 and P (90.2, 88.25, and 83.5, respectively) although
small increase in AnnV+ cells were observed with P when
compared to C#1 and Mock (Supplementary Information S1). The Expression of MERTK Was
Differentially Modulated With JUNV
Variants The TAM family tyrosine kinase receptors TYRO3, AXL,
and MERTK (TAM) receptors have been assigned to have a
prominent role in the following: regulating the innate immune
response (17); phagocytosis and macrophage polarization by
acting in coordination with cytokine signaling (18); and in
several aspects of the host response to viral infection (19, 20). Considering our observation that JUNV modulates macrophage
polarization and that AXL and MERTK are differentially
expressed in pro-inflammatory M1 and anti-inflammatory JUNV Strains Differentially Activate
Macrophages and Cytokine Production
We have analyzed the expression pattern of co-stimulatory
markers such as CD80 and CD86, and the antigen presentation JUNV Strains Differentially Activate Our
results showed that while TYRO3+ or AXL+ macrophages
showed a similar response to infection with the two strains,
the percentage of MERTK+ cells was down-regulated by C#1
strain and up-regulated by P strain infection, highlighting again
a differential macrophage response depending on virus strain
(Figures 5A,B). M2 macrophages, respectively (21), we next evaluated TAM
expression in HMDM after infection with both strains. Our
results showed that while TYRO3+ or AXL+ macrophages
showed a similar response to infection with the two strains,
the percentage of MERTK+ cells was down-regulated by C#1
strain and up-regulated by P strain infection, highlighting again
a differential macrophage response depending on virus strain
(Figures 5A,B). was interpreted as macrophage maturation and/or activation. In addition, we observed similar infectivity titers in the
supernatants and a comparable percentage of infected monolayer
cells. This stands in contrast with the reported minimal
replication of JUNV-XJ (pathogenic) and XJ-Cl3 (attenuated)
strains in macrophage cells from adult rats (23), a fact that
may be attributed that macrophages were from a different
donor species. Previous
studies
of
human
macrophage
infection
by
mammarenavirus have been shown that the non-pathogenic
MOPV both replicates and activates macrophages (24) whereas
pathogenic LASV replicates, but fails to activate macrophages
(25). The lack of activation of LASV-infected macrophages was
later associated with sequence differences in viral protein N
(26) and involved CXCL10 (27). On the other hand, it has been
reported that the non-pathogenic TCRV replicates less efficiently
in macrophages than the pathogenic JUNV, but induces a
cytokine release not observed in JUNV-infected cells (28). More
recently, a differential inhibition of macrophage activation by
LCMV and PICV, mediated by the N-terminal domain (NTD)
of viral Z protein, has been reported (29). Moreover, LCMV
Z NTD leads to increased viral replication and inhibition of
IFN responses in macrophages (30), a fact that has been more Since activation of MERTK triggers the induction of the
suppressor of cytokine signaling 1 (SOCS1) and SOCS3, we next
analyzed the transcription level of these genes by RT-qPCR. We
also analyzed interferon regulatory factor 1 (IRF-1) as a target
gene of infection and a member of the interferon regulatory
factor family (22). As expected, we observed higher transcription
levels of IRF-1, SOCS1, and SOCS3 concomitant with lower levels
of IFN-β in macrophages infected with P strain as compared with
mock conditions after 24 hpi (Figures 6A–D). JUNV Strains Differentially Activate JUNV Strains Differentially Activate
Macrophages and Cytokine Production g
We have analyzed the expression pattern of co-stimulatory
markers such as CD80 and CD86, and the antigen presentation October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org 4 Arenavirus Modulates Macrophage Polarization Ferrer et al. FIGURE 1 | Human macrophages are equally susceptible to both JUNV variants. Human monocyte-derived macrophages (HMDM) cells were infected at a multiplicity
of infection (MOI = 1) with the attenuated C#1 or the pathogenic P strains of JUNV. (A) Morphological observation of the cells. JUNV-infected cells become more
flattened and extended. (B) Infective titration of cell supernatants was performed in Vero cells for 6 days by a standard PFU method. (C) Viral antigens were detected
by immunofluorescence with a pool of antibodies and anti-JUNV-FITC, and nuclei were counterstained with DAPI. (D) HMDM cells were infected with C#1 or P strains
and at 3 dpi cells were stained with anti-JUNV-FITC and the percentage of viral antigen positive cells were analyzed by flow cytometry. The percentage of infected
cells was compared using the Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test. The results are graphed as the median (min-max,
horizontal line indicates the median). At least three independent donors were used in each assay. FIGURE 1 | Human macrophages are equally susceptible to both JUNV variants. Human monocyte-derived macrophages (HMDM) cells were infected at a multiplicity
of infection (MOI = 1) with the attenuated C#1 or the pathogenic P strains of JUNV. (A) Morphological observation of the cells. JUNV-infected cells become more
flattened and extended. (B) Infective titration of cell supernatants was performed in Vero cells for 6 days by a standard PFU method. (C) Viral antigens were detected
by immunofluorescence with a pool of antibodies and anti-JUNV-FITC, and nuclei were counterstained with DAPI. (D) HMDM cells were infected with C#1 or P strains
and at 3 dpi cells were stained with anti-JUNV-FITC and the percentage of viral antigen positive cells were analyzed by flow cytometry. The percentage of infected
cells was compared using the Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test. The results are graphed as the median (min-max,
horizontal line indicates the median). At least three independent donors were used in each assay. M2 macrophages, respectively (21), we next evaluated TAM
expression in HMDM after infection with both strains. DISCUSSION (C) Expression level of CD71 analyzed as mean fluorescent intensity (MFI) in graph. Non-parametric One-way ANOVA followed by Dunn’s multiple
comparison test was used to detect significant differences between groups; *P < 0.05. The results are graphed as the median (min-max, horizontal line indicates the
median) of at least four independent donors in each assay. recently assigned to the Z protein from pathogenic arenaviruses
only (31). virus in the absence of disease in the host. By contrast, the
pathogenic P strain elicits a more attenuated activation state of
macrophages by decreasing the prototypical pro-inflammatory
cytokines. However, it also remarkably induces higher levels of
IL-6, a cytokine also associated with immunomodulation (33),
and increases the percentage of CD14+HLA-DR++, two signals
that indicate an anti-inflammatory response that might allow
early immune evasion, facilitating viral dissemination in the host
and subsequent disease. Our results partially support to the hypothesis that OW and
NW arenaviruses may have different pathogenic mechanisms, at
least in macrophages cells (4, 32). We have previously shown that JUNV infection up-regulates
TfR1 in CD34+ hematopoietic stem cells (15). Here, we
show that both JUNV strains also increased the expression
of CD71 in infected HMDM, with P showing higher values. This supports the hypothesis that JUNV promotes its own
dissemination not only in undifferentiated hematopoietic cells
but also in a differentiated lineage and that P exploits this
mechanism more. It has been demonstrated that most of the acute viral
infections of pathogenic viruses are associated with macrophage
activation to a M1 status promoting inflammation (34). Regarding M2, the first studies were carried out with viruses
associated with chronic infections, and the first accepted
paradigm was that viral infection activates macrophages inducing
a M1 profile during the acute phase and an M2 profile
emerged during the eventual chronic phase of the disease (34,
35). In many of these studies, the M2-prone response related
to an enhanced production of IL-10, which indirectly exerts
potent immunosuppressive effects (36–38). Moreover, some
viruses, such as herpesviruses and poxviruses, encode functional
orthologs of IL-10 (vIL-10s) (39) or IL-6 (33). The viral IL-6 could The observed differential maturation and activation markers
and the cytokine expression profile depending on which JUNV
strain infects the macrophages strongly support the notion
that C#1 and P strains are able to elicit differential immune
responses. DISCUSSION In the present study, we showed that both attenuated C#1
and pathogenic P JUNV strains induced a phenotypic change
in primary human macrophages as early as 1 dpi, that October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org 5 Arenavirus Modulates Macrophage Polarization Ferrer et al. FIGURE 2 | JUNV enhances CD71 expression in human macrophages with P showing the highest values. HMDM cells were infected at a multiplicity of infection (MOI
= 1) with C#1 or P strains of JUNV. After 3 dpi, cells were double stained with anti-JUNV and anti-CD71 and analyzed by flow cytometry. (A) Representative dot-plots
showing positive signal for viral antigen in macrophages. (B) Representative histogram showing CD71 expression after gating in positive cells for JUNV antigen and
negative controls. (C) Expression level of CD71 analyzed as mean fluorescent intensity (MFI) in graph. Non-parametric One-way ANOVA followed by Dunn’s multiple
comparison test was used to detect significant differences between groups; *P < 0.05. The results are graphed as the median (min-max, horizontal line indicates the
median) of at least four independent donors in each assay. FIGURE 2 | JUNV enhances CD71 expression in human macrophages with P showing the highest values. HMDM cells were infected at a multiplicity of infection (MOI
= 1) with C#1 or P strains of JUNV. After 3 dpi, cells were double stained with anti-JUNV and anti-CD71 and analyzed by flow cytometry. (A) Representative dot-plots
showing positive signal for viral antigen in macrophages. (B) Representative histogram showing CD71 expression after gating in positive cells for JUNV antigen and
negative controls. (C) Expression level of CD71 analyzed as mean fluorescent intensity (MFI) in graph. Non-parametric One-way ANOVA followed by Dunn’s multiple
comparison test was used to detect significant differences between groups; *P < 0.05. The results are graphed as the median (min-max, horizontal line indicates the
median) of at least four independent donors in each assay. FIGURE 2 | JUNV enhances CD71 expression in human macrophages with P showing the highest values. HMDM cells were infected at a multiplicity of infection (MOI
= 1) with C#1 or P strains of JUNV. After 3 dpi, cells were double stained with anti-JUNV and anti-CD71 and analyzed by flow cytometry. (A) Representative dot-plots
showing positive signal for viral antigen in macrophages. (B) Representative histogram showing CD71 expression after gating in positive cells for JUNV antigen and
negative controls. Frontiers in Immunology | www.frontiersin.org DISCUSSION The results are graphed as the
median (min-max, horizontal line indicates the median) of at least four independent donors in each assay. FIGURE 4 | JUNV modulates macrophage M1/M2 polarization. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and at 3 dpi the cells expressing
CD64+ (M1) and CD206+ (M2) on CD11b+ were evaluated by flow cytometry. (A) Representative dot-plots of CD64 vs. CD206 after gating in CD11b of each
condition are shown. (B) Independent data are graphed showing a shift to M1 after C#1 infection and to M2 after P infection. Non-parametric One-way ANOVA
followed by Dunn’s multiple comparison test was used to detect significant differences between groups; *P < 0.05. The results are graphed as the median (min-max,
horizontal line indicates the median) of five independent donors. We had set the quadrant threshold based on the FMO of each marker. FIGURE 4 | JUNV modulates macrophage M1/M2 polarization. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and at 3 dpi the cells expressing
CD64+ (M1) and CD206+ (M2) on CD11b+ were evaluated by flow cytometry. (A) Representative dot-plots of CD64 vs. CD206 after gating in CD11b of each
condition are shown. (B) Independent data are graphed showing a shift to M1 after C#1 infection and to M2 after P infection. Non-parametric One-way ANOVA
followed by Dunn’s multiple comparison test was used to detect significant differences between groups; *P < 0.05. The results are graphed as the median (min-max,
horizontal line indicates the median) of five independent donors. We had set the quadrant threshold based on the FMO of each marker. Very little is known about M2 macrophage polarization
during acute viral infection since the early anti-viral response is
normally associated with a pro-inflammatory immune response
(45). In this regard, a recently transcriptomic analysis of
macrophages infected with attenuated or virulent influenza virus
strains showed an early and clear profile of genes associated with
the M2 phenotype triggered by a pathogenic influenza virus (46). also inhibit antiviral immunity through inhibition of type I IFN,
which allows HHV8 to evade immune detection (40). Our results clearly show a potent pro-inflammatory response
was elicited when macrophages were infected with C#1 strain,
concordant with a M1 phenotype. Frontiers in Immunology | www.frontiersin.org DISCUSSION In this sense, a higher level of pro-inflammatory
cytokines (IL-12 and TNF-α) together with an increased level
of a co-stimulatory marker (CD86) demonstrates the ability of
the C#1 strain to mount an adequate inflammatory response. This allows the generation of protective immunity against the October 2019 | Volume 10 | Article 2499 6 Arenavirus Modulates Macrophage Polarization Ferrer et al. GURE 3 | JUNV activates human macrophages and selectively modulates cytokine production. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1)
d at 3 dpi the activation and co-stimulation CD80, CD86, or antigen presentation HLA-DR expression were evaluated in CD14+ cells by flow cytometry. (A)
presentative dot-plots showing CD14+ and each marker are shown. (B) The percentage of double positive CD14+CD86+, CD14+CD80+, and
(Continued) FIGURE 3 | JUNV activates human macrophages and selectively modulates cytokine production. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1)
and at 3 dpi the activation and co-stimulation CD80, CD86, or antigen presentation HLA-DR expression were evaluated in CD14+ cells by flow cytometry. (A)
Representative dot-plots showing CD14+ and each marker are shown. (B) The percentage of double positive CD14+CD86+, CD14+CD80+, and
(Continued) (Continued) October 2019 | Volume 10 | Article 2499 7 Frontiers in Immunology | www.frontiersin.org Arenavirus Modulates Macrophage Polarization Ferrer et al. FIGURE 3 | CD14+HLA-DR++ are graphed. We had set the threshold for each marker based on the FMO. The expression level of TNF-α and IL-1β (C), IL-12 (D),
IL-10 (E), IL-6 (F), and IL-1β (G) were measured in the supernatant of infected macrophages after 72 h, employing commercial ELISA kits. Non-parametric One-way
ANOVA followed by Dunn’s multiple comparison test was used to detect significant differences between groups, *P < 0.05, **P < 0.01. The results are graphed as the
median (min-max, horizontal line indicates the median) of at least four independent donors in each assay. FIGURE 3 | CD14+HLA-DR++ are graphed. We had set the threshold for each marker based on the FMO. The expression level of TNF-α and IL-1β (C), IL-12 (D),
IL-10 (E), IL-6 (F), and IL-1β (G) were measured in the supernatant of infected macrophages after 72 h, employing commercial ELISA kits. Non-parametric One-way
ANOVA followed by Dunn’s multiple comparison test was used to detect significant differences between groups, *P < 0.05, **P < 0.01. DISCUSSION However, the P strain elicited
a more anti-inflammatory M2 response, associated with a higher
level of IL-6, but not IL-10, an increase in CD11b+CD206+
and HLA-DR cell expression suggesting that the P strain shifts
the macrophage response to a regulatory program (41). In
macrophages, IL-10 as well as pro-inflammatory cytokines (TNF-
α, IL-12 and IL-6) are produced in response to activation of
TLRs 2, 3, 4, 7, and 9, via MyD88 or TRIF, NF-kB, and MAPK
pathways (42). The strain P did not stimulate the production of
IL-10 neither pro-inflammatory ones such as IL-12 or TNF-α,
instead of that, a large amount of IL-6, a cytokine also associated
with immunomodulation, was specifically induced by P and
not by C#1 strain. Interestingly, in a different model, the IL-6
cytokine has recently been associated with the promotion of the
M2 phenotype (43) and described as a potent inducer of SOCS3
(33). Furthermore, the generation of human immunosuppressive
myeloid cell populations in human IL-6 transgenic NOG mice
has been demonstrated (44). p
yp
gg
y
p
g
It has been shown that AXL and MERTK receptors are
differentially modulated in cytokine-induced M1 and M2
macrophages, where enhanced levels of MERTK were associated
with M2 polarization (21). IRF-1 was initially described as a
regulator of type I IFN and MHC-I expression by binding to
regulatory regions of their promoters (47). However, IRF-1 is
one of the most important IFN-stimulated genes for innate and
adaptive antiviral immunity, making a complex network with
other transcription factors to finally have a specific response
(48–50). In this sense, IRF-2 and 8 can both inhibit IRF-1–
mediated induction of transcription competing by promoter
binding sites (51, 52), or by blocking protein:protein interactions
(53, 54), supporting the hypothesis that viruses can manipulate
the induction of IFN and ISGs to enhance their replication. In this sense, the selective modulation of the MERTK, as October 2019 | Volume 10 | Article 2499 Frontiers in Immunology | www.frontiersin.org 8 Arenavirus Modulates Macrophage Polarization Ferrer et al. FIGURE 5 | JUNV selectively modulates TAM expression in human macrophages. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and at 3 dpi the
expression level of TYRO3, AXL, and MERTK receptors was evaluated by flow cytometry. (A) Representative dot plot of each receptor is shown and the percentage of
macrophages expressing TYRO3, AXL, and MERTK receptors are graphed in (B). DATA AVAILABILITY STATEMENT well as higher levels of IRF-1, SOCS1, and SOCS3 during P
strain infection highlight not only the skewing property of
this strain, but also its ability to usurp immunomodulatory
pathways (TAM/SOCS1 and 3) and potentially use them for
immune evasion. The datasets generated for this study are available on request to
the corresponding authors. DISCUSSION Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test was
(Continued)
Frontiers in Immunology | www.frontiersin.org
9
October 2019 | Volume 10 | Article 2499 FIGURE 5 | JUNV selectively modulates TAM expression in human macrophages. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and at 3 dpi the
expression level of TYRO3, AXL, and MERTK receptors was evaluated by flow cytometry. (A) Representative dot plot of each receptor is shown and the percentage of
macrophages expressing TYRO3, AXL, and MERTK receptors are graphed in (B). Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test was
(Continued) October 2019 | Volume 10 | Article 2499 9 Frontiers in Immunology | www.frontiersin.org Arenavirus Modulates Macrophage Polarization Ferrer et al. FIGURE 5 | Used to detect significant differences between groups; **P < 0.01. The results are graphed as the median (min-max, horizontal line indicates the median)
of seven independent donors. We had set the threshold based on the FMO of each receptor. FIGURE 6 | JUNV P enhances transcription levels of IRF-1, IFN-β, SOCS1, and SOCS3. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and after
1 dpi the transcription levels of (A) IRF-1, (B) SOCS1, (C) SOCS3, and (D) IFN-β, were studied by RT-qPCR. The Comparative Ct method (2−1CT) was used to
analyze the expression level of the target genes. Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test was used to detect significant
differences between groups; *P < 0.05. The results are graphed as the median (min-max, horizontal line indicates the median) of five independent donors. FIGURE 6 | JUNV P enhances transcription levels of IRF-1, IFN-β, SOCS1, and SOCS3. HMDM cells were infected with C#1 or P strains of JUNV (MOI =1) and after
1 dpi the transcription levels of (A) IRF-1, (B) SOCS1, (C) SOCS3, and (D) IFN-β, were studied by RT-qPCR. The Comparative Ct method (2−1CT) was used to
analyze the expression level of the target genes. Non-parametric One-way ANOVA followed by Dunn’s multiple comparison test was used to detect significant
differences between groups; *P < 0.05. The results are graphed as the median (min-max, horizontal line indicates the median) of five independent donors. AUTHOR CONTRIBUTIONS Our results show that JUNV triggers differential macrophage
activation and modulates polarization according to viral strain
pathogenicity, inducing distinct cell responses that might
facilitate correct immune surveillance or viral evasion and
dissemination events that end in disease. Thus, the results
provide important mechanistic insights into the understanding
of JUNV pathogenesis and the multi-faceted host immune
responses in arenavirus infection. MF did most of the experiments. PT performed IF studies. AL
and NC made qPCR analysis. AE participated in the generation of
macrophages and flow cytometry analysis. VR, JG, and MR edited
the manuscript. EC and RG designed the experiments, discussed
results, and wrote the manuscript. Taking our results together with the above-mentioned recent
findings by others, it may be speculated that in some acute
viral infections, subversion of the conventional M1 pro-
inflammatory response to a M2 anti-inflammatory response by
acute pathogenic viruses will be more frequent than previously
described, and thus is deserving of further study since this
may allow the development of potential new candidates for
therapeutic targets. Frontiers in Immunology | www.frontiersin.org REFERENCES The regulation role of
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(ANPCyT), Roemmers 2017 (RG), PPID X037 (MF), and
P11/X854 (VR) from UNLP, PIP 2015-0567 (AE) and PIP 0680
(VR) from Consejo Nacional de Investigaciones Científicas y This work was supported by the grants PICT 2016-1740
(RG), PICT 2015-2573 (EC), and PICT 2016-2608 (MF) from
the Agencia Nacional de Promoción Científica y Tecnológica
(ANPCyT), Roemmers 2017 (RG), PPID X037 (MF), and
P11/X854 (VR) from UNLP, PIP 2015-0567 (AE) and PIP 0680
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in
preparation
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of
cellular cultures. Técnicas (CONICET), Argentina. The funders had no role in
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to submit the work for publication. Técnicas (CONICET), Argentina. The funders had no role in
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to submit the work for publication. ACKNOWLEDGMENTS The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02499/full#supplementary-material We
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https://dro.deakin.edu.au/articles/journal_contribution/High_burden_of_complicated_skin_and_soft_tissue_infections_in_the_Indigenous_population_of_Central_Australia_due_to_dominant_Panton_Valentine_leucocidin_clones_ST93-MRSA_and_CC121-MSSA/20840413/1/files/37098136.pdf
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High burden of complicated skin and soft tissue infections in the Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-MRSA and CC121-MSSA
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High burden of complicated skin and soft tissue infections in the Indigenous
population of Central Australia due to dominant Panton Valentine leucocidin
clones ST93-MRSA and CC121-MSSA AUTHOR(S) AUTHOR(S) High burden of complicated skin and soft tissue infections in the Indigenou
population of Central Australia due to dominant Panton Valentine leucocidi
clones ST93-MRSA and CC121-MSSA
AUTHOR(S)
S A J Harch, E MacMorran, S Y C Tong, D C Holt, J Wilson, Eugene Athan, S Hewagama
PUBLICATION DATE
01-01-2017
HANDLE
10536/DRO/DU:30101636
Downloaded from Deakin University’s Figshare repository
Deakin University CRICOS Provider Code: 00113B High burden of complicated skin and soft tissue infections in the Indigenous
population of Central Australia due to dominant Panton Valentine leucocidin
clones ST93-MRSA and CC121-MSSA High burden of complicated skin and soft tissue infections in the Indigenous
population of Central Australia due to dominant Panton Valentine leucocidin clones
ST93-MRSA and CC121-MSSA
Citation:
Harch, Susan AJ, MacMorran, Eleanor, Tong, Steven YC, Holt, Deborah C, Wilson, Judith, Athan,
Eugene and Hewagama, Saliya 2017, High burden of complicated skin and soft tissue infections in the
Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-
MRSA and CC121-MSSA, BMC infectious diseases, vol. 17, Article number: 405, pp. 1-7.
DOI: 10.1186/s12879-017-2460-3 High burden of complicated skin and soft tissue infections in the Indigenous
population of Central Australia due to dominant Panton Valentine leucocidin clones
ST93-MRSA and CC121-MSSA 10536/DRO/DU:30101636 Downloaded from Deakin University’s Figshare repository
Deakin University CRICOS Provider Code: 00113B DOI: 10.1186/s12879-017-2460-3 High burden of complicated skin and soft tissue infections in the Indigenous
population of Central Australia due to dominant Panton Valentine leucocidin clones
ST93-MRSA and CC121-MSSA Citation:
Harch, Susan AJ, MacMorran, Eleanor, Tong, Steven YC, Holt, Deborah C, Wilson, Judith, Athan,
Eugene and Hewagama, Saliya 2017, High burden of complicated skin and soft tissue infections in the
Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-
MRSA and CC121-MSSA, BMC infectious diseases, vol. 17, Article number: 405, pp. 1-7. Harch, Susan AJ, MacMorran, Eleanor, Tong, Steven YC, Holt, Deborah C, Wilson, Judith, Athan,
Eugene and Hewagama, Saliya 2017, High burden of complicated skin and soft tissue infections in the
Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-
MRSA and CC121-MSSA, BMC infectious diseases, vol. 17, Article number: 405, pp. 1-7. Harch, Susan AJ, MacMorran, Eleanor, Tong, Steven YC, Holt, Deborah C, Wilson, Judith, Athan,
Eugene and Hewagama, Saliya 2017, High burden of complicated skin and soft tissue infections in the
Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-
MRSA and CC121-MSSA, BMC infectious diseases, vol. 17, Article number: 405, pp. 1-7. DOI: 10.1186/s12879-017-2460-3 DOI: 10.1186/s12879-017-2460-3 © 2017, The Authors © 2017, The Authors Reproduced by Deakin University under the terms of the Creative Commons Attribution Licence Downloaded from DRO:
http://hdl.handle.net/10536/DRO/DU:30101636 Downloaded from DRO: DRO
Deakin Research Online,
Deakin University’s Research Repository
Deakin University CRICOS Provider Code: 00113B Deakin University CRICOS Provider Code: 00113B Harch et al. BMC Infectious Diseases (2017) 17:405
DOI 10.1186/s12879-017-2460-3 RESEARCH ARTICLE
Open Access
High burden of complicated skin and soft
tissue infections in the Indigenous
population of Central Australia due to
dominant Panton Valentine leucocidin
clones ST93-MRSA and CC121-MSSA
Susan A.J. Harch1,4*, Eleanor MacMorran1, Steven Y.C. Tong2,5, Deborah C. Holt2, Judith Wilson2, Eugene Athan3
and Saliya Hewagama1 Open Access * Correspondence: susan.harch@sa.gov.au
1Alice Springs Hospital, Alice Springs, Northern Territory, Australia
4SA Pathology, PO Box 14, Rundle Mall, Adelaide, South Australia 5000,
Australia
Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Superficial skin and soft tissue infections (SSTIs) are common among the Indigenous population
of the desert regions of Central Australia. However, the overall burden of disease and molecular epidemiology
of Staphylococcus aureus complicated SSTIs has yet to be described in this unique population. Methods: Alice Springs Hospital (ASH) admission data was interrogated to establish the population incidence
of SSTIs. A prospective observational study was conducted on a subset of S. aureus complicated SSTIs (carbuncles
and furuncles requiring surgical intervention) presenting during a one month period to further characterize the
clinical and molecular epidemiology. High resolution melting analysis was used for clonal complex discrimination. Real-time polymerase chain reaction identifying the lukF component of the Panton Valentine leucocidin (pvl)
gene determined pvl status. Clinical and outcome data was obtained from the ASH medical and Northern
Territory shared electronic health records. Results: SSTIs represented 2.1% of ASH admissions during 2014. 82.6% occurred in Indigenous patients (n = 382)
with an estimated incidence of 18.9 per 1, 000 people years compared to the non-Indigenous population of 2.9
per 1000, with an incident rate ratio of 6.6 (95% confidence interval 5.1–8.5). Clinical and molecular analysis was
performed on 50 isolates from 47 patients. Community-associated methicillin-resistant S. aureus (CA-MRSA)
predominated (57% of isolates). The high burden of SSTIs is partly explained by the prevalence of pvl positive
strains of S. aureus (90% isolates) for both CA-MRSA and methicillin-susceptible S. aureus (MSSA). ST93-MRSA and
CC121-MSSA were the most prevalent clones. SSTIs due to ST93-MRSA were more likely to require further
debridement (p = 0.039), however they also more frequently received inactive antimicrobial therapy (p < 0.001). Conclusions: ST93-MRSA and CC121-MSSA are the dominant causes of carbuncles and furuncles in Central
Australia. Both of these virulent clones harbor pvl but the impact on clinical outcomes remains uncertain. The
high prevalence of CA-MRSA supports empiric vancomycin use in this population when antimicrobial therapy is
indicated. Prompt surgical intervention remains the cornerstone of treatment. Keywords: Staphylococcus aureus, Methicillin resistance, Abscess, Panton Valentine leucocidin © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. * Correspondence: susan.harch@sa.gov.au High burden of complicated skin and soft
tissue infections in the Indigenous
population of Central Australia due to
dominant Panton Valentine leucocidin
clones ST93-MRSA and CC121-MSSA Susan A.J. Harch1,4*, Eleanor MacMorran1, Steven Y.C. Tong2,5, Deborah C. Holt2, Judith Wilson2, Eugene Athan3
and Saliya Hewagama1 Background Data pertaining to the Diagnosis-Related Group (DRG)
‘cellulitis, boil, furuncle, carbuncle and abscess’ for the
calendar year 2014 at ASH was queried to establish the
incidence of hospital admissions due to SSTIs [13]. Australian Bureau of Statistics (ABS) population data [14]
was utilised to calculate the regional incidence of SSTIs. The ABS estimate the total population for the Alice
Springs, Barkly, Central Desert and MacDonnell Local
Government Areas to be 48, 079 persons, with 20,262 per-
sons identifying as Indigenous and 27,817 persons identi-
fying as non-Indigenous [14]. Indigenous populations worldwide suffer from a high
burden of infectious diseases. As with Indigenous popu-
lations elsewhere, Indigenous Australians of Central
Australia share many of the same issues of remoteness,
overcrowding, poverty and poor access to sanitation and
health care [1–3]. Skin and soft tissue infections (SSTIs) are observed to
be extremely common within the Indigenous population
of Central Australia. The combination of socioeconomic
disadvantage, high rates of communicable infections
such as scabies and non-communicable diseases inclu-
ding diabetes mellitus provide a highly vulnerable host
for SSTIs [2, 3]. Staphylococcus aureus infection is
responsible for significant morbidity and mortality
amongst this population, with SSTIs the leading cause
of S. aureus bacteraemia [4] and frequently associated
with prolonged hospital admissions for wound care
with subsequent community dislocation. Despite this,
the burden of disease due to superficial SSTIs in the
Central Australian Indigenous population has not pre-
viously been investigated. We conducted a prospective, observational cohort
study of a subset of patients presenting to ASH with a
complicated, abscess related SSTI during a 1 month
period (October 2014). We identified cases via operating
theatre lists and included patients with: (1) spontaneous
SSTI due to a carbuncle or furuncle, (2) surgical inter-
vention required, and (3) available S. aureus isolate. Ex-
clusion criteria were: (1) secondary infection related to a
wound, and (2) polymicrobial infections. We obtained microbiological cultures from routine clin-
ical specimens, with preference for surgical specimens. S. aureus was identified by routine laboratory protocols
(morphology, catalase and Staphaurex tests (Oxoid, 2011)),
with organism confirmation and antimicrobial susceptibility
testing performed on the Vitek 2 (Biomerieux, version 7.01)
using the Clinical Laboratory Standards Institute M100-S24
Performance Standards. We used real-time polymerase
chain reaction (PCR) to detect the presence of the nucA,
mecA and lukSF genes to determine the status of S. aureus,
methicillin-resistance and PVL respectively. Background High resolution
melting (HRM) analysis in conjunction with real-time PCR
was used to discriminate the different clonal complexes
[15]. We used ST93 rather than CC93 for those inferred to
be ST93, as ST93 is a singleton sequence type with no iden-
tified related sequence types in the MLST database. We de-
fined CA-MRSA as resistant to <3 non β–lactam antibiotic
classes, and multi-resistant MRSA (mMRSA) as resistant to
≥3 non β–lactam antibiotic classes. Clinical data of cases
was obtained from ASH medical records and included
demographics, co-morbidities, recent hospitalization, infec-
tion, treatment and outcome data. Community-associated methicillin-resistant S. aureus
(CA-MRSA) and Panton Valentine leucocidin (PVL)
positive infection are prevalent and seemingly on the
increase in regional studies on S. aureus bacteraemia
[4–7]. The rise of this S. aureus phenotype may be
contributing to the burden of SSTIs related disease
[8]. While the molecular epidemiology of S. aureus
has been described in Indigenous Australians in tro-
pical northern Australia [9], data on the circulating S. aureus clones associated with SSTIs is lacking for the
geographically and ethnically distinct desert regions of
Central Australia. Furthermore, the correlates between
the molecular epidemiology and clinical characteristics
including outcomes of complicated S. aureus SSTIs is
unknown. Additionally, the prevalence of a newly defined species,
S. argenteus [10] has not been established. This is of
interest as S. argenteus, formerly known as S. aureus
CC75, is common in the Indigenous population of tro-
pical northern Australia [11]. We analyzed differences between clonal types, clinical
and outcome data using Analysis of Variance for con-
tinuous data and Fisher’s exact test for categorical va-
riables in R version 3. 1. 2 (R Development Core Team
2014). Incident rate ratios were calculated in Stata ver-
sion 14.2 (StataCorp, College Station, Texas). Methods Alice Springs Hospital (ASH) is the sole hospital provi-
ding tertiary health services for Central Australia, a
population of approximately 60, 000 over a catchment
area of 1.6 million square kilometers [12]. The catch-
ment region is predominantly within the Northern Ter-
ritory but also extends into Western Australia, South
Australia and Queensland. Approximately 44% of the
population identify as Indigenous but represent over
70% of ASH inpatients. Ethics approval was granted by the Central Australian
Human Research Ethics Committee (HREC-14-223). Abstract 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 7 Page 2 of 7 Harch et al. BMC Infectious Diseases (2017) 17:405 Discussion Key findings of this study include the very large burden
of SSTIs experienced in Central Australia and the vast
disparity
between
Indigenous
and
non-Indigenous
Australians. The incidence of SSTIs requiring hospitali-
sation within the Indigenous population of Central
Australia is estimated to be 18.8 per 1000 people years. This is almost seven times the incidence for the local
non-Indigenous population. More concerning, the inci-
dence is over 30 times the estimated national Australian
hospitalisation rate for cutaneous abscesses of 62 per
100, 000 people years [16]. Moreover the incidence is
significantly higher than other developed nations, with
an American retrospective survey describing an inpatient
admission SSTI incidence of 2.19 per 1, 000 person
years [8]. Forty-nine cases for the prospective, observational
cohort study were identified following screening and
application of exclusion criteria. Most identified as
Indigenous (90%), median age was 30 years and approxi-
mately 60% were female (Table 1). Despite the young
population age, comorbidities with diabetes mellitus
(50%), chronic kidney disease (25%), obesity (27%) and
scabies infection (25%) were common. Almost half (49%)
the population lived in remote communities. All cases had community onset disease, 36.7% had
been admitted to ASH in the preceding 6 months and
38% had CA-MRSA previously isolated (clinical or
screening specimens). CA-MRSA was the predominant
phenotype (57%). Many patients received inactive empi-
rical antimicrobial therapy (36.7%) prior to surgical
intervention. The most frequently prescribed empiric
antimicrobials were flucloxacillin (50%), vancomycin
(20%) and cephazolin (14%). Surgical intervention was
generally performed within 1 day of admission. Blood
cultures were obtained in 16 cases (33%) and were posi-
tive for MSSA in just one instance. There continues to be a rise in CA-MRSA particularly
within Australian Indigenous communities [7] and our
study reflects this trend. We have demonstrated that
CA-MRSA is now the dominant S. aureus phenotype in
carbuncle and furuncle related SSTIs in the desert re-
gions of Central Australia. The prevalence of CA-MRSA
in this SSTI subset (57%) well exceeds the prevalence
rates
of
approximately
20%
described
in
previous
Australian S. aureus studies [6, 7, 17]. CA-MRSA is in-
creasingly responsible for SSTIs in Australian Indigen-
ous populations, and in particular the dominant ST93-
MRSA clone has continued its spread within Australia
into the desert regions of Central Australia. MSSA
continues to contribute substantially to the burden of
carbuncle and furuncle related SSTIs with CC121 the
major MSSA clone. Results Skin and soft tissue infections represented by the DRG
code ‘cellulitis, boil, furuncle, carbuncle and abscess’ Harch et al. BMC Infectious Diseases (2017) 17:405 Page 3 of 7 further debridement (p = 0.039) but this group also
more frequently received inactive antimicrobial therapy
prior to debridement (p < 0.001). further debridement (p = 0.039) but this group also
more frequently received inactive antimicrobial therapy
prior to debridement (p < 0.001). constituted 2.1% of all ASH admissions during 2014. Consistent with previous years, it was the most frequent
reason for admission under the General Surgical Unit. Overall, there were 462 ASH admissions attributed to
SSTIs with the majority occurring in Indigenous patients
(n = 382, 82.6%). The estimated incidence of SSTIs
within the Indigenous population was 18.9 per 1, 000
people years and within the non-Indigenous population
was 2.9 per 1, 000 people years, within an incident rate
ratio of 6.6 (95% confidence interval 5.1–8.5). constituted 2.1% of all ASH admissions during 2014. Consistent with previous years, it was the most frequent
reason for admission under the General Surgical Unit. Overall, there were 462 ASH admissions attributed to
SSTIs with the majority occurring in Indigenous patients
(n = 382, 82.6%). The estimated incidence of SSTIs
within the Indigenous population was 18.9 per 1, 000
people years and within the non-Indigenous population
was 2.9 per 1, 000 people years, within an incident rate
ratio of 6.6 (95% confidence interval 5.1–8.5). Discussion Notably, both ST93 and CC121 are
pvl positive clones. Clonal complex and pvl status was successfully deter-
mined for 50 isolates from 47 patients. Eight clonal com-
plexes were identified (Table 2). The clonal complexes
with the highest frequency were ST93 (48%) and CC121
(30%). Of the paired isolates, one case had two MSSA
strain types (CC5 and CC121) and two cases had con-
current MSSA and CA-MRSA strain types (CC5 and
CC8, and CC1 and ST93). pvl positive disease domi-
nated the cohort with 90% of isolates carrying the lukSF
gene, occurring in both MSSA and CA-MRSA isolates
and across all clonal complexes except for CC1 and
CC88. S. argenteus was not identified. Despite the geographic isolation, high Indigenous popu-
lation and substantial burden of disease, the molecular
epidemiology of SSTI isolates in Central Australia had a
number of similarities to other Australian regions. Clonal
diversity is a unique feature of S. aureus in Australia, with
a recent prevalence survey identifying more than 30
clones in CA-MRSA alone [17]. There are six major
MRSA clones including ST93-IV, ST30-IV, ST1-IV,
ST45-V, ST78-IV and ST5-IV [17]. ST93 is notable
for causing abundant [9, 17, 18] and possibly more
virulent disease in Australia [19] . We also observed
significant clonal diversity with 8 clonal complexes
identified within 50 isolates, including CC1, CC5,
CC8, CC22, CC30, ST93, CC121 and CC88. There was clear correlation between clones and anti-
microbial phenotype. The majority of ST93 isolates were
CA-MRSA (96%), whilst all CC121 isolates were MSSA
(p < 0.001). 60% of CC121 isolates were resistant to
clindamycin, compared to 12.5% of ST93 isolates being
clindamycin resistant (p = 0.006). Trimethoprim/ sulfa-
methoxazole resistance occurred in two pvl positive CC5
isolates (4%). Like other regions in Australia, we observed ST93 to
be the most frequent strain (48%) and strongly asso-
ciated with CA-MRSA (96%). ST93 has been found to There were some differences between S. aureus clones
and clinical variables (Table 1). On univariate analysis,
infections due to ST93 were more likely to require Harch et al. BMC Infectious Diseases (2017) 17:405 Page 4 of 7 Table 1 Correlation of clinical variables and S. Discussion aureus clonal complexes
Variable
Overall n = 49
n (%a), median (IQR)
At least one isolate
ST93 n = 24
n (%), median (IQR)
At least one isolate
CC121 n = 15
n (%), median (IQR)
Other clonal
complexes n =
n (%), median (
Demographics
Ethnicity: Indigenous
44 (89.8)
22 (91.7)
14 (93.3)
8 (80.0)
Gender: Female
29 (59.2)
12 (50.0)
9 (60.0)
8 (80.0)
Age (years)
30.0 [10.0, 46.0]
28.5 [11.2, 47.0]
34.0 [24.5, 45.5]
20.5 [4.5, 42.2]
Residence
Town camp
12 (24.5)
6 (25.0)
4 (26.7)
2 (20.0)
Remote community
24 (49.0)
12 (50.0)
6 (40.0)
6 (60.0)
Prison
1 (2.0)
1 (6.7)
Visitor
2 (4.1)
1 (4.2)
1 (10.0)
House in Alice Springs
10 (20.4)
5 (20.8)
4 (26.7)
1 (10.0)
Co-morbidities
Diabetes mellitus
22 (44.9)
12 (50.0)
6 (40.0)
4 (40.0)
Chronic kidney disease
12 (24.5)
6 (25.0)
3 (20.0)
3 (30.0)
Haemodialysis
2 (4.1)
2 (8.3)
0 (0.0)
0 (0.0)
Excess alcohol use
6 (12.2)
3 (12.5)
2 (13.3)
1 (10.0)
Obesity (BMI ≥30)
13 (26.5)
3 (12.5)
7 (46.7)
3 (30.0)
Cardiac failure
6 (12.2)
2 (8.3)
3 (20.0)
1 (10.0)
Ischaemic heart disease
4 (8.2)
2 (8.3)
1 (6.7)
1 (10.0)
Scabies
12 (24.5)
5 (20.8)
4 (26.7)
3 (30.0)
Asthma
5 (10.2)
1 (4.2)
1 (6.7)
3 (30.0)
Healthcare exposure
Admission in previous 6 months
18 (38.3)
9 (39.1)
5 (33.3)
4 (44.4)
Previous CA- MRSA
18 (38.3)
10 (43.5)
3 (20.0)
5 (55.6)
Clinical presentation
Size (cm)
4.0 [3.0, 5.0]
4.0 [2.5, 5.0]
4.0 [3.0, 6.0]
5.5 [3.5, 9.0]
Duration (days)
5.0 [2.0, 7.0]
5.5 [3.2, 7.0]
4.0 [2.0, 6.5]
3.0 [2.0, 7.0]
Site of infection
Head & neck
8 (16.3)
5 (20.8)
3 (20.0)
Upper limb
9 (18.4)
7 (29.2)
1 (6.7)
1 (10.0)
Torso
6 (12.2)
3 (12.5)
13.3 (2)
Buttock/groin/thigh
12 (24.5)
6 (25.0)
2 (13.3)
1 (10.0)
Lower leg
11 (22.4)
2 (8.3)
26.7 (4)
3 (30.0)
Multiple
3 (6.1)
1 (4.2)
13.3 (2)
5 (50.0)
Treatment
Time to surgery (days)
1.0 [1.0, 1.0]
1.0 [1.0, 1.0]
1.0 [1.0, 1.0]
1.0 [1.0, 1.0]
Antimicrobials prior surgery
Inactive
18 (36.7)
13 (54.2)
5 (50.0)
Active
30 (61.2)
10 (41.7)
15 (100.0)
5 (50.0)
Planned oral antibiotic duration (days)
7.0 [2.0, 7.0]
6.5 [3.2, 7.0]
5.0 [0.0, 7.0]
7.0 [5.5, 7.0]
Clinical outcomes
Length of stay (days)
3.0 [2.0, 7.0]
3.0 [3.0, 6.0]
2.0 [2.0, 6.5]
3.0 [2.2, 7.0]
Progression to deep structures
5 (11.9)
3 (15.0)
0 (0.0)
2 (25.0) Table 1 Correlation of clinical variables and S. Discussion aureus clonal complexes Harch et al. BMC Infectious Diseases (2017) 17:405 Page 5 of 7 Table 1 Correlation of clinical variables and S. aureus clonal complexes (Continued)
Secondary infection
1 (2.5)
0 (0.0)
1 (7.7)
0 (0.0)
Return to theatre
6 (12.5)
6 (26.1)
0 (0.0)
0 (0.0)
Readmission within 30 days
4 (8.3)
2 (8.7)
2 (13.3)
0 (0.0)
Unscheduled medical review
8 (21.1)
4 (23.5)
2 (15.4)
2 (25.0)
Repeat antibiotics required
10 (26.3)
3 (17.6)
6 (46.2)
1 (12.5)
Antimicrobial susceptibility
Methicillin resistance
28 (57.1)
23 (95.8)
0 (0.0)
5 (50.0)
Clindamycin resistance
3 (12.5)
9 (60.0)
Trimethoprim/sulfamethoxazole resistance
0 (0.0)
0 (0.0)
aCalculation of percentages takes into account missing data points Table 1 Correlation of clinical variables and S. aureus clonal complexes (Continued) cause abscesses and furuncles and further studies of non-
complicated SSTI will be required to determine whether
S. argenteus is truly absent from Central Australia. hyperexpress the exotoxin α-haemolysin which enhances
the virulence of the clone, particularly in SSTIs [20]. The high virulence and expression of α-haemolysin in
ST93 may be a driving factor in the substantial burden
of disease seen in Central Australia [19–21]. g
y
The dominance of pvl positive S. aureus isolates (90%)
may be contributing to the very high incidence of ab-
scess related SSTIs in Central Australia [9] and is not-
able for two reasons. Firstly, it was widespread in both
MSSA and CA-MRSA isolates. The association between
PVL and abscess related SSTIs is well described [25],
however the prevalence of pvl in our cohort was sub-
stantially higher than in other Australian urban settings
(MSSA 15%, CA-MRSA 42%) [18] and the Northern
Territory Top End (MSSA 40%, CA-MRSA 54%). Se-
condly, the presence of pvl appears to have limited
impact on clinical outcomes following surgical interven-
tion. Controversy continues regarding the pathogenic
role of PVL, with laboratory and animal model data sug-
gesting it is not a major virulence factor [26, 27]. In our
study, detailed clinical outcome comparisons were li-
mited by the small number of pvl negative cases (10%). Despite this, most patients achieved good clinical out-
comes with only one episode of bacteraemia and a mi-
nority experiencing progression to deep tissue structures
(12%) and secondary infection (2.5%). Funding
N No external financial support was received for this work. Authors’ contributions SAJH Study design, acquisition of data, analysis of data and preparation of
manuscript. EM. Study design and review of manuscript. SYCT Study design,
acquisition of data, analysis of data and review of manuscript. DCH Acquisition
of data and review of manuscript. JW. Acquisition of data and review of
manuscript. EA Study design, analysis of data and review of manuscript. SH
Study design, analysis of data and review of manuscript. All authors read and
approved the final manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request. The small sample size of cases, microbiological isolates
and patients with PVL negative disease is the major limi-
tation of this study and prevented detailed molecular
and clinical correlations. Similarly, our sampling oc-
curred over 1 month and if seasonal variation was
present, we would not have identified this. Review of
the geographic distribution of clonal complexes was
not possible due to use of broad residential categories
and the highly mobile nature of the local Indigenous
population. Given
asymptomatic
patients
colonized Acknowledgements Philip Giffard Menzies School of Health Research, Darwin and James McLeod
Microbiology Department, Alice Springs Hospital for assistance with handling
of microbiological isolates. Jacob Jacob, Head of General Surgical Unit, Alice Springs Hospital for assistance
with case identification. Stephen E Lane: Barwon Health, Geelong, Victoria for assistance with statistical
analysis. Abbreviations ABS: Australian Bureau of Statistics; ASH: Alice Springs Hospital; CA-
MRSA: Community-associated methicillin-resistant Staphylococcus aureus;
DRG: Diagnosis-related group; HRM: High resolution melting (analysis);
mMRSA: Multi-resistant methicillin-resistant Staphylococcus aureus;
MSSA: Methicillin-susceptible Staphylococcus aureus; PCR: Polymerase chain
reaction; Pvl: Panton Valentine leucocidin (gene); PVL: Panton Valentine
leucocidin (toxin); SSTIs: Skin and soft tissue infections This study is limited by its observational design with
reliance on coding data to determine SSTI incidence
rates and retrieval of clinical data from medical records. It was not possible to obtain ABS population data for
the entire ASH catchment given it includes partial Local
Government
Areas
of
Western
Australia,
South
Australia and Queensland. Consequently, the population
incidence rate may be overestimated. Correlation of the
estimated ASH catchment population of 60, 000 with
44% identifying as Indigenous determines an incidence
of SSTIs within the Indigenous population as 14.5 per 1,
000 people years and within the non-Indigenous popula-
tion as 2.4 per 1000 people years. Discussion It was not possible
to determine if these complications were related to the
presence of pvl or a delay in presentation to health-
services or poor wound care upon discharge. Therefore,
we believe the typical rapid response to surgical inter-
vention observed in our study provides further support
to the findings of Shallcross et al. that provided ‘appro-
priate surgical treatment and the correct antibiotics’ are
received, PVL appears to have little impact on clinical
outcomes [25]. Following ST93, the next major clone was CC121
(30%), which was exclusively methicillin-susceptible. The
association of CC121 with SSTI is consistent with the
international literature and CC121 is phenotypically
usually MSSA [22]. The molecular epidemiology of
MSSA infections has been studied in less detail than
CA-MRSA [23], but CC121 is commonly associated with
SSTIs in Australia. However, the high prevalence of
CC121 in our study (30%) is greater than that reported
elsewhere in the Northern Territory ‘Top End’ and
metropolitan centers [9, 18], and may be a unique fea-
ture of the Central Australian region. The absence of S. argenteus is an interesting finding
of this study and key difference to other Australian
regions. S. argenteus was initially reported in the
Northern Territory ‘Top End’ [9, 24], but it is now
clear it has a global distribution [10]. The clinical
niche of S. argenteus appears to differ from S. aureus
[21]. S. argenteus may have less of a predilection to Table 2 S. aureus isolates: Frequency of clonal complexes and
carriage of pvl and mecA genes
Clonal complex
n (%)
pvl positive
n (%)
mecA positive
n (%)
Overall (n = 50)
45 (90.0)
28 (56.0)
CC1
1 (2.0)
0 (0)
0 (0)
CC5
4 (8.0)
3 (75.0)
3 (75.0)
CC8
3 (6.0)
1 (33.3)
1 (33.3)
CC22
1 (2.0)
1 (100.0)
0 (0)
CC30
1 (2.0)
1 (100.0)
1 (100.0)
ST93
24 (48.0)
24 (100.0)
23 (95.8)
CC121
15 (30. 0)
15 (100.0)
0 (0)
CC88
1 (2.0)
0 (0)
0 (0) Table 2 S. aureus isolates: Frequency of clonal complexes and
carriage of pvl and mecA genes Patients with CA-MRSA frequently received inactive
empiric antimicrobial therapy with antibiotic prescrip-
tion often in accordance with the Australian Therapeutic
Guidelines recommendation of di/flucloxacillin for em-
pirical therapy [28]. Conclusions In conclusion, we observed extremely high rates of com-
plicated
SSTIs
requiring
hospitalization
in
Central
Australia, with the Indigenous population disproportio-
nately affected. Detailed review of the molecular epi-
demiology of carbuncle and furuncle related SSTIs
revealed the dominance of two clones ST93-MRSA and
CC121-MSSA. These two virulent clones both possess
the pvl gene but the impact of PVL toxin on clinical out-
comes remains uncertain. The prevalence of CA-MRSA
continues to rise locally, with significant ramifications for
antimicrobial prescribing. Empiric intravenous vanco-
mycin or oral trimethoprim-sulfamethoxazole is recom-
mended for this population when antimicrobial therapy is
indicated. Prompt
surgical
intervention
remains
the
cornerstone of treatment. Further studies correlating
clonal complex type, asymptomatic pvl carriage as well as
the presence of other virulence factors will help improve
our understanding of the pathogenesis of SSTIs. Associations between clonal complexes and antibio-
grams were noteworthy. Clindamycin resistance occurred
in 32% of isolates, substantially higher than previous stu-
dies [17]. There was also a significant difference in clinda-
mycin resistance between the major strains CC121 (60%)
and ST93 (12.5%), p = 0.006. The impact on antimicrobial
prescribing is attenuated by CC121 isolates being exclu-
sively MSSA but this highlights the evolution to a more
broadly resistance antibiogram. The rise in clindamycin
resistance may be driven by high use of macrolide anti-
biotics in Indigenous communities of Central Australia,
typically indicated for Chlamydia trachomatis or chronic
suppurative lung disease. There are now reports of resistance to trimethoprim-
sulfamethoxazole in CC5 strains from Indigenous com-
munities in the neighboring jurisdiction of Western
Australia
[29]. We
found
two
CC5
trimethoprim-
sulfamethoxazole resistant MRSA strains that were also
pvl positive. Therefore, ongoing surveillance for changes
in resistance and clonal patterns of S. aureus are re-
quired to update prescribing recommendations. Discussion It follows then that infections due to
ST93 were more likely to receive inactive antimicrobials
compared to CC121 related infections (54.2% vs 100%, Harch et al. BMC Infectious Diseases (2017) 17:405 Page 6 of 7 p < 0.001). More importantly, all cases requiring further
debridement (n = 6) were ST93 (p = 0.039) and 50% of
these received inactive empiric antimicrobials. The high
rates of CA-MRSA and potential impact on clinical
outcomes lead us now to recommend the empiric use
of vancomycin for patients in Central Australia pre-
senting with SSTIs requiring surgical management. Trimethoprim-sulfamethoxazole may be a preferred
empiric oral agent given the high rates of observed
clindamycin resistance (32%) and dosing ease. with pvl positive S. aureus were not included, associa-
tions with pvl positivity and clinical disease are only
epidemiological in nature and not causal. Other recog-
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et al. Novel staphylococcal species that form part of a Staphyloccus aureus-
related complex: the non-pigmented Staphylococcus argenteus sp nov and
the non-human primate-associated Staphylococcus schweitzeri sp nov. Int J
Syst Evol Microbiol. 2015;65:15–22. 11. Tong SYC, Bishop EJ, Lilliebridge RA, Cheng AC, Spasova-Penkova Z, Holt DC,
et al. Community- Associated Strains of Methicillin- Resistant Staphylococcus
aureus and Methicillin- Susceptible S.aureus in Indigenous Northern Australia:
Epidemiology and Outcomes. J Infect Dis. 2009;199:1461–70. 12. Northern Territory Department of Health: Alice Springs Hospital: https://
health.nt.gov.au/professionals/medical-officers/teaching-hospitals/alice-
springs-hospital. Accessed 3 June 2017. 13. Competing interests Steven Y C Tong: Member on Editorial Board (Associate Editor), BMC
Infectious Diseases. Page 7 of 7 Page 7 of 7 Harch et al. BMC Infectious Diseases (2017) 17:405 All other authors have no conflicts of interest to disclose. 14. Australian Bureau of Statistics: Local Government Areas - Alice Springs,
Barkly, Central Desert, MacDonnell: Data by Region. Edited by Australian
Bureau of Statistics. Canberra, Australia; 2015. Author details
1 1Alice Springs Hospital, Alice Springs, Northern Territory, Australia. 2Menzies
School of Health Research, Charles Darwin University, Darwin, Northern
3
4 1Alice Springs Hospital, Alice Springs, Northern Territory, Australia. 2Menzies
School of Health Research, Charles Darwin University, Darwin, Northern 1Alice Springs Hospital, Alice Springs, Northern Territory, Australia. 2Menzies
School of Health Research, Charles Darwin University, Darwin, Northern
Territory, Australia. 3Deakin University, Geelong, VIC, Australia. 4SA Pathology,
PO Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia. 5Royal
Darwin Hospital, Darwin, Northern Territory, Australia. Territory, Australia. 3Deakin University, Geelong, VIC, Australia. 4SA Pathology,
PO Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia. 5Royal
Darwin Hospital, Darwin, Northern Territory, Australia. 20. Chua K, Monk I, Lin Y-H, Seemann T, Tuck KL, Porter J, et al. Hyperexpression
of α-haemolysin explains enhanced virulence of sequence type 93
community-associated methicillin-resistant Staphylococcus aureus. BioMedCentral Microbiol. 2014:14(31). Received: 9 November 2016 Accepted: 11 May 2017 28.
Moulds R, Anderson T, Daly C, Daskalakis S, Evill C, Grayson L, Korman T:
Boils and carbuncles. In: Therapeutic Guidelines: Antibiotics. Edited by Moulds
R, etg45 Therapeutic Guidelines; 2014. References 1. Tong SYC, McDonald MI, Holt DC, Currie BJ. Global implications of the
emergence of community-associated methicillin-resistant Staphylococcus
aureus in Indigenous populations. Clin Infect Dis. 2008;46:1871–8. 1. Tong SYC, McDonald MI, Holt DC, Currie BJ. Global implications of the
emergence of community-associated methicillin-resistant Staphylococcus
aureus in Indigenous populations. Clin Infect Dis. 2008;46:1871–8. 22. Monecke S, Coombs GW, Shore AC, Coleman DC, Akpaka P. A field
guide to pandemic, epidemic and sporadic clones of methicillin-
resistant Staphylococcus aureus. PLoS One. 2011;6(4):e17936. 2. Tong SYC, van Hal S, Einsiedel LJ, Currie BJ, Turnidge J. Impact of ethnicity
and socioeconomic status on Staphylococcus aureus bacteraemia incidence
and mortality: a heavy burden in Indigenous Australians. BioMedCentral
Infec Dis. 2012;12:9. 2. Tong SYC, van Hal S, Einsiedel LJ, Currie BJ, Turnidge J. Impact of ethnicity
and socioeconomic status on Staphylococcus aureus bacteraemia incidence
and mortality: a heavy burden in Indigenous Australians. BioMedCentral
Infec Dis. 2012;12:9. 23. Williamson DA, Coombs GW, Nimmo GR. Staphylococcus aureus 'Down
Under': contemporary epidemiology of S. aureus in Australia, New Zealand,
and the South West Pacific. Clin Microbiol Infect. 2014;20:597–604. 24. Ng J, Holt DC, Lilliebridge RA, Stephens AJ, Huygens F, Tong SYC, et
al. Phylogenetically distinct Staphylococcus aureus lineage prevalent
among Indigenous communities in northern Australia. J Clin Microbiol. 2009;47(7):2295–300. 3. Einsiedel LJ, Fernandes L, Joseph S, Brown A, Woodman RJ. Non-
communicable diseases, infection and survival in a retrospective cohort
of Indigenous and non-Indigenous adults in Central Australia. BMJ
Open. 2013;3:e003070. 3. Einsiedel LJ, Fernandes L, Joseph S, Brown A, Woodman RJ. Non-
communicable diseases, infection and survival in a retrospective cohort
of Indigenous and non-Indigenous adults in Central Australia. BMJ
Open. 2013;3:e003070. 25. Shallcross L, Fragazy E, Johnson AM, Hayward AC. The role of Panton-
Valentine leucocidin toxin in staphylococcal disease: a systematic review
and meta-analysis. Lancet Infect Dis. 2013;13:43–54. 4. Hewagama S, Spelman T, Einsiedel L. Staphylococcus aureus Bacteraemia at
Alice Springs Hopsital, Central Australia, 2003–2006. Intern Med J. 2011:505–12. 4. Hewagama S, Spelman T, Einsiedel L. Staphylococcus aureus Bacteraemia at
Alice Springs Hopsital, Central Australia, 2003–2006. Intern Med J. 2011:505–12. 5. Hewagama S, Spelman T, Woolley M, McLeod JE, Gordon D, Einsiedel LJ. The
Epidemiology of Staphylococcal aureus and Panton-Valentine Leucocidin (pvl)
in Central Australia, 2006–2010. BioMedCentral Infect Dis. 2016;16(382):1–6. 5. Hewagama S, Spelman T, Woolley M, McLeod JE, Gordon D, Einsiedel LJ. The
Epidemiology of Staphylococcal aureus and Panton-Valentine Leucocidin (pvl)
in Central Australia, 2006–2010. References Alice Springs Hospital: Diagnosis-Related Group J64B - Cellulitis- CSCC: Alice
Springs Hospital Separation Data. 2015.
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Robotics: development factor or social isolation of the child
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Robotics: development factor or social isolation
of the child
Irina Kulikovskaya1, Raisa Chumicheva1, and Ivan Panov 2
1South Federal University 344006, Rostov-on-Don, Russia
2MBDOU № 312, 344093Rostov-on-Don, Russia Abstract. In this article, dealing with robotics is defined as a factor
driving the development of preschool children. What was created by
science fiction writers has become a natural space for the child. Children
do not know a world where there are no drones, smart phones, and
computers. Robotics is becoming one of the leading activities for children,
which determines the development of creativity, initiative, and
independence. Joint design acts as a team work environment where
children learn to agree on a project topic, discuss problems in its
implementation, look for information from different sources, and use
digital technologies. However, immersing a child into the world of robotics
can isolate him from the society; immerse him into the virtual world. “Digital flashing” of a child‟s brain can affect its cognitive methods,
affecting the neural mechanisms responsible for communicating with other
people. This problem is being studied by scientists from around the world. Today the world is doubling - life in two spaces - material, objective and
virtual, ideal. That is why the determination of the common ground for
these worlds determines the harmonization of the children development in
modern space. One of this common ground could be robotics classes. In
preschool education, the development of technical creativity occurs
through the designers of LegoEdu. The logic of designing cognitive-
research activities of children is presented. Corresponding author: iekulikovskaya@sfedu.ru https://doi.org/10.1051/shsconf /20197203008 https://doi.org/10.1051/shsconf /20197203008 , 0
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3008 © 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/). 3 Research methods Theoretical systematic comparative analysis of scientific literature over the past 5 years,
content analysis of scientific literature, analysis of products of children's constructive
activities are used. 2 Problem formulation In the process of studying this problem, the team of authors posed a number of tasks: 1. To actualize the problem of robotics in the system of preschool education, to stu
the degree of its development in modern pedagogical research. 2. To identify the pedagogical developing potential of robotics in the cognitive
development of the child and his research activities. 3. To identify the pedagogical risks of social self-isolation of the child in the process of
robotic design. 1 Introduction The world in which modern children live is filled with various technical devices -
household (home appliances), entertainment (game consoles, tablets, etc.), industrial
(devices for building, repair, etc.), medical (research devices activities of organs and
systems of the body, biotechnology), research (computing devices and tools). The challenge
for a child entering the techno world is more relevant than ever, since robots are no longer
something fantastic, but are in direct contact with humans. At various times, such scientists
and philosophers as M.I. Makarov, S.L. Zenkevich, O.A. Tyagunov, V.V. Tarapata, Yu.I. Topcheev, K. Fu, R. Gonzalez, N. Braga, V. G. Bykov, R. M. Luchin, I. V. Tuzikova, S. A. Filippov, K. Schwab, A. S. Yushchenko and others studied the issue. Turning to the future, © 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/). , 0
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3008 https://doi.org/10.1051/shsconf /20197203008 where human-robot interaction will become a natural phenomenon, was the thought subject
of world famous writers - A. Azimov, R. Heinlein, S. Lem, D. Adams and many others. The urgency of the robotics problem is enhanced by the fact that it is attractive for its
unusualness, originality, stimulates the child to interact with it, giving rise to a new problem
of his social self-isolation. 4 Cases of educational robotics Unmatched interest in most is caused by robotics, games with
robots and designers, for example, LEGO Education designers, which are designed for
preschool children. of the XXI century, which must be developed in children, using the resources of activities
that are of interest to them. Unmatched interest in most is caused by robotics, games with
robots and designers, for example, LEGO Education designers, which are designed for
preschool children. However, robots and computer games can act as a factor in the social isolation of the
child. The lack of mutual understanding with adults, the child being „unnecessary‟
determines the immersion in the virtual world, hiding from the frightening reality. Speaking
about social isolation, scientists emphasize that this condition of a person causes his
suffering, a sense of insecurity, and fear of the outside world (K. Rubin, 1993). Partial or
complete refusal to communicate with peers and adults, weakening of emotional reactions,
narrowing of the sphere of interests are the consequences of the child‟s social isolation. Robots are interesting in themselves - as partners in interaction and games. Being in the
virtual space, the child “falls out” of reality. He has virtual friends and heroes. Spending a
lot of time in this world, he satisfies his needs for interaction and mutual understanding. Case 2. Challenges of Robotics in Modern Preschool Education Robotics has a wide range of applications in the system of preschool education: as a
means of personality development in the educational mode (learning in the game) -
initiative and independence, communication methods, constructive, research skills. Work on robotics begins long before the child connects his mechanism to the USB port,
his mechanism begins to move in the direction he sets and make the sounds the creator
needs. It begins with the task of a teacher for a group of 3-year-old children in combining
two or more details to create something new. Preschool years are very important for
socialization, learning about the world by children, and acquiring ways to discover
something new, since it was at this time that the development of their abilities was much
faster and more intense than in any other period of life [8, 9, 10]. 4 Cases of educational robotics Case 1. Robotics as a scientific problem and a means for a child’s development Case 1. Robotics as a scientific problem and a means for a child’s development
Educational robotics is an interdisciplinary phenomenon combining such fields as physics,
mechatronics, mathematics, cybernetics, etc. In education, robotics ceases to be in the status
of innovation. The child lives in a rapidly changing world where the robot occupies its
unique place, acting as a car driver, a seller in a store or cafe, a surgeon, etc. Robots are
increasingly becoming part of everyday life; cheap sensors, powerful computers, artificial
intelligence programs make them more independent. They help people by replacing them,
transforming healthcare and caring for the elderly. Robotics provides a change in the status
of engineering professions [6]. The subjectivity of the child manifests itself in the fact that
he independently discovers a problem (game), takes the initiative in solving it, and
embodies his plan. Robotics is becoming a factor in the development of children, as they can not only
participate in solving those issues, topics and problems that adults formulate, but also offer
their own, interesting questions, topics and forms of work for them. Activities with
different objects provide the child with the development of cognitive competencies and the
ability to independently learn the meaning of objects and the interdependence between
them, which determines the development of mental actions — comparison, analysis,
inference, and conclusions. In an ever-changing world, it is necessary to teach children to
cope with unexpected situations and solve problems that they have not encountered before. We can only guess what professions will be in demand in the future, but we know for sure
that emotional self-control, the ability to think positively, the realization of creative energy,
the willingness to adapt to constantly changing living conditions and learn all life will
remain significant, as well as a creative economy based on integration of intellectual capital
and creativity (STEAM-competence) [12]. The abbreviation STEAM is deciphered as
follows: S - natural sciences (science), T - technology (technology), E - engineering
(engineering), A - art (art), M - mathematics (mathematics). These are human competences 2 , 0
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3008 https://doi.org/10.1051/shsconf /20197203008 of the XXI century, which must be developed in children, using the resources of activities
that are of interest to them. 4 Cases of educational robotics Several children can get involved in
the game at once: they design, discuss ideas and roles together, think and communicate
with the help of elements acting as a means of forming respect for the opinions and
contributions of other participants. They are looking for different options for solving the
tasks, learn to understand how different solutions are applied in practice, program and
design their own robots. Science becomes a holiday, captivates and fascinates children. Young children, playing in Lotto LEGO DUPLO, learn the names of animals, their cubs,
parts of their bodies, behavior patterns. This game also develops mindfulness, design
abilities and fine motor skills. Kids remember colors, get acquainted with geometric shapes,
learn how to analyze and work according to a pattern, to develop communication skills,
playing in turn, to negotiate and share the results. They can interact in pairs, teams,
subgroups. Artistic and aesthetic development through LEGO designers is due to bright, colorful
images. Through various elements, children learn to create ornaments, different designs -
urban, suburban landscapes, to equip home space. Realizing original ideas, little builders
develop constructive thinking, an aesthetic attitude to the world, and creative projects are
being implemented. Colorful "bricks" are a fascinating tool for the productive development
of creative skills in preschool children. Case 3. Robotic engineering: the social position of the child in it The designer sets of LEGO Education have become a means for children to express
their own role in the peer community. The child‟s social position represents his place,
position in the group, regulating behavior, helping or limiting opportunities to realize
fantasies, be creative and create his own project. Robotic engineering as a cultural practice
becomes a unique source of optimizing the social positions of children, the manifestation of
each as a leader in any activity. Materials help them improve their knowledge; activate
communication, as children always enthusiastically describe the results of their creative
activity. The development of speech, cognitive, creative, artistic and aesthetic development
in unity stimulates the joint activity of children who intuitively compose stories, adding
unique details to the overall end result. Children are provided with unlimited opportunities
for research, so they realize that for creativity there are no limits in self-expression with the
help of a designer, they realize incredible ideas (“City residents of LEGO DUPLO.” All the
characters are dressed in clothing or uniforms corresponding to their profession). 4 Cases of educational robotics The prerequisites for the development of robotics are the psychological characteristics
of the child - curiosity development, the need to learn new things, to experiment, to find
answers to the questions that underlie the future successes of the child in the study of
mathematics, technology, physics, engineering design, and even the fine arts. The social and communicative development of children by means of LEGO games is
determined by joint design, the development of non-standard solutions due to the
development of the plot, positive attitudes towards interaction, and the importance of safe
behavior in games. Communications on a variety of occasions, in particular, on the role of
robots in modern life, contribute to the development of children's social and emotional
intelligence. Such philosophical dialogues require teachers to pay attention to the children's
way of thinking, expressing their emotions and feelings, and open up unusual
interpretations of future scenarios. Children are interested in communicating with each
other and adults, pondering the idea of a robot that performs actions inherent in people. Kids become more confident when they start to express their thoughts, and the moderator
teacher supports the thinking process (“Have you expressed an interesting thought, does
anyone have other ideas?”, “But how can I say it differently?”, “Oh, this thought sounds
great. Can I see it from the other side?" "Are there any other solutions?"), Contributing to
the social and communicative development of children. Designer LEGO DUPLO provides
the emotional development of kids from 3 years. Cognitive development in the process of
engaging in robotics is associated with the discovery of various spheres of life - urban,
outer space, the life of wild and domestic animals, the world of professions, etc. The
absence of emotionally negative assessments and constant indications of errors in the
design process become the motivators that captivate children. For example, the LEGO
Education “STEAM Planet” set in a playful way develops children's natural curiosity about
the principles of gears, movement and measurements, etc. 3 3 , 0
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3008 LEGO WeDo ensures the achievement of the Federal Governmental Standard of
Preschool Education targets: development of initiative and independence; teaches to model;
develops engineering competencies, stimulates research activities, etc. Thanks to the
designer, the speech development of children occurs. 4 Cases of educational robotics This
constructor allows you to explore situations like "What happens if? ..", "How to do that? ..",
etc. Joint thinking over possible options for the development of the plot determines a
positive change in the social positions of children. Conclusion Robotics in preschool education is not only a way of introducing a child to modern trends
in society, science, culture, but also an excellent tool for the development of children, their
social and STEAM competencies. These competencies provide children with the
opportunity to participate in team projects, express their opinions taking into account the
creative environment, perceive and focus on constructive criticism, and carry out self-
presentations. Modern didactic tools, such as LEGO Education, cultivate and develop such
abilities of children. Classes in robotics ensure the holistic development of children - their playful,
communicative, cognitive-research, constructive, aesthetic activities. Investigations of
objects in the surrounding world and experimenting with them are significant for modern 4 4 https://doi.org/10.1051/shsconf /20197203008 , 0
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3008 https://doi.org/10.1051/shsconf /20197203008 children, for their possible future technical education. In the context of civilization
development, mastery of the basics of robotics acquires special significance. We are
surrounded by mechanisms without which it is impossible to imagine everyday life: talking
navigators; voice applications that easily answer any question; interactive toys that
remember the owner‟s habits and enter into communication with him; a robot vacuum
cleaner that allows the child to understand who is responsible for cleanliness in the house,
etc. For modern children, a fascination with technology does not mean the ability to
engineering and exact sciences. Today, technology is becoming an area of life in which the
adherents of the exact sciences and the humanities must be competent. The task of the
education system is to provide the child with a relevant set of competencies corresponding
to the real spirit of the times, in which robots are not the future, but the present. References 1. Sberknoledge, 8, 3-10 (2017). 2. D. Markoff, Homo roboticus, LLC “Alpina non-fiction”, (2017). 3. Information on www.2045.ru/manifest. 4. I.V. Ladygina, Social and Ethical Problems of Robotics, Bulletin of Vyatka State
University, 30 (2017). 5. V.V. Tarapata, Five lessons in robotics, Informatics-First of September 2014, 11, 12-
25 (2014). 6. I.V. Tuzikova, The study of robotics - the path to engineering specialties, School and
production, 46 (2013). 7. K. Schwab, The Fourth Industrial Revolution (Eksmo, 2016). 8. Lego “More than a Cube” (Base Edition 2017). 9. I.E. Kulikovskaya, Theory of Personal World Outlook Evolution: Categories,
Provisions, Proofs, Middle East Journal of Scientific Research, 15(5), 698-706 (2013)
DOI: 10.5829 / idosi.mejsr.2013.15.5.11320 10. M. Rollins, LEGO technic robotics [Electronic resource]: design and build custom
LEGO bots with LEGO technic, (New York, NY: Apress, 2013). (Technology in
Action). Information on http://link.springer.com/book/10.1007%2F978-1-4302-4981-8. 11. The Robotics Divide. A New Frontier in the 21st Century?, ed. A. L. Peláez
(Springer-Verl.,
London,
2014). Information
on
https://link.springer.com/book/10.1007%2F978-1-4471-5358-0. 12. A.V. Frolov, Reform of the US Innovation System: from STEM to STEAM
Education, Alma mater (Bulletin of Higher Education), 9, 101-105 (2013). 13. K.H. Rubin, J.B. Asendorpf, N.J. Hillsdale, Social withdrawal, inhibition and
shyness in childhood (Lawrence Erlbaum Ass., 1993). 5 5
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Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses
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University of Groningen
Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human
Macrophages; Balancing Higher Fusion against Antiviral Responses
Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-
Inchima, Silvio; Smit, Jolanda M.
Published in:
Scientific Reports University of Groningen University of Groningen Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human
Macrophages; Balancing Higher Fusion against Antiviral Responses
Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-
Inchima, Silvio; Smit, Jolanda M. Published in:
Scientific Reports
DOI:
10.1038/srep29201
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record
Publication date:
2016
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Flipse, J., Diosa-Toro, M. A., Hoornweg, T. E., van de Pol, D. P. I., Urcuqui-Inchima, S., & Smit, J. M. (2016). Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages;
Balancing Higher Fusion against Antiviral Responses. Scientific Reports, 6, Article 29201. https://doi.org/10.1038/srep29201 ntibody-Dependent Enhancement of Dengue Virus Infection in Primary Human
acrophages; Balancing Higher Fusion against Antiviral Responses Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-
; Smit, Jolanda M. DOI:
10.1038/srep29201 DOI:
10.1038/srep29201 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record Publication date:
2016 Publication date:
2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Flipse, J., Diosa-Toro, M. A., Hoornweg, T. E., van de Pol, D. P. I., Urcuqui-Inchima, S., & Smit, J. M. (2016). Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages;
Balancing Higher Fusion against Antiviral Responses. Scientific Reports, 6, Article 29201. https://doi.org/10.1038/srep29201 Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
amendment. Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim. Antibody-Dependent Enhancement
of Dengue Virus Infection in
Primary Human Macrophages;
Balancing Higher Fusion against
Antiviral Responses
Jacky Flipse1, Mayra A. Diosa-Toro1,2, Tabitha E. Hoornweg1, Denise P. I. van de Pol1,
Silvio Urcuqui-Inchima2 & Jolanda M. Smit1 received: 13 January 2016
accepted: 14 June 2016
Published: 06 July 2016 Jacky Flipse1, Mayra A. Diosa-Toro1,2, Tabitha E. Hoornweg1, Denise P. I. van de Pol1,
Silvio Urcuqui-Inchima2 & Jolanda M. Smit1 The dogma is that the human immune system protects us against pathogens. Yet, several viruses,
like dengue virus, antagonize the hosts’ antibodies to enhance their viral load and disease severity; a
phenomenon called antibody-dependent enhancement of infection. This study offers novel insights
in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in
primary human macrophages. No differences were observed in the number of bound and internalized
DENV particles following infection in the absence and presence of enhancing concentrations of
antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The
higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher
infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong
effects on infection, virus production and immune response. Importantly, and in contrast to other
studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive
to interferon. Additionally, this study gives insight in how human macrophages interact and respond to
viral infections and the tight regulation thereof under various conditions of infection. Through the evolutionary process, viruses have acquired many mechanisms to hijack the host cell machinery and
to suppress antiviral responses within infected cells. More intriguingly, viruses have found ways to antagonize
the host immune system by using the hosts’ antibodies to enhance infection and disease: a phenomenon called
antibody-dependent enhancement (ADE)1,2. In vitro, ADE has been shown for influenza A virus3, Coxsackievirus
B4, respiratory syncytial virus5, Ebola virus6, human immunodeficiency virus (HIV)7, and many other viruses8. In vivo ADE has been linked with the severity of HIV7 and dengue virus (DENV) infection9,10. DENV infection is the most prevalent arthropod-borne viral infection worldwide with approximately 390
million infections and 96 million symptomatic cases in 201011. Four serotypes of DENV exist (DENV1–4) and
symptomatic infection with any DENV serotype leads to mild dengue fever or to the life-threatening dengue
haemorrhagic fever and dengue shock syndrome9.h The question why some patients develop dengue fever, and others dengue haemorrhagic fever or dengue shock
syndrome is continuously under investigation and debate. University of Groningen
Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human
Macrophages; Balancing Higher Fusion against Antiviral Responses
Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-
Inchima, Silvio; Smit, Jolanda M.
Published in:
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If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Download date: 24-10-2024 www.nature.com/scientificreports www.nature.com/scientificreports received: 13 January 2016
accepted: 14 June 2016
Published: 06 July 2016 Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 Methods
A tib di Antibodies. Human monoclonal antibodies (#753 C6 and #751 A2) against the DENV2 E protein were a kind
gift of J. Mongkolsapaya and G. Screaton (Imperial College, UK). Human IgG Fc-fragments were used as control
(Jackson immunoResearch, USA). Antibodies for the phenotyping of the macrophages, and corresponding iso-
types, were obtained through an unrestricted grant (Immunotools, DE). Cell lines. Baby Hamster Kidney cells (BHK-15), gift of Richard Kuhn, Purdue University, were propagated
in Dulbecco’s minimal essential medium (DMEM) (Gibco, NL) supplemented with 10% Fetal Bovine Serum
(FBS) (Lonza, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (PAA, Switzerland), 0.75 g/L sodium
bicarbonate (Gibco). The green monkey-derived Vero WHO cell line (WHO RCB 10–87), gift of James Brien, was
cultured as described for the BHK-15, yet at 5% FBS. P338D1 cells (ATCC, #CCL-46) were cultured as described
for BHK-15, with addition of 1mM sodium pyruvate (Gibco). Macrophages. Buffy coats were obtained from anonymous donors with informed consent from Sanquin
blood bank (Groningen, the Netherlands), in line with the declaration of Helsinki. The flavivirus-immune status
of the 23 blood donors that we used is unknown but it is unlikely that a major proportion is flavivirus-positive. PBMCs were isolated by Ficoll-Paque (GE Healthcare, Belgium) gradient, and stored in 90% FBS, 10% DMSO on
liquid nitrogen till use. Monocytes were isolated from total PBMCs, as described previously31, and differentiated
into macrophages by culturing in 12-wells plates for 6 days at 37 °C, 5% CO2 in RPMI with HEPES (Gibco), sup-
plemented with 20% FBS and 100 ng/mL recombinant human M-CSF (Prospec-Tany, Israel). On alternate days,
75% of the medium was replaced with medium and the full amount of M-CSF. Virus stocks. Dengue virus serotype 2, strain 16681 (DENV2) was propagated on C6/36 cell line, as described
previously34,35. The specific infectivity of the virus stock was 79 genomes per PFU. Purification was performed as
described by Ayala et al.35. Vesicular Stomatitis Virus, Indiana serotype, strain San Juan A (VSV) was propagated
on Vero WHO cells. Vero WHO cells were infected at MOI 0.1 in cell culture medium with 2% FBS. After 24 h,
the culture medium was harvested, clarified, and mixed with HEPES (PAA, Switzerland) to 10 mM final concen-
tration. All virus samples were aliquoted and snap frozen in liquid nitrogen prior to storage at −80 °C. Infectious virus titers were determined by plaque assay. DENV2 titers were determined on BHK-15 cells. Antibody-Dependent Enhancement
of Dengue Virus Infection in
Primary Human Macrophages;
Balancing Higher Fusion against
Antiviral Responses
Jacky Flipse1, Mayra A. Diosa-Toro1,2, Tabitha E. Hoornweg1, Denise P. I. van de Pol1,
Silvio Urcuqui-Inchima2 & Jolanda M. Smit1 Epidemiologic research showed that severe dengue
disease is strongly associated with primary infection of infants with waning maternal anti-dengue immunity10,12,
and with secondary, heterotypic dengue infection13–16. Patients with severe dengue disease often present high viral
loads early in infection17–19. In line with this, heterotypic sub-neutralizing antibodies, or waning concentrations of
homotypic antibodies have been found to enhance DENV infectivity in vitro and in vivo12,20,21. yp
y
So far, little is known about how antibodies enhance DENV infection and disease20,22. Previously, antibodies
were found to increase the number of infected cells and, subsequently, facilitate higher virus production once the
concentration of the antibody falls below the neutralization threshold23–26. Furthermore, it has been suggested 1Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, the
Netherlands. 2Grupo Immunovirología, Facultad de Medicina, Universidad de Antioquia UdeA, Medellín, Colombia. Correspondence and requests for materials should be addressed to J.M.S (email: Jolanda.smit@umcg.nl) Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 1 www.nature.com/scientificreports/ that DENV immune-complexes would boost virus production per infected cell (burst size) by suppressing intra-
cellular antiviral responses22,27–29. The latter is also called intrinsic ADE to stress putative involvement of an intra-
cellular mechanism. Consequently, enhancement of the infected cell mass is named extrinsic ADE. q
y
DENV replicates in macrophages, monocytes, and dendritic cells in vitro24,30,31 and in vivo32. During second-
ary infection, i.e. in the presence of antibodies, monocytes and macrophages actively support ADE, whereas
immature dendritic cells do not23,24,33. Indeed, studies with primary macrophages and monocytes found that ADE
can enhance DENV burst sizes in the order of 5.3- to 7.2-fold23–25. Here, we focused on one cell type, i.e. primary
human macrophages, and attempted to discern the mechanism of enhanced DENV infection within this cell type. p
g
p
yp
We discovered that, in primary human macrophages, antibody-mediated cell entry of DENV enhances the
fusion potential of the virus. No enhanced binding and entry was seen. Also, we observed that ADE does not
induce an increased antiviral response early in infection. We propose that the increase in membrane fusion activ-
ity initiates an aggravating cascade leading to the typical enhancement of (i) infection, (ii) burst size, and (iii)
disproportionally stronger anti-inflammatory responses. Importantly, no antibody-mediated immunosuppressive
signalling was detected in primary human macrophages. Our data suggests that DENV antagonizes the host anti-
body as a vehicle to enhance its fusion efficiency within macrophages and consequently enhance disease severity. Methods
A tib di For the entry studies, cells were gently rocked for 2 min with high-salt-high-pH
shaving buffer (1M NaCl, pH 9.5) prior to lysis in AVL buffer. gf
p
p
yf
Flow cytometric of DENV-infected macrophages was performed using 4G2 antibody (Millipore, UK) and
donkey anti-mouse IgG-coupled to AF647 (Molecular Probes). Hereto, trypsinized macrophages were fixed with
4% paraformaldehyde in PBS and permeabilized with saponin (Sigma, USA). Flow cytometry was performed on
a FACScalibur (BD Biosciences) and analysed using FlowJo 7.6.2 or Kaluza 1.1.l y
g
Viral protein translation was determined using the mean fluorescence intensity of infected cells, Hereto,
infected cells were gated in Kaluza and the mean fluorescence intensity of AF647 was determined for each sam-
ple. Values were expressed as percentage of the maximal fluorescence intensity observed within the donor/exper-
iment. Extracellular virus particles did not contribute to the fluorescence signal. pl
g
Macrophage IFNαβR signalling was blocked using the mouse anti-human IFNαβR2 (Millipore) by
pre-incubating the cells for 2 h with 0.5 μg/well of antibody. Then, cells were infected and cultured as usual, in the
presence of the antibody (1 μg/mL). Infection was scored at 26 hpi and 48 hpi by qRT-PCR. Specificity of IFNαβR
blocking was confirmed by quantifying surface expression of CD14 and MHC class I by flow cytometry. gi
y q
y
g
p
yl
y
y
For the interferon-alpha add-on experiments; macrophages were infected as described above. At the desig-
nated time points, 1 unit/mL of recombinant interferon α2a (Prospec-Tany, 2.7 · 108 IU/mg) was added to the cul-
ture and the concentration was maintained throughout the remainder of the experiment. At 26 hpi, virus particle
production was determined by qRT-PCR. Fusion assay on primary human macrophages. Isolation and culture of macrophages was initiated as
described above. On the second day of culture, the cells were gently dissociated by incubation at 4 °C for 15 min-
utes followed by gentle pipetting. Subsequently, cells were reseeded at 2 ∙ 105 per quadrant in 500 μL in CELLview
dishes (Greiner Bio-One, DE). Subsequent culture and infection was done as above. At 30 min post-infection,
extracellular virus was removed by gentle washing the cells with shaving buffer followed by fixation with 4%
PFA-PBS. Fusion activity was determined as described before35. Methods
A tib di Briefly, wide-field microscopy analysis was done
by taking 15–30 random snapshots using both differential interference contrast and DiD-channels in a Leica
Biosystems 6000B instrument with a 635-nm helium-neon laser. Acquired images were analysed with ImageJ
using an in house macro35 measuring the total fluorescent signal per field of view with the “Particle analyzer”
plugin of ImageJ. Fluorescence intensity was normalized relative to MOI 1. The fraction of fusion-positive cells,
with at least one bright fluorescent spot, was blindly scored. Antiviral bio-assay. The antiviral response in culture supernatants was determined using VSV and Vero
WHO cells as described before25. Briefly, Vero WHO cells were seeded in 12-wells plates at 2 ∙ 105 per mL per
well. After adherence for 8 h, cells were incubated for 12 h with UV-inactivated supernatant, followed by infection
with VSV at an MOI of 0.1 in 100 μL. At 1 hpi, inoculum was removed and cells were washed. Incubation was
continued in fresh UV-inactivated supernatant. At 6 hpi, medium was collected and VSV titers were determined
by plaque assay. Experimental results are reported as percentage of mock-medium. Recombinant human inter-
feron–alpha (Prospec-Tany) served as positive control. Microarray. Macrophages were infected at MOI 1, MOI 1-ADE and MOI 1-IgG. Matched infection controls
represent conditions resulting in similar fractions of infected cells; i.e. infection at MOI 5 or MOI 2½ (3 and 1
donor at 2 hpi, 2 and 1 donor at 24 hpi, respectively). Cellular RNA was extracted with RNeasy Plus (Qiagen)
as per manufacturer’s instructions. RNA was processed in house and randomly annealed to Human HT-12 V3
BeadChip array (Illumina, USA), as per manufacturers’ protocol. Data was converted using GeneSpring (Agilent
Technologies, USA). Probe values were normalized against the total signal intensity of the sample and subse-
quently, the fold change of the probes were expressed relative to mock condition of the same donor. For each time
point, the donors were pooled and averaged. Probes with an absolute, average fold-change of ≥1.5 relative to the
mock were considered differentially regulated. Probes were manually curated to single gene level, independent of
the potential isoforms, and Venn diagrams were drawn based on overlaps between infection conditions. All data
is been freely available through ArrayExpress ID: E-MTAB-3138. DAVID. [Database for Annotation, visualization, and Integrated Discovery]37 Pathway analysis was performed
to functionally annotate gene groups using standard criteria and an EASE of 0.1. Methods
A tib di VSV was titrated on Vero WHO cells. The limit of detection is 40 PFU/mL. In brief, for both plaque assays, cells
were seeded the day before infection (1 ∙ 105 per well in 24-wells plates). Serial dilutions of virus supernatant were
added to the cells followed by 2 h incubation at 37 °C prior to placing an overlay of 1% seaplaque agarose (Lonza,
Switzerland) in MEM (Gibco). Cells were fixed at 1 (VSV) or 6 days post infection (DENV2). Plaques were visu-
alized with crystal violet (TCS biosciences, UK). y
The number of genome-containing particles was determined as previously described34 with minor
modifications. Virus labelling. DENV2 was labelled with DiD (Molecular Probes, USA) as described before35. Virus label-
ling with PKH67 (Sigma, USA) was performed based on Balogh et al.36. Briefly, 1.5 ∙ 108 GCPs of tartrate-purified
DENV2 was diluted in PBS to 100 μL and mixed with 1 μL PKH67 dye in 99 μL Diluent C (Sigma, USA). At 30
seconds post addition, 300 μL of pure FBS was added to stop the labelling. PKH67-labelled virus was used directly
after labelling. Infection of macrophages. Prior to infection, two wells were trypsinized using 10xTrypsin/EDTA (Gibco)
and counted to determine the required amount of virus for each multiplicity-of-infection (MOI). To standardize
the opsonisation in ADE experiments, a fixed ratio of antibodies to virus particles was used; per 1 · 105 PFU of
DENV2, we added 3 ng of antibody in 75 μL total volume. Thereafter, viruses and antibodies were incubated for
1 h at 37 °C prior to addition to the cells. Cells were washed with warm RPMI and infected at the indicated MOI in
200 μL per well. At 2 hpi, cells were washed to remove extracellular virus and incubation was continued in culture
medium (RPMI, 20% FBS, and 10 ng/mL M-CSF). For virus titrations, culture samples were snap-frozen in liquid
nitrogen and stored at −80 °C until analysis.t g
y
For binding and entry studies, the virus was added to the cells and left to incubate for the designated time peri-
ds. At the end, the inoculum was removed, and the cells were lysed in AVL buffer (Qiagen) to determine the tota Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 2 www.nature.com/scientificreports/ number of particles by qRT-PCR. Methods
A tib di Interferome database38 was used
to identify an IFN signature in the gene selections, using standard criteria with absolute fold change of ≥2 and
limited to the haematopoietic system38. Statistical analysis. Statistical analysis was performed using Prism 5.00 (Graphpad, USA). A two-sided stu-
dent’s t-test was used throughout the paper to determine the significance of enhancement after antibody-mediated
infection. The antiviral response was analysed using One-way ANOVA with Bonferroni compensation. A p-value
of ≤0.05 was considered significant for both tests. Results Figure 1. Antibody-dependent enhancement of DENV2 infection of primary human macrophages is dose-
dependent. Macrophages were incubated with DENV2, strain 16681 at MOI 1 which had been pre-incubated
for 1 h with increasing concentrations of monoclonal human antibody (#753 C6; light grey, or #751 A2; dark
grey). At 48 hpi, virus production was determined by plaque assay on BHK-15 cells. Shown is the SEM of
duplicates. The figure is representative for two independent experiments. These antibodies are described more
in-depth in a previous publication42. Figure 1. Antibody-dependent enhancement of DENV2 infection of primary human macrophages is dose-
dependent. Macrophages were incubated with DENV2, strain 16681 at MOI 1 which had been pre-incubated
for 1 h with increasing concentrations of monoclonal human antibody (#753 C6; light grey, or #751 A2; dark
grey). At 48 hpi, virus production was determined by plaque assay on BHK-15 cells. Shown is the SEM of
duplicates. The figure is representative for two independent experiments. These antibodies are described more
in-depth in a previous publication42. expression pattern (Fig. A1). In line with previous literature31,41, primary human macrophages were
susceptible to DENV2 infection in an MOI-dependent manner (between MOI 0.2 and MOI 10)
(Fig. A2, and Table 1). To test for ADE, we used human monoclonal antibodies against distinct epitopes of the DENV envelope
protein; e.g. domains (D) I/DII (#753 C6), and the DII fusion loop (#751 A2). These antibodies were previously
shown to be cross-reactive against all four serotypes of DENV42. Notably, we found that the power of enhance-
ment was similar between the antibodies (Fig. 1) and independent of the MOI (Fig. A2). Given the overlapping
results between the antibodies, we decided to focus on the E DII fusion loop antibody #751 A2 for the remaining
experiments. Peak enhancement was observed at an antibody concentration of 40 ng/mL, giving 6.8 ± 1.3-fold
enhancement (N = 9) at MOI 1 (Fig. 1). The observed power of enhancement is comparable to other studies using
primary human cells23–25. p
y
In line with the current hypothesis of ADE, ADE enhanced both the infected cell mass and the burst size
(Table 1). The percentage of infection was scored at 26 h post infection (hpi) by flow cytometry. The burst size was
calculated by dividing the virus titer as measured with plaque assay by the number of infected cells. Results Human monoclonal antibodies enhance dengue virus infection of primary human
macrophages. Primary macrophages are considered important players in ADE of DENV infec-
tion12,39, yet most of the mechanistic studies conducted so far have been conducted in cell lines26,27,40. Although working with primary cells is more challenging than cell lines given the inherent varia-
bility between blood donors and the less pronounced ADE effects in primary cells, we feel that it is
important to dissect the fundaments of ADE in cells that are thought to contribute to ADE during
natural infection. Primary human macrophages were generated by culturing isolated blood mono-
cytes for 6 days in the presence of 100 ng/mL M-CSF. The resulting cells showed a typical macrophage Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 3 www.nature.com/scientificreports/ MOI
Infected cells (%)
Burst size (PFU/cell)
Particles/PFU
1
2.0 ± 0.5
2.8 ± 1.0
255 ± 34
1-ADE
3.7 ± 0.9
8.9 ± 2.4
212 ± 19
10
11.3 ± 2.3
17.2 ± 4.7
268 ± 24 Table 1. Antibodies enhance both infection and burst size, but not the specific infectivity of progeny
virions. Primary human macrophages were infected at MOI 1 or 10, and MOI 1-ADE. For MOI 1-ADE DENV
was pre-incubated for 1 h with 40 ng/mL of antibody #751 A2. The number of infected cells was determined by
flow cytometry at 26 hpi. Concurrently, the virus titer was determined by both qRT-PCR (physical particles)
and plaque assay (infectious particles). The burst size was calculated by dividing the virus titer by the number
of infected cells and the specific infectivity resembles the number of physical particles divided by the number of
infectious particles. All values are SEM of duplicates of 4 donors. Table 1. Antibodies enhance both infection and burst size, but not the specific infectivity of progeny
virions. Primary human macrophages were infected at MOI 1 or 10, and MOI 1-ADE. For MOI 1-ADE DENV
was pre-incubated for 1 h with 40 ng/mL of antibody #751 A2. The number of infected cells was determined by
flow cytometry at 26 hpi. Concurrently, the virus titer was determined by both qRT-PCR (physical particles)
and plaque assay (infectious particles). The burst size was calculated by dividing the virus titer by the number
of infected cells and the specific infectivity resembles the number of physical particles divided by the number of
infectious particles. All values are SEM of duplicates of 4 donors. Results In absence
of antibodies, 2.0% ± 0.5 of the cells were infected with DENV at MOI 1. Under conditions of MOI 1-ADE, the
fraction of infected cells increased to 3.7% ± 0.9 (Table 1). For individual donors, the average enhancement was
2.3 ± 0.6-fold (MOI 1 versus MOI 1-ADE, p = 0.023, N = 4 donors). At the same time, the burst size increased
4.2 ± 0.4-fold (N = 4, p = 0.005). The increase in burst size indeed suggests that intrinsic ADE mechanisms are
involved. Interestingly though, infection at MOI 10 in the absence of antibodies also increased the burst size
(Table 1). Increased burst sizes were already observed at MOI 2½, and infection at MOI 5 closely mimicked MOI
1-ADE in terms of burst size and infected cell mass (Table A1).li To determine whether ADE influenced the specific infectivity of the progeny virus (particles per PFU), we
quantified the number of genome-containing particles (qRT-PCR) and the number of infectious particles (plaque
assay). In contrast to results from cell lines26, we found that neither antibodies nor MOI influenced the particle/ Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 4 www.nature.com/scientificreports/ Figure 2. Antibodies and infection at high MOI enhance virus translation. Viral translation was determined
per cell on the intracellular pool of envelope protein. Macrophages were infected with DENV2. At 24–26 h,
cells were stained with the anti-envelope antibody 4G2 and analysed by flow cytometry. Mean fluorescence
intensities were normalized to the sample with the highest intensity of the donor. Up to 11 blood donors were
used and six donors were used twice. Statistical analysis was done by One way ANOVA with Bonferroni post-
test correction; *(p ≤ 0.05), **(p ≤ 0.01) ***(p ≤ 0.0001), n.s.: non-significant. Figure 2. Antibodies and infection at high MOI enhance virus translation. Viral translation was determined
per cell on the intracellular pool of envelope protein. Macrophages were infected with DENV2. At 24–26 h,
cells were stained with the anti-envelope antibody 4G2 and analysed by flow cytometry. Mean fluorescence
intensities were normalized to the sample with the highest intensity of the donor. Up to 11 blood donors were
used and six donors were used twice. Statistical analysis was done by One way ANOVA with Bonferroni post-
test correction; *(p ≤ 0.05), **(p ≤ 0.01) ***(p ≤ 0.0001), n.s.: non-significant. Figure 2. Antibodies and infection at high MOI enhance virus translation. Results Viral translation was determined
per cell on the intracellular pool of envelope protein. Macrophages were infected with DENV2. At 24–26 h,
cells were stained with the anti-envelope antibody 4G2 and analysed by flow cytometry. Mean fluorescence
intensities were normalized to the sample with the highest intensity of the donor. Up to 11 blood donors were
used and six donors were used twice. Statistical analysis was done by One way ANOVA with Bonferroni post-
test correction; *(p ≤ 0.05), **(p ≤ 0.01) ***(p ≤ 0.0001), n.s.: non-significant. PFU ratio of DENV2 in primary human macrophages (Table 1). This suggests that the observed ADE effect
occurs prior to assembly, maturation and secretion of progeny virions. Enhanced transcription and translation during ADE, yet the replication efficiency is unaf-
fected. We next studied the effect of antibodies on protein translation and viral genome replication using flow
cytometry and qRT-PCR, respectively. Protein translation was determined on a per-cell-basis by measuring the
mean fluorescence intensity (MFI) of the envelope proteins within the cell. Figure 2 shows that the MFI is clearly
enhanced under conditions of ADE at 24 hpi (N = 11, p ≤ 0.0001), suggesting that ADE may enhance viral protein
translation. Higher concentrations of virus (MOI 2½ , MOI 5, and MOI 10), in line with the increased burst size,
also resulted in higher E protein content per cell yet protein translation did not differ among the higher MOI’s
(Fig. 2). ( g
)
Next, the replication efficiency of the virus was determined at 24 hpi by measuring the intracellular ratio of
positive-sense and negative-sense RNA. ADE leads to 10-fold higher numbers of both negative- (9.8 ± 4.4, N = 3)
and positive-sense (9.8 ± 3.5, N = 4) RNA. Yet, the ratio of positive versus negative RNA was comparable between
MOI 1 and MOI 1-ADE (4.9 ± 1.0 versus 4.8 ± 2.7, respectively (N = 4)), suggesting that antibody-mediated
infection does not influence the replication efficiency of DENV. Contrary to this, infection at MOI 5 showed a
ratio of 2.5 ± 1.5 (N = 4), indicating that the replication efficiency is negatively affected under conditions of higher
MOI’s. Thus, the increased burst size during ADE is associated with enhanced transcription and translation while the
replication efficiency is similar to MOI 1. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 Results Antibodies do not alter the efficiency of dengue virus to bind or enter into primary human
macrophages, whilst promoting fusion within primary macrophages. (A) DENV2 binding and uptake in
primary macrophages was determined at 1 hpi by qRT-PCR using template-specific primers in combination
with RNAse A treatment. Extracellular virus was removed by shaving the cells with a high-salt-high-pH buffer
for 2 min. Squares show the total number of virus particles that had bound or entered cells. Circles depict
entered viral genomes, while triangles show negative-sense RNA genomes. Shown are 4 donors with each
condition in duplicate. (B) Macrophages were infected with PKH67-labelled DENV at MOI 1 (red), MOI
1-ADE (blue) or MOI 5 (green). At 1 h (filled) or 2 h (striped) of incubation, the cells were shaved and fixed
prior to analysis by flow cytometry. Cell entry was normalized to MOI 1, and the mean fluorescence intensity
(MFI) was normalized to the negative control. Shown are the SEM of 5 donors. At MOI 1, 13 ± 3% of the cells
were positive for PKH67-labelled DENV entry. (B–E) The fusion activity of DENV2 within primary human
macrophages was determined at 30 min by microscopy using the self-quenching fluorophore DiD. Pictures
were taken randomly and analysed for fusion activity (C), and the fraction of fusion-positive cells (D). All
values were normalized to MOI 1 of the same donor (C) or infection in absence of antibodies (D,E). At MOI
1, the average number of fusion-positive cells was 17 ± 2.2%. (E) An overview of connected values of fusion
activity when primary human macrophages are infected in the presence of enhancing antibodies (ADE), and
when infected in the presence of an isotype (IgG). The inset shows the correlation between enhancement of
fusion activity at 30 min post infection and the subsequent enhancement in virus production at 26 hpi. All
values are given as percentage of the results obtained at MOI 1. Shown are the normalized SEM of up to 12
donors (A–C) and 5 experiments (D,E). Statistical analysis was done by 2-tailed t-test; *(P ≤ 0.05), **(P ≤ 0.01),
***(P ≤ 0.0005). Figure 3. Antibodies do not alter the efficiency of dengue virus to bind or enter into primary human
macrophages, whilst promoting fusion within primary macrophages. (A) DENV2 binding and uptake in
primary macrophages was determined at 1 hpi by qRT-PCR using template-specific primers in combination
with RNAse A treatment. Results Extracellular virus was removed by shaving the cells with a high-salt-high-pH buffer
for 2 min. Squares show the total number of virus particles that had bound or entered cells. Circles depict
entered viral genomes, while triangles show negative-sense RNA genomes. Shown are 4 donors with each
condition in duplicate. (B) Macrophages were infected with PKH67-labelled DENV at MOI 1 (red), MOI
1-ADE (blue) or MOI 5 (green). At 1 h (filled) or 2 h (striped) of incubation, the cells were shaved and fixed
prior to analysis by flow cytometry. Cell entry was normalized to MOI 1, and the mean fluorescence intensity
(MFI) was normalized to the negative control. Shown are the SEM of 5 donors. At MOI 1, 13 ± 3% of the cells
were positive for PKH67-labelled DENV entry. (B–E) The fusion activity of DENV2 within primary human
macrophages was determined at 30 min by microscopy using the self-quenching fluorophore DiD. Pictures
were taken randomly and analysed for fusion activity (C), and the fraction of fusion-positive cells (D). All
values were normalized to MOI 1 of the same donor (C) or infection in absence of antibodies (D,E). At MOI
1, the average number of fusion-positive cells was 17 ± 2.2%. (E) An overview of connected values of fusion
activity when primary human macrophages are infected in the presence of enhancing antibodies (ADE), and
when infected in the presence of an isotype (IgG). The inset shows the correlation between enhancement of
fusion activity at 30 min post infection and the subsequent enhancement in virus production at 26 hpi. All
values are given as percentage of the results obtained at MOI 1. Shown are the normalized SEM of up to 12
donors (A–C) and 5 experiments (D,E). Statistical analysis was done by 2-tailed t-test; *(P ≤ 0.05), **(P ≤ 0.01)
***(P ≤ 0.0005). Figure 3. Antibodies do not alter the efficiency of dengue virus to bind or enter into primary human
macrophages, whilst promoting fusion within primary macrophages. (A) DENV2 binding and uptake in
h
d
d
h
b
l
fi
b assess if antibody-mediated DENV entry increases the fusion potential of the virus, we employed a microscopic
fusion assay involving DiD-labelled DENV. Fusion is observed as a sudden increase in fluorescence intensity due
to dilution of the probe in the target membrane34,35,44. Results In absence of antibodies, similar infectivity, burst size and translation
can be attained using higher MOIs yet at the cost of the replication efficiency. Antibodies enhance fusion while maintaining the same cellular dose of virus particles. It is
assumed that the higher number of genome copies and increased protein translation is a consequence of higher
virus cell binding and/or increased uptake of particles into cells. Therefore, we quantified virus binding and cell
entry using qRT-PCR. To measure viral entry, extracellular virions were removed by washing the cells with a
high-salt-high-pH buffer43. g
g
pf
Overall, virus cell binding (squares) and entry (circles) was not enhanced under conditions of ADE (N = 4
donors) (Fig. 3A). We were surprised by these findings, yet we were not able to use qRT-PCR at later time points
due to the initiation of replication as visualized by the negative sense RNA (triangles, Fig. 3A). Therefore, we next
used flow cytometry to assess the binding and entry dynamics of DENV2 in individual cells using PKH67-labelled
DENV particles. PKH67 is a fusion-independent lipophilic dye that intercalates into the viral membranes36. This approach allows us to measure viral uptake and reveals the population of cells that are positive for PKH67. DENV2 was successfully labelled with PKH67 (Fig. A3).i Figure 3B shows the extent of DENV2 uptake against the cell population positive for uptake at 1 hpi (filled
shape) and 2 hpi (striped shape). The extent of virus uptake per cell as well as the fraction of positive cells
increased over time. Comparable results were obtained for MOI 1 (blue) and MOI 1-ADE (red). Yet, at MOI 5
(green) both the fraction of PKH67-positive cells and the extent of viral uptake per cell were higher than MOI
1/1-ADE at both time points (Fig. 3B). These results are in line with the qRT-PCR data (Fig. 3A), and confirm that
ADE does not enhance the binding- or entry-efficiency of DENV2 in primary macrophages. g
yfi
y
y
g
Hence, we hypothesized that antibodies enhance a step downstream of entry and prior to replication. For
example, antibodies may direct the virus to an organelle and/or cellular location that is more beneficial for mem-
brane fusion and infection. Alternatively, antibodies may enhance the intrinsic fusion capacity of the virus. To Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 5 www.nature.com/scientificreports/ Figure 3. Results Figure 3C shows that this assay is specific and robust since
the extent of fusion was MOI-dependent and inhibited by ammonium chloride (Fig. 3C, ref. 34). Importantly,
membrane fusion activity of DENV under conditions of ADE was enhanced with 65% (Fig. 4A). Furthermore,
the number of fusion-positive macrophages increased with 40% (Fig. 3D). The extent to which fusion is enhanced
is variable between donors and ranged from 115% to 336% (Fig. 3E inset). Importantly, when the extent of fusion
enhancement is plotted against those for the PFU production, the results are correlating with each other, indicat-
ing a causal relationship between the two parameters (Fig. 3E). Recently, we performed similar experiments in the macrophage-like cell line P338D1 (Ayala et al., accompa-
nying manuscript). In contrast to our results for primary human macrophages, we did find enhanced binding/
entry of DENV in P338D1 cells under conditions of ADE and subsequent higher fusion activity (3.85 ± 0.5 and
3.52 ± 0.64 fold change, respectively). This suggests that the qRT-PCR is able to detect differences in binding and
entry where they occur, but also that the mechanism of ADE is cell-type-specific.l i
We next attempted to assess if antibodies itself influence the fusion potential of the virus using a cell-free lipo-
somal system. However, despite our experience in virus-liposome fusion studies45–47, we were not able to detect
fusion of DENV with negatively-charged liposomes (data not shown). Thus we were not able to examine whether
antibodies intrinsically promote membrane fusion of DENV. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 6 www.nature.com/scientificreports/ Figure 4. Dengue virus infection of macrophages induces an antiviral response, which depends on the viral
load. (A,D) Primary human macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, and matched
infection (MOI 5 or MOI 2½). Total RNA was isolated at 24 h (A) and 2 h (D). Gene expression was investigated
by microarray. Genes whose average expression had an absolute fold change of at least 1.5-fold over the mock
were selected. The Venn diagram shows how these genes are connected with the various infection conditions. (A) is based on 3 donors and (D) on 4 donors. (B,C,E) Gene ontology of DAVID pathway analysis was used to
annotated genes at 24 h (B,C) and at 2 h (E). Results (A) is based on 3 donors and (D) on 4 donors. (B,C,E) Gene ontology of DAVID pathway analysis was used to
annotated genes at 24 h (B,C) and at 2 h (E). (B) shows the ontological analysis of the genes that were shared
among the three conditions (see A, triangle, 100 genes).(C) shows the analysis of the ADE-effect (see A, 460
genes total; 318 + 142). (E) shows the ontological analysis of the genes that were shared among the three
conditions at 2 h (see D, triangle, 160 genes). The x-axis shows the enrichment of the term within our selection
relative to the DAVID database. The y-axis shows the significance calculated with 1-tailed Fisher Exact statistical
analysis. Gene ontology terms with at least 5-fold enrichment and a p-value of ≤1 ∙ 10−4 are considered relevant. To summarize, ADE in both models led to more fusion-positive cells and more fusion activity per cell (i.e. higher genome delivery). The relatively small increase in fusion activity probably initiates a cascade leading to
higher infection rates and burst sizes. In primary human macrophages, the efficiency of infection and the burst
size is however not solely dependent on membrane fusion activity since MOI 5 and ADE had a similar infected
cell mass and burst size, while at MOI 5 a five-fold higher fusion activity was seen compared to ADE. This sug-
gests that the infection process is negatively influenced at MOI 5 compared to MOI 1-ADE. Therefore, we next
wished to better understand what happens within the cell to-be-infected. Gene profiles of DENV-infected macrophages discriminate between high and low infection. To identify the cellular responses during DENV infection, we profiled the gene expression patterns at 2 and
24 hpi. Macrophages were infected at MOI 1, and MOI 1-ADE to study the biological process of ADE. Also,
infection-matched controls, with a similar fraction of infected cells (MOI 2½ or MOI 5, depending on the donor),
were included to better understand the increased burst size observed at high MOI. As controls, we included
non-infected cells and infections at MOI 1 in the presence of non-relevant antibodies (1-IgG). All donors and
conditions were normalized to the mock, and genes with a fold-change of ≥1.5 of the mock were selected. Venn
diagrams were drawn to visualize the overlaps between various conditions of infection. Results (B) shows the ontological analysis of the genes that were shared
among the three conditions (see A, triangle, 100 genes).(C) shows the analysis of the ADE-effect (see A, 460
genes total; 318 + 142). (E) shows the ontological analysis of the genes that were shared among the three
conditions at 2 h (see D, triangle, 160 genes). The x-axis shows the enrichment of the term within our selection
relative to the DAVID database. The y-axis shows the significance calculated with 1-tailed Fisher Exact statistical
analysis. Gene ontology terms with at least 5-fold enrichment and a p-value of ≤1 ∙ 10−4 are considered relevant. Figure 4. Dengue virus infection of macrophages induces an antiviral response, which depends on the viral
load. (A,D) Primary human macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, and matched
infection (MOI 5 or MOI 2½). Total RNA was isolated at 24 h (A) and 2 h (D). Gene expression was investigated
by microarray. Genes whose average expression had an absolute fold change of at least 1.5-fold over the mock
were selected. The Venn diagram shows how these genes are connected with the various infection conditions. (A) is based on 3 donors and (D) on 4 donors. (B,C,E) Gene ontology of DAVID pathway analysis was used to
annotated genes at 24 h (B,C) and at 2 h (E). (B) shows the ontological analysis of the genes that were shared
among the three conditions (see A, triangle, 100 genes).(C) shows the analysis of the ADE-effect (see A, 460
genes total; 318+142). (E) shows the ontological analysis of the genes that were shared among the three Figure 4. Dengue virus infection of macrophages induces an antiviral response, which depends on the v Figure 4. Dengue virus infection of macrophages induces an antiviral resp Figure 4. Dengue virus infection of macrophages ind Figure 4. Dengue virus infection of macrophages induces an antiviral response, which depends on the viral
load. (A,D) Primary human macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, and matched
infection (MOI 5 or MOI 2½). Total RNA was isolated at 24 h (A) and 2 h (D). Gene expression was investigated
by microarray. Genes whose average expression had an absolute fold change of at least 1.5-fold over the mock
were selected. The Venn diagram shows how these genes are connected with the various infection conditions. Results g
p
First, we studied the biological process of ADE at 24 hpi.The Venn diagram for this time point shows that ADE
induces a strong alteration in the transcriptional profile of infected human macrophages; 101 genes were shared
between MOI 1 and MOI 1-ADE, while 460 genes were uniquely for ADE. Contrary to this, only 2 genes were
unique to infection at MOI 1 without antibodies. q
Yet, a comparison of MOI 1-ADE with the infection-matched control shows that a large majority of the
ADE-genes are associated with high infection (319 genes, Fig. 4A). Thus, the transcriptional response upon ADE
is likely induced by the larger infected cell mass and/or higher viral load in the supernatant (2-fold and 8-fold
higher than MOI 1, respectively). DAVID pathway analysis37 was used to functionally annotate the gene patterns
(Fig. 5B,C). We were interested in two sets of genes: the shared genes (100 genes, Fig. 4A) and the ADE-associated
genes (141 + 319 genes, Fig. 4A). Gene ontology identified the term “antiviral defense” as the most significant
term for the shared genes (Fig. 4B). For the ADE-associated genes, the sole significant term was “inflammatory
responses” (Fig. 4C), hence antibodies did not induce a long-lasting immune-suppressive state in our macrophage
cultures. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 7 www.nature.com/scientificreports/ Figure 5. Early type-I-interferon (IFN)-mediated responses significantly determine the outcome of
infection. (A) Primary macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, MOI 5, and MOI
10. Culture supernatants were harvested at 24 h and UV-inactivated. Inactivated supernatants were used to
pre-activate Vero cells prior to infection with VSV at MOI 0.1. Shown is the SEM of 3 donors, with infection
performed in duplicate and duplicates of this assay. Statistical analysis was done with One way ANOVA with
post-test Bonferroni compensation; **(p ≤ 0.01) and n.s.; non-significant. (B) IFNα protects primary human
macrophages against DENV2 infection when applied during the early (0–2 hpi) time points, and not during
late (8 hpi) time points. Macrophages were infected with DENV2 at MOI 1, MOI 1-ADE and MOI 5. At the
designated time points, 1 IU of recombinant human IFNα2a was added to the culture. At 26 hpi, the viral titre
in the supernatant was determined by qRT-PCR and normalized relative to the unperturbed condition (No
IFNα). Shown is the SEM of 4–5 donors, each condition in duplicate. Results (C) Type I IFN signalling was blocked by
pre-incubating macrophages for 2 h with an antibody against the IFNαβR. After incubation, cells were infected
at MOI 1, MOI 1-ADE and MOI 5. Supernatants were sampled at 26 hpi and virus production was determined
by qRT-PCR. Addition of 10 units of IFNα at 0 hpi served as a control for IFNαβR-blocking. Shown is the
normalized SEM of 2 donors, each condition in duplicate. Figure 5. Early type-I-interferon (IFN)-mediated responses significantly determine the outcome of
infection. (A) Primary macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, MOI 5, and MOI
10. Culture supernatants were harvested at 24 h and UV-inactivated. Inactivated supernatants were used to
pre-activate Vero cells prior to infection with VSV at MOI 0.1. Shown is the SEM of 3 donors, with infection
performed in duplicate and duplicates of this assay. Statistical analysis was done with One way ANOVA with
post-test Bonferroni compensation; **(p ≤ 0.01) and n.s.; non-significant. (B) IFNα protects primary human
macrophages against DENV2 infection when applied during the early (0–2 hpi) time points, and not during
late (8 hpi) time points. Macrophages were infected with DENV2 at MOI 1, MOI 1-ADE and MOI 5. At the
designated time points, 1 IU of recombinant human IFNα2a was added to the culture. At 26 hpi, the viral titre
in the supernatant was determined by qRT-PCR and normalized relative to the unperturbed condition (No
IFNα). Shown is the SEM of 4–5 donors, each condition in duplicate. (C) Type I IFN signalling was blocked by
pre-incubating macrophages for 2 h with an antibody against the IFNαβR. After incubation, cells were infected
at MOI 1, MOI 1-ADE and MOI 5. Supernatants were sampled at 26 hpi and virus production was determined
by qRT-PCR. Addition of 10 units of IFNα at 0 hpi served as a control for IFNαβR-blocking. Shown is the
normalized SEM of 2 donors, each condition in duplicate. Shared
MOI 5 unique
Shared MOI 5 & 1-ADE
2 hpi
30.9% (160 genes)
25.7% (152 genes)
xxx
24 hpi
79.8% (100 genes)
xxx
49.2% (319 genes)
Table 2. High viral load stimulates IFN-regulated genes early in infection. Genes were selected from
the Venn diagrams in Fig. 4A (24 h) and 4E (2 h). Selected genes were analysed by mining the Interferome
database38 for interferon-regulated genes in haematopoietic cells which are reported as the percentage of the
selected genes. Results Table 2. High viral load stimulates IFN-regulated genes early in infection. Genes were selected from
the Venn diagrams in Fig. 4A (24 h) and 4E (2 h). Selected genes were analysed by mining the Interferome
database38 for interferon-regulated genes in haematopoietic cells which are reported as the percentage of the
selected genes. Hence, we focused on the transcriptional profiles shortly after infection. At 2 hpi, both MOI 1 and MOI 1-ADE
had very similar transcriptional profiles (Fig. 4D). Contrary to this, MOI 5 showed a much stronger shift in
its transcriptional profile. The lack of difference between MOI 1 and MOI 1-ADE at 2 hpi is in contrast with
the 24 hpi time point (Fig. 4A,D, respectively), suggesting that antibodies do not induce specific transcriptional
profiles. A comparison of the profiles induced by MOI 1, MOI 1-IgG and MOI 1-ADE indeed revealed that
infection in the presence of DENV antibodies does not induce specific pathways (Fig. A4). At 2 hpi, 160 of the
genes were shared between the three conditions (triangle, Fig. 4D). Gene ontology analysis of these 160 shared
genes identified an inflammatory response (Fig. 4E). No specific suppressive pathway was identified in the MOI
1-ADE cluster (Fig. 4E). Thus, at both time points of infection, antibody-mediated infection did not activate
immune-suppressive pathways. Rather, we found high induction of the cytokines IFNβ, TNFα, IL1β, IL6 and little
to no IL10 (Fig. A4B). Moreover, the fraction of interferon (IFN)-regulated genes38 is quite substantial (Table 2),
suggesting that there was an antiviral state in our cultures despite the antibody-dependent infection mechanism. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 8 www.nature.com/scientificreports/ Antibodies enhance fusion while avoiding additional antiviral responses. Microarrays provide a
snap-shot of the expression profile at the mRNA level. To confirm the antiviral state, we next analysed the antivi-
ral activity of the macrophage supernatants described above (Fig. 5A). As different cytokine profiles can confer
the same protection48, we used a viral bio-assay25 combining the IFN-sensitive VSV49 with the IFN-deficient yet
-sensitive Vero WHO cell line50. At 24 hpi, no antiviral activity towards VSV was observed in the supernatants of
macrophages infected at MOI 1 of DENV without antibodies. Yet, following infection at MOI 1-ADE, MOI 5 and
MOI 10, antiviral activity was seen (Fig. 5A). This indicates that antibodies per se do not induce a virus-tolerant
environment by 24 hpi. Results Moreover, antiviral responses appear to increase with the percentage of DENV2-infected
macrophages (Fig. 5A, Table 1 and A1), indicating a dose-dependent response. p
g
( g
)
g
p
p
At 24 hpi, MOI 5 had a comparable number of infected cells, antiviral activity and burst size as MOI 1-ADE. Yet, MOI 5 had a 5-fold higher fusion activity and a 2-fold lower replication efficiency. Moreover, more genes
are potentially IFN-regulated (Table 2). Earlier studies in human cell lines showed that the sensitivity of DENV
to IFN is most pronounced during early stages of the viral life cycle, and over time the virus becomes resist-
ant to the antiviral activity of IFN43. Hence, we hypothesized that a high MOI of DENV2 induces a stronger,
type-I-IFN-mediated response early in infection thereby lowering the replication efficiency. To confirm our
hypothesis, we first attempted to quantify the concentrations of IFNα and IFNβ at 2 hpi in the macrophage super-
natants from the screen by ELISA. The IFNβ concentration was below the limit of detection for all conditions, and
the IFNα concentration fluctuated between the donors and conditions likely due to the very low concentrations
measured (Table A2). (
)
To evaluate if DENV also becomes resistant to IFN in primary macrophages, we added exogenous IFN to
cells at different time points post-infection and assessed virus production. Indeed, and in line with earlier results,
the antiviral activity of IFNα is most pronounced when added early in infection (Fig. 5B). At late time points,
the effect of exogenous IFN diminishes indicating that once infection is established the antiviral effect of IFN is
limited. Importantly, the effect of exogenous IFN was distinct between the conditions tested with the lowest effect
at MOI 5 and the strongest effect at MOI 1. The higher resistance at MOI 5 is indicative for an endogenous IFN
response early in infection, since the added IFN had a lower contribution to the antiviral state already present in
the culture. Interestingly, Fig. 5B also shows that the addition of just one unit of IFNα to the cells at the time of
infection significantly reduces ADE (4-fold reduction in virus production), indicating that ADE is sensitive to
IFN. If, at MOI 5, the early endogenous IFN response reduces virus particle production, then blocking the IFNαβR
prior to infection should lead to an increase in virus production. Discussion Antibody-mediated cell entry of DENV is known to increase the infected cell mass and virus particle production,
but little is known about the underlying mechanisms. Our results show that, in primary macrophages, antibod-
ies enhance DENV infection by promoting fusion (Fig. 3C,E), while no altered uptake of virus particles is seen
(Fig. 3A,B). The higher fusion potential triggers a cascade of events leading to enhanced infection, replication,
translation, and burst size. Furthermore, the presence of enhancing concentrations of antibodies did not trigger
pro- or anti-viral programs early in infection (Fig. 4E). At high MOI, however, increased binding, uptake, fusion
and an increased antiviral response is seen early in infection (Figs 3A–D and 5C).i y
g
Our results show that fusion is the first step within primary macrophages where antibodies have a positive
influence on the life cycle of DENV. Indeed, not only the total extent of virus cell binding but also the over-
all distribution of virus particles among cells was comparable in the absence and in the presence of antibodies
(Fig. 3A,B). The increase in membrane fusion activity does, however, not directly translate into the observed
increase in the burst size. Yet, it is interesting to note that the extent of membrane fusion activity correlates with
virus production (Fig. 3E inset). This is suggestive for a causal relationship between fusion activity and virus pro-
duction in primary macrophages. Further research is required to investigate the downstream effects of ADE more
in-depth. Important steps to elucidate herein are for example the effect on nucleocapsid delivery, nucleocapsid
uncoating and subsequent initiation of protein translation. We propose that the “normal” binding and entry characteristics of ADE is key to the success of ADE in pri-
mary macrophages as this avoids extensive activation of antiviral signalling early in infection (Fig. 5B). Indeed,
blocking IFNαβR-signalling rescued DENV production at MOI 5 but had no effect at MOI 1 or 1-ADE (Fig. 5C). Contrary to MOI 5, ADE displays enhanced fusion activity while maintaining low antiviral responses (Fig. 5C). Collectively, our results suggest that ADE of DENV infection in primary macrophages involves a novel mech-
anism that is tightly balanced between the extents of binding, entry, fusion and the antiviral responses early in
infection (Fig. 6). In contrast to our results in primary macrophages, we did observe enhanced binding of DENV-
immune-complexes in macrophage-like P338D1 cells (Ayala et al., accompanying manuscript). Results Indeed, specific blocking type-I-IFN receptor
signalling significantly enhanced virus production at MOI 5 (Fig. 5C and A5). Interestingly, and in contrast,
infection at MOI 1 or MOI 1-ADE was not enhanced by the IFNαβR antibody, confirming that MOI 1 and MOI
1-ADE do not trigger IFNαβR-signalling during the early stages of infection. This suggests that the efficiency of
DENV2 infection in primary human macrophages is determined by the balance between fusion activity and anti-
viral responses early in infection. Once DENV infection and replication is established, the antiviral response no
longer determines virus production (Fig. 5A(+24 hpi) and 5B(+8 hpi)), likely due to the viral proteins51. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 Discussion The presence of an early IFN response significantly reduces virus replication and production. ADE
is thus based on higher fusion but due to the absence of an early IFN response, it remains unnoticed by the cell
allowing virus replication to higher titres. Figure 6. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus
infection in primary human macrophages. Antibody-dependent infection does not enhance binding or
entry of the virus to the cells. Yet, the membrane fusion potential within the endosomes of the macrophage
is increased. Thanks to the unaltered characteristics of binding and entry, ADE does not trigger endogenous
interferon-responses which thus allows the virus to replicate freely during the early stages of infection. ADE
can be mimicked in terms of the number of infected cells and burst size by infection at high MOI in absence
of antibodies. Yet high MOI leads to more binding, entry, fusion, and as a consequence induction of an IFN
response. The presence of an early IFN response significantly reduces virus replication and production. ADE
is thus based on higher fusion but due to the absence of an early IFN response, it remains unnoticed by the cell
allowing virus replication to higher titres. seems unlikely as the overall distribution of virus particles was comparable in the presence and in the absence of
antibodies (Fig. 3B). ADE could be mimicked in terms of infected cell mass and burst size by infection at high MOI. Furthermore,
strong overlapping transcriptional profiles and antiviral responses were observed late in infection. This sug-
gests that antibodies do not trigger specific intrinsic pathways. Indeed, no antibody-specific anti-inflammatory
program was found in primary human macrophages (Fig. 5D and A4). This in agreement with a recent whole
blood transcriptome analysis52 and in contrast with studies using PBMCs19 or cell lines53. Furthermore, other
studies described a “muted” response54,55, but argued that the actual response could have waned by the time
of sampling54,56. Therefore, the antibody-dependent immune suppression could be specific for the cell type(s)
investigated. g
In monocytes, the anti-inflammatory cytokine IL10 is considered to be one of the driving forces of intrinsic
ADE29,53. In macrophages, however, we and others found low induction of IL10 after ADE (Fig. 5F, refs 25,57),
and only at late time points for conditions with high infection and viral load (Fig. 5F, ref. 58). Hence, IL10 does
not directly influence ADE of DENV infection in macrophages. Discussion In P338D1 cells,
we noted 3.85-fold higher binding/uptake of DENV into the cells and 3.52-fold higher fusion activity, which sug-
gests that ADE in P338D1 cells is facilitated by an increased binding efficiency of the virus-immune-complexes to
the cell. These results suggests that alternative, cell-type-specific, mechanisms exists to facilitate ADE. An alter-
native explanation for our findings in primary macrophages is that virus particles are non-randomly distributed
and cells that support ADE are more permissive than other cells. While this notion deserves further research, it Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 9 www.nature.com/scientificreports/ Figure 6. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus
infection in primary human macrophages. Antibody-dependent infection does not enhance binding or
entry of the virus to the cells. Yet, the membrane fusion potential within the endosomes of the macrophage
is increased. Thanks to the unaltered characteristics of binding and entry, ADE does not trigger endogenous
interferon-responses which thus allows the virus to replicate freely during the early stages of infection. ADE
can be mimicked in terms of the number of infected cells and burst size by infection at high MOI in absence
of antibodies. Yet high MOI leads to more binding, entry, fusion, and as a consequence induction of an IFN
response. The presence of an early IFN response significantly reduces virus replication and production. ADE
is thus based on higher fusion but due to the absence of an early IFN response, it remains unnoticed by the cell
allowing virus replication to higher titres. Figure 6. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus
i f
ti
i
i
h
h
A tib d
d
d
t i f
ti
d
t
h
bi di Figure 6. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus
infection in primary human macrophages. Antibody-dependent infection does not enhance binding or
entry of the virus to the cells. Yet, the membrane fusion potential within the endosomes of the macrophage
is increased. Thanks to the unaltered characteristics of binding and entry, ADE does not trigger endogenous
interferon-responses which thus allows the virus to replicate freely during the early stages of infection. ADE
can be mimicked in terms of the number of infected cells and burst size by infection at high MOI in absence
of antibodies. Yet high MOI leads to more binding, entry, fusion, and as a consequence induction of an IFN
response. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 Discussion In line with this, IL10-primed macrophages
are highly susceptible to DENV infection but do not produce progeny virus59. This manuscript focused only on
primary human macrophages and future research will have to elucidate the role of IL10 in DENV-ADE in other
cell types. Based on our results, we propose that the enhanced burst size, as observed in human macrophages, is
dependent on the effective MOI (fusion extent) and is not antibody-specific. fi
Dengue haemorrhagic fever is characterized by vascular leakage, which on its turn has been linked to the high
inflammatory response in patients (reviewed in: ref. 60) and the action of the DENV NS161,62. Here we show that
antibody-dependent DENV infection of primary human macrophages results in 7-fold higher virus titres, and
subsequently triggers strong inflammatory responses (Figs 5C,F and 6A,25). The link between viral burden and
vascular permeability has been described before24,52,61,62 and our data further strengthens the notion that dengue
haemorrhagic fever might be alleviated by reducing the viral load. Indeed, we show that application of IFNα
suppressed virus production by human macrophages, even under conditions of ADE. An important remaining question is how the antibodies enhanced the fusion efficiency of the internal-
ized particles. There are two options; (i) antibodies directly enhance the fusion potential of the virus, or
(ii) the phagosomal environment of primary macrophages is more favourable for fusion. Although we can-
not disprove the first hypothesis, it seems unlikely as no epitope-specific effects were seen in ADE (data not
shown). Antibody-mediated cell entry is facilitated through interaction of the antibody with the Fc receptor. Due to this interaction, DENV-immune-complexes are internalized via phagocytosis (Ayala et al., accompanying
manuscript). DENV fusion is critically dependent on pH and negatively charged lipids34,63. These factors are Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 10 www.nature.com/scientificreports/ differentially regulated and distributed between distinct organelles63,64, and may explain why enhanced fusion is
seen in primary macrophages. p
y
p
g
In conclusion; antibodies enhance DENV infection of human macrophages by promoting fusion of the virus
particles within endosomes. Our work suggests that the higher effective MOI (fusion, translation, replication)
causes the enhanced burst size, not the interaction of antibodies with the FcR. We show that even modest enhance-
ment of fusion activity initiates a cascade with increasingly aggravating impacts. Importantly, DENV2-ADE does
not suppress antiviral responses, but rather avoids induction hereof. References 11. Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013). . Bhatt, S. et al. The global distribution and burden of dengue. Natu h
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JF and JMS were supported by Dutch Scientific Organization (NWO) VIDI-grant to JMS. Part of the work was
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tracking. J. Virol. 81, 12019–12028 (2007). Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 11 www.nature.com/scientificreports/ Author Contributions J.F., M.A.D.T., J.M.S.: designed experiments, J.F., M.A.D.T., T.E.H., D.P.I.v.d.P.: performed experiments, J.F. and
J.M.S.: wrote the manuscript. All authors analysed results and approved the manuscript. Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 12 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Scientific Reports | 6:29201 | DOI: 10.1038/srep29201 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Flipse, J. et al. Antibody-Dependent Enhancement of Dengue Virus Infection in
Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses. Sci. Rep. 6, 29201; doi:
10.1038/srep29201 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
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Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats
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Development and evaluation of recombinant GRA8
protein for the serodiagnosis of Toxoplasma gondii
infection in goats Development and evaluation of recombinant GRA8
protein for the serodiagnosis of Toxoplasma gondii
infection in goats
Charoonluk Jirapattharasate
(
charoonluk.jir@mahidol.edu
)
Mahidol University
Ruenruetai Udonsom
Faculty of tropical medicine, Mahidol University
Apichai Prachasuphap
Medical Life Science Institute, Department of Medical Sciences
Kodcharad Jongpitisub
Medical Life Science Institute, Department of Medical Sciences
Panadda Dhepakson
Medical Life Science Institute, Department of Medical Sciences
Research article
Keywords: Toxoplasma gondii, GRA8, Serodiagnosis, Goat, Gene synthesis
Posted Date: December 11th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-46170/v3
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at BMC Veterinary Research on January 9th,
2021. See the published version at https://doi.org/10.1186/s12917-020-02719-3. Charoonluk Jirapattharasate
(
charoonluk.jir@mahidol.edu
) Research article Keywords: Toxoplasma gondii, GRA8, Serodiagnosis, Goat, Gene synthesis
Posted Date: December 11th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-46170/v3
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Version of Record: A version of this preprint was published at BMC Veterinary Research on January 9th,
2021. See the published version at https://doi.org/10.1186/s12917-020-02719-3. Page 1/23 Abstract Background The development of sensitive and specific methods for detecting Toxoplasma gondii
infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently,
various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The
production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory
animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in
recombinant protein production. For the first time, we demonstrated the production of the recombinant
protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The
recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein
was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent
assay (iELISA) and the latex agglutination test (LAT). Results Western blotting using known positive serum samples from goats identified a single antigen at
the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were
positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity,
with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using
LAT. Conclusion Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene
and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8
demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera Background Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, and this infection is widespread
in humans and animals, occurring in approximately 25%–30% of the human population [1]. Most people
infected with T. gondii are asymptomatic; however, fatal encephalitis caused by this protozoan can be
observed in immunocompromised patients [2]. Infection in humans and animals, as the intermediate
hosts, occurs mainly through the ingestion of raw or undercooked meat containing viable tissue cysts or
through exposure to soil, food, or water contaminated with oocysts passed in the feces of infected cats or
other felines [3]. Normally, farm animals display no clinical symptoms, although T. gondii infection may
induce abortion, leading to reproductive losses in the livestock industry [4]. Serological methods play a major role in the diagnosis of T. gondii infection in humans and animals [5,6]. Several serological tests have been developed using either live tachyzoites or native soluble antigens;
however, they are expensive, laborious, and nonspecific [7]. Recently, recombinant T. gondii antigens were
identified as good candidates for replacing native antigens because they are easily produced in large
volumes using standardized methods [8,9]. Dense granule antigens (GRAs) of T. gondii are secreted in the Page 2/23 Page 2/23 parasitophorous vacuole (PV), and they are involved in survival and virulence of the parasite [10]. Several
studies demonstrated the diagnostic potential of numerous GRAs such as GRA2 [11], GRA5 [12], GRA6
[13], and GRA7 [14,15]. GRA8 is a 38-kDa praline-rich (24%) protein that is released from PVs shortly after
invasion. GRA8 is a 269-amino acid polypeptide with a terminal signal peptide, three degenerate proline-
rich repeats in the central region, and a potential transmembrane domain near the carboxy-terminal
region [16]. Previous studies used the recombinant GRA8 protein in specific IgM and IgG enzyme-linked
immunosorbent assay (ELISA) in humans [17, 18, 19]. However, little information is available concerning
the use of recombinant GRA8 protein-based ELISA for the serodiagnosis of T. gondii infection in animals. Regarding recombinant protein production in T. gondii, the mRNA of target genes is extracted from live
tachyzoites and recombinant plasmid is transformed to bacteria for protein expression. However, the
transfer of gene sequences between organisms may not be successful, leading to low level protein
expression because of differences in codon usage [20, 21]. Recently, synthetic gene synthesis has been
used to design and create genes without an existing DNA template [22]. Construction of the recombinant TgGRA8 plasmids The 582-bp GRA8 gene was PCR-amplified from synthetic TgGRA8 (Fig.1). The PCR product was purified
and double digested with NdeI and AgeI. The digested product (25 ng/ml) was used for ligation. E. coli
DH5α-competent cells were transformed with recombinant pET-21a vectors and cultured with 2XTY agar
containing ampicillin. Positive colonies were identified by colony PCR. Sequence analysis of the clone
revealed 100% homology with the sequence of recombinant TgGRA8. Background In addition, gene synthesis tools
do not require access to a pathogen, thus preventing the exposure of research staff to harmful living
parasites [23]. In this study, we used a synthesized T. gondii GRA8 gene (designated TgGRA8) as a DNA
template for recombinant protein production. Furthermore, the purified protein was used in specific IgG
indirect ELISA (iELISA) in the diagnosis of T. gondii infection using goat sera. The latex agglutination test
(LAT) was used to validate the detection system in this study. Purification and Western blot analysis of expressed protein Production of recombinant TgGRA8 was optimized by altering various incubation periods, and expression
levels were analyzed by SDS-PAGE as shown in Fig 2. A 27 kDa band was observed in the induced
bacteria. Expression of this protein increased up to two hours after induction and remained constant after
overnight. To confirm the protein expression, the induced bacteria exhibited a protein expression band of
27 kDa in size after purification using anti-FLAG tag affinity resin (Fig. 3A). The purified protein was analyzed by Western blotting using peroxidase-conjugated anti-FLAG tag
antibody (GenScript, USA) diluted 1:1000 in blocking buffer. The result illustrated that the TgGRA8 fusion
protein was specifically recognized by anti-FLAG tag antibody (Fig. 3B). However, the specific band size
was slightly larger than the estimated size of 22.55 kDa (amino acids 24–217 plus the 2X FLAG tag). The Page 3/23 Page 3/23 Page 3/23 concentration of the protein was measured as 1.26 mg/ml by BSA assay (Pierce Biotechnology, Inc.,
USA). The specific reactivity and purity of TgGRA8 was checked using known positive and negative
serum samples from goats. Western blotting revealed that the TgGRA8 fusion protein was recognized by
the known positive serum (Fig. 4). Evaluation of the serodiagnostic potential of recombinant TgGRA8 by indirect ELISA (iELISA) The serodiagnotic potential of recombinant TgGRA8-iELISA was evaluated for its potential utility in
serological testing using known positive (N = 10) and negative (N = 21) goat sera. The cut-off value was
calculated as the average OD450 plus three standard deviations of standard T. gondii-negative control
goat sera. The cut-off value for goats in this study was determined as 0.61 (Fig. 5). Base on recombinant
TgGRA8-iELISA, 15.35% goat sera samples were positive for T. gondii-specific IgG antibodies. Confirmation of TgGRA8 protein The identity of protein expressed and purified recombinant protein was confirmed by mass spectrometry
(MS) analysis. The partial sequence of TgGRA8 in this study shared 98.95-100% identities with database
sequences (XP002369526, KFG46645, RQX68523 and AAD55381). Therefore, we confirmed that our
expressed recombinant protein was T. gondii GRA8. Discussion Recently, recombinant DNA technology and synthetic DNA have played important roles in high-quality
recombinant antigenic protein production for the serological diagnosis of T. gondii infection. Several
recombinant proteins have been produced and applied for the detection of T. gondii infection. These
proteins include rhoptry proteins, matrix proteins, microneme proteins, surface antigens, and GRAs [8]. Among them, the GRA proteins have been considered potential diagnostic antigens and have been used
to differentiate the stages of infection [24]. Generally, the method of recombinant protein production
requires cDNA extracted from live pathogens as a template to amplify target genes. However,
unsuccessful recombinant protein production using natural gene sequences, including no or low
expression, inclusion body formation, and protein inactivity, has been described [25]. To overcome these
problems, we demonstrated the production of recombinant protein from a synthetic TgGRA8 gene and
tested the immunodiagnostic potential of the produced protein via iELISA. Although codon optimization was used to optimize and enhance protein expression in the present study,
we failed to produce the recombinant protein using the full-length TgGRA8 gene. Previous studies
described a transmembrane region of the GRA8 gene encoding amino acids 223–242 using
bioinformatic prediction [16] and reported that the region can affect host cell growth and decrease protein
yield [26]. Therefore, we selected the specific region of the TgGRA8 protein based on the prediction of
transmembrane helices in proteins using an online program (http://www.cbs.dtu.dk/services/TMHMM/). After removing the transmembrane region, the gene fragment encoding amino acids 24–217 was used to
express the protein, and a specific 27-kDa band was observed on SDS-PAGE. Our result was similar to
that of Babaie et al. [18], who designed and expressed a recombinant protein from the GRA8 gene
fragment corresponding to amino acids 23–169. However, a difference in size between the apparent
band and the calculated molecular weight of TgGRA8 (22.55 kDa) was observed in the present study. The
predicted protein encoded by the TgGRA8 gene featured high proline content (54 amino acids). The
presence of excessive proline residues in proteins causes structural rigidity in the primary sequence,
thereby decreasing the electrophoretic mobility [16]. Regarding the yield of TgGRA8, our production
gained lower yields than the method of Babaie and colleague [18]. Therefore, the attempt of using codon-
optimisation DNA is not considered advantageous for recombinant GRA8 expression. Numerous GRA proteins, both single and combinations of proteins, have been applied for the
serodetection of animal toxoplasmosis. Comparison of iELISA and LAT The diagnostic performance of recombinant TgGRA8-iELISA was evaluated with reference to LAT [14]. The sensitivity and specificity of the recombinant protein and kappa values at 95% confidence interval
(95% CI) were calculated. The seropositivity rate of goat samples in LAT was 17.0%. The sensitivity and
specificity of LAT for the recombinant protein were 71.1 and 96.0%, respectively. Substantial agreement
between the two methods was indicated by κ = 0.69 (Table 1). Table 1. Comparison of LAT and TgGRA8 recombinant protein-based iELISA for the detection
IgG antibodies against Toxoplasma gondii infection Table 1. Comparison of LAT and TgGRA8 recombinant protein-based iELISA for the detection
IgG antibodies against Toxoplasma gondii infection Table 1. Comparison of LAT and TgGRA8 recombinant protein-based iELISA for the detection
IgG antibodies against Toxoplasma gondii infection
TgGRA8 iELISA
LAT
Sensitivity
(95% CI)
Specificity
(95% CI)
Kappa value
Positive
Negative
Total
Positive
37
10
47
71.17
96.06
0.69
Negative
15
244
259
(56.72–82.45)
(92.65–97.98)
Total
52
254
306 TgGRA8 iELISA
LAT LAT, latex agglutination test; TgGRA8, T. gondii dense granular antigen 8; iELISA, indirect
enzyme-linked immunosorbent assay; CI, confidence interval LAT, latex agglutination test; TgGRA8, T. gondii dense granular antigen 8; iELISA, indirect
enzyme-linked immunosorbent assay; CI, confidence interval Page 4/23 Discussion In cats, a single GRA7 recombinant protein [27] and a mixture of
recombinant GRA2, GRA6, GRA7, and GRA15 [28] were used to determine the prevalence of T. gondii
infection in China and Japan, respectively. Moreover, recombinant GRA7 protein-based ELISA has been
used in seroprevalence studies of farm animals in Egypt [29] and Thailand [30]. To date, only one study
describing the use of TgGRA8 together with recombinant GRA7 to detect specific IgG antibodies against
the parasite was published in domestic turkeys [31]. The potential utility of recombinant TgGRA8 protein in serodiagnosis was assessed using known positive
and negative goat sera. The result of Western blotting indicated that the protein is a potential marker for Page 5/23 Page 5/23 Page 5/23 detecting T. gondii infection in goats. A previous immunochemical evaluation of TgGRA8 using ELISA
recorded high reactivity for the recombinant protein using human sera [19], in line with the present result. A possible explanation for the high OD in this study could be the unspecific epitopes of this antigen in the
amino-terminal region [32] The infection rate in our study was lower than that of 27.9% in a previous report on goats in Satun
province, Thailand [30]. The difference of the seroprevalence rate may be attributable to the use of
different serological diagnosis techniques (iELISA and LAT) and different sampling areas. The sensitivity
and specificity obtained using recombinant TgGRA8 in this study indicated that the recombinant protein
could be used as an antigen for serological tests of T. gondii. However, the use of recombinant proteins
for the serodetection of animal toxoplasmosis may be affected by the immune system in different animal
species. Therefore, the antibody response in various animals and the epitope structures of recombinant
TgGRA8 should be confirmed. Gene synthesis of TgGRA8 The complete GRA8 coding sequence (accession number: TGME49_054720) was obtained from an
online database (http://ToxoDB.org) [34]. The TgGRA8 sequence consists of 810 nucleotides that encode
a 269-amino acid protein. A signal peptide (SPs) of GRA8 was determined using online program, SignalP
4.1 (http://www.cbs.dtu.dk/services/SignalP/). The results showed that small fragments of amino acids
1-23 were expressed as a signal sequence. Therefore, encoding amino acid 24-269 was constructed and
inserted into a pET-21a vector using NdeI and XhoI as the cloning sites (General Biosystems, USA). Conclusion Our study produced a recombinant protein from a synthetic TgGRA8 gene. The sensitivity and specificity
of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in
goat sera. Commercial gene synthesis is an alternative tool to support recombinant protein expression in
the absence of pathogen access. Expression of TgGRA8 The recombinant TgGRA8 plasmids were transformed into E. coli strain Rosetta (DE3) cells and cultivated
in 2XTY supplemented with 1% glucose and 200 ng/ml ampicillin at 37℃ with shaking at 200 rpm. E. coli carrying recombinant TgGRA8 was measured at an optimal density at 600 nm (OD600) of 0.5 and
induced with isopropyl-β-D-thiogalactopyranoside at a final concentration of 1 mM 20 ℃ for various
incubation periods (2, 4 and overnight) with shaking at 250 rpm. The induced bacteria were harvested via
centrifugation at 4400 × g for 20 min at 4℃, and the bacterial pellet was resuspended in 20 ml of pre-
chilled lysis buffer (150 mM NaCl, 50 mM Tris-HCL [pH 9.5], 1% Triton X-100, 1 mM EDTA [pH 8.0], and 1%
NP 40) and then incubated at 4℃ for 30 min. After incubation, the bacterial cells were lysed via
sonication on ice for 10 min, and 1 ml of 1× Protease Inhibitor Cocktail was added (Promega, USA). TgGRA8 expression was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Construction of recombinant TgGRA8 The potential transmembrane regions (TMs) of TgGRA8 were predicted by online server
(http://www.cbs.dtu.dk/services/TMHMM/). The encoding amino acids 218-269 was transmembrane
region. Therefore, only an antigenic fragment of recombinant TgGRA8 encoding amino acids 24–217
was PCR-amplified (Fig. 6). The primers used for amplification of the sequence by PCR was T7 promoter-
(FW), 5′-TAA TACG ACT CAC TAT AG-3′ (New England Biolabs, UK); and TgGRA8-RW, 5′-AGT acc ggt GGT
GGC GGT TGC CGG CTG-3′. The reverse primer was designed to contain the AgeI restriction site. PCR was
performed using PCR Q5® High-Fidelity DNA Polymerase (New England Biolabs) using the following Page 6/23 program: 98℃ for 1 min, followed by 30 cycles of 98℃ for 10 s, 58℃ for 20 s, and 72 ℃ for 20 s, and
final extension at 72℃ for 2 min. The PCR amplicon was digested using NdeI and AgeI. After digestion, the PCR product was ligated into
the modified pET-21a vector harboring a C-terminal fusion protein linker (GGGS) and 2X FLAG tag
(DYKDDDDKDYKDDDDK) (General Biosystems, USA) and transformed into Escherichia coli DH5α-
competent cells. Ten colonies were selected and expanded in overnight cultures, and DNA was extracted using a QIAprep
Spin Miniprep Kit (Qiagen, Germany). The insert of TgGRA8 in the purified plasmid was sequenced using
a Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and the 3500xL genetic analyzer
(Applied Biosystems). The TgGRA8 sequences were determined using Bioedit version 7.2.5 (Tom Hall Ibis
Biosciences, USA). Protein purification Anti-DYKDDDDK G1 affinity resin (GenScript, USA) was used for protein purification. The debris was
centrifuged at 10,000 × g for 30 min at 4℃, after which the supernatant was transferred to a clean tube. The resin suspension (600 µl) was loaded into an empty gravity flow column (Bio-Rad, USA) and washed
with Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Protein was eluted from the resin using
alkaline elution buffer (0.1 M Tris, 0.5 M NaCl, pH 12.0) and neutralized with 1 M HCl. The protein
concentration was measured using NanoDrop ND-1000 UV/Vis spectrophotometer (Thermo Fisher
Scientific, USA). The eluted fractions were dialyzed using SnakeSkin Dialysis Tubing, 10 kDa cut-off (Thermo Fisher
Scientific) against phosphate-buffered saline (PBS, pH 7.2) at 4℃. The debris formed during dialysis was
removed via centrifugation at 10,000 × g for 5 min at 4℃, and the concentration of the purified Page 7/23 Page 7/23 Page 7/23 recombinant protein was assayed using both SDS-PAGE and a Coomassie protein assay reagent kit using
BSA according to the manufacturer’s protocol (Pierce Biotechnology, Inc., USA). recombinant protein was assayed using both SDS-PAGE and a Coomassie protein assay reagent kit using
BSA according to the manufacturer’s protocol (Pierce Biotechnology, Inc., USA). To confirm the sequence of the recombinant protein, the mass spectrometry (MS) analysis was carried
out by Proteomics Services Center, Faculty of Medical Technology, Mahidol University. Western blotting Five micrograms of recombinant TgGRA8 was resolved by 12% SDS-PAGE and then electrotransferred
(Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA). The membrane was washed three
times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h with constant shaking. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20 (PBS-T)
and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using antibody or
known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory
and incubated at 37℃ for 1 h with constant shaking. The monoclonal antibody (mAb) against Flag-tag
(GenScript, USA) was diluted 1:1000, while polyclonal mouse anti-goat immunoglobulin/HRP (Dako,
Denmark) was diluted 1:2000 in blocking buffer. After incubation, the membrane was washed three times
with PBS-T. The protein band was developed according to peroxidase activity using 3,3′,5,5′-
tetramethylbenzidine (KPL, Gaithersburg, MD, USA). Goat serum samples The process of sample collection was reviewed and approved by the Animal Care and Use Committee of
the Faculty of Veterinary Science, Mahidol University, Thailand (Approval No. MUVS-2018-03-09). A total
of 306 serum samples were obtained from a goat farm in Kanchanaburi province, Thailand. The goats
were restrained by holding the base of the horn and blood was collected from the jugular vein and
immediately transferred into 10 ml vacuum blood tubes without anticoagulant. The animals were not
allowed returned to their cage until complete hemostasis has been achieved. All blood samples were kept
in cooled box with ice pack and sent to the laboratory at Faculty of Veterinary Science, Mahidol
University. The sera were separated after sedimentation of blood cells and stored at −20 °C until
examination. LAT The negative and positive control sera were confirmed using MAST® TOXOREAGENT (Mast Group,
Liverpool, UK). Positive samples were considered when agglutination was observed at a dilution of 1:32
or greater. The negative and positive control sera were confirmed using MAST® TOXOREAGENT (Mast Group,
Liverpool, UK). Positive samples were considered when agglutination was observed at a dilution of 1:32
or greater. Abbreviation ELISA, Enzyme-linked immunosorbent assay; LAT, Latex agglutination test; PBS, Phosphate-buffered
saline; PV, Parasitophorous vacuole Statistical analyses The results of iELISA and LAT were calculated using online software (http://vassarstats.net) to determine
the percentage of agreement, sensitivity, specificity, and the kappa values with 95% confidence intervals. The strength of agreement was graded as fair (κ = 0.21–0.40), moderate (κ = 0.41–0.60), and substantial
(κ =0.61–0.80). Consent for publication Not applicable. Ethics approval and consent to participate The process of sample collection was reviewed and approved by the Animal Care and Use Committee of
the Faculty of Veterinary Science, Mahidol University, Thailand (approval no. MUVS-2018-03-09). The
animals were handled humanely in strict accordance with the requirements of the Animal Ethics
Procedures and Guidelines of Institute of Animals for Scientific purpose Development (IAD), Thailand. Before signing a consent, the owners of the selected farms were informed of the study and provided their
approval for sampling of the goats. All samples collection process was conducted by local authorities
and veterinarians. IgG iELISA Purified recombinant TgGRA8 was diluted at a final concentration of 0.1 µg/ml in coating buffer (50 mM
bicarbonate, pH 9.6) and added to separate wells of the ELISA plates (Nunc, Denmark). The coated plates
were incubated overnight at 4℃. The next day, the plates were washed five times with PBS-T and blocked
with 5% PBS-skimmed milk (PBS-SM) for 1 h at 37°C. After washing with PBS-T, duplicate serum samples
were diluted 1:250 in PBS-SM, and 50 µl of diluted serum were added to each well. The plates were
incubated at 37 °C for 1 h and washed with PBS-T five times. Specific IgG antibody was detected using
horseradish-peroxidase-conjugated anti-goat IgG antibodies (Invitrogen, USA). The conjugate was diluted
1:5000 with PBS, and 50 µl of diluted conjugates were added. After incubation at 37 °C for 1 h, the plates Page 8/23 Page 8/23 were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to
develop the color. After 15 min, the reaction was stopped by adding 50 µl of 0.1 M HCl. OD450 was read
using a microplate reader (model ELx808, Biotex, VT, USA). were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to
develop the color. After 15 min, the reaction was stopped by adding 50 µl of 0.1 M HCl. OD450 was read
using a microplate reader (model ELx808, Biotex, VT, USA). were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to
develop the color. After 15 min, the reaction was stopped by adding 50 µl of 0.1 M HCl. OD450 was read
using a microplate reader (model ELx808, Biotex, VT, USA). were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to
develop the color. After 15 min, the reaction was stopped by adding 50 µl of 0.1 M HCl. OD450 was read
using a microplate reader (model ELx808, Biotex, VT, USA). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author
on reasonable request Competing interests Competing interests Page 9/23 Funding This research is supported by Mahidol University (grant number: A30/5261). The funding body has not
participated in the research design, collection, analysis, interpretation of data and writing the manuscript. A th
'
t ib ti Acknowledgements We thank Dr. Sarawut Taksinoros and Dr. Raweenipa Tohkwankeaw, for their significant contribution
during sample collection, and veterinarians from Kanchanaburi provincial livestock office for their general
support. The authors thanks Dr. Phirom Prompiram for technical assistance. In addition, we would like to
express our appreciation to Asst. Prof. Dr, Onrapak Reamtong for mass spectrometry analysis. The
authors would like to thank Enago (https://www.enago.com) for the professional English language
review. The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Availability of data and materials Not applicable. Authors' contributions CJ conceived the present idea in this paper, artwork, perform data analysis and drafted the first
manuscript. RU, AP, KJ carried out the laboratory work. PD approved the manuscript. All authors read and
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product after digestion with NdeI and AgeI. Figure 1
Screening of the PCR product of the TgGRA8 gene. Lane M, 10
product after digestion with NdeI and AgeI. Figure 1
Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; L
product after digestion with NdeI and AgeI. Figures Page 12/23 Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR
product after digestion with NdeI and AgeI. Figure 1
Screening of the PCR product of the TgGRA8 gene. Lane M, 10
product after digestion with NdeI and AgeI. Figure 1
Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; L
product after digestion with NdeI and AgeI. Figure 1 Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR
product after digestion with NdeI and AgeI. Page 13/23 Figure 1 Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR
product after digestion with NdeI and AgeI. Page 14/23 Figure 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Figure 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expres
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular w
Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgG
and overnight) Figure 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Page 15/23 Figure 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Figure 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized express
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Figure 1 Lane M: protein molecular we
Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr a
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgG
and overnight) Figure 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of
TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 ℃ after induction with 1.0 mM IPTG (4,8 hr and
overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 hr
and overnight) Page 16/23 Figure 3
A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant
Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker
Lane 1, the soluble recombinant TgGRA8 protein was purified using anti-FLAG tag affinity resin. B)
Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1,
the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody. electrophoresis analysis of expression of recombinant
gGRA8) protein. Lane M, protein molecular weight marker;
n was purified using anti-FLAG tag affinity resin. B)
gGRA8. Lane M, protein molecular weight marker; Lane 1,
ed using an anti-FLAG tag antibody. Figure 3 Figure 3 A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant
Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker;
Lane 1, the soluble recombinant TgGRA8 protein was purified using anti-FLAG tag affinity resin. B)
Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1,
the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody. Page 17/23 Page 17/23 Page 17/23 Page 17/23 Figure 3
A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant
Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker
Lane 1, the soluble recombinant TgGRA8 protein was purified using anti-FLAG tag affinity resin. B)
Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1,
the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody. ectrophoresis analysis of expression of recombinant
gGRA8) protein. Lane M, protein molecular weight marker;
was purified using anti-FLAG tag affinity resin. B)
gGRA8. Lane M, protein molecular weight marker; Lane 1,
d using an anti-FLAG tag antibody. Figure 3 A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant
Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker;
Lane 1, the soluble recombinant TgGRA8 protein was purified using anti-FLAG tag affinity resin. B)
Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1,
the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody. Page 18/23 Figure 4
Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Figure 4 Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Page 19/23 Page 19/23 Figure 4
Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Figure 4 Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and
negative goat sera. A) Lane M, protein molecular weight marker; Lane 1, strong reactivity with known
positive serum; B) Lane M, protein molecular weight marker; Lane 1, result for negative serum. Page 20/23 Figure 5 Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense
granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays. Figure 5 Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense
granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays. Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense
granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays. Page 21/23 Figure 6 The complete nucleotide and amino acid sequence of T. gondii GRA8 (accession number:
TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-
residual peptide). Page 22/23 TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-
residual peptide). Figure 6 The complete nucleotide and amino acid sequence of T. gondii GRA8 (accession number:
TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-
residual peptide). TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-
residual peptide). TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-
residual peptide). Page 23/23
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[Corrigendum] Protective effects of exendin‑4 on hypoxia/reoxygenation‑induced injury in H9c2 cells
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Molecular Medicine Reports
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cc-by
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This work is licensed under a Creative Commons
Attribution 4.0 International (CC BY 4.0) License. Molecular Medicine REPORTS 22: 1090, 2020 corrigendum Figure 8. Effects of exendin‑4 on GLUT‑1 and GLUT‑4 translocation in
H/R‑treated H9c2 cells. H9c2 cells were pre‑treated with exendin‑4 (200 nM)
for 30 min prior to H/R (12/4 h). Two inhibitors of p38 mitogen‑activated pro‑
tein kinase, BIRB796 (1 μM) and SB203580 (5 μM), were added to the culture
medium and kept for 10 min before the cells were treated with exendin‑4. GLUT‑1 and GLUT‑4 translocation was investigated by measuring GLUT
expression on the cytomembrane following H/R treatment using western
blot analysis. Relative expression levels of GLUT‑1/GLUT4 were analyzed. Values are expressed as the mean ± standard deviation (n=4). *P<0.05 vs. control group; #P<0.05 vs. H/R group; &P<0.05 vs. exendin‑4 + HR group. H/R, hypoxia/reoxygenation; GLUT, glucose transporter. DOI: 10.3892/mmr.2020.11159 DOI: 10.3892/mmr.2020.11159 Kai Lu, Guanglei Chang, Lin Ye, Peng Zhang,
Yong Li and Dongying Zhang Mol Med Rep 12: 3007‑3016, 2015; DOI: 10.3892/mmr.2015.3682 Following the publication of the above article, the authors
noted that an incorrect version of Fig. 8 had been included. Essentially, the data presented as panel (B) in this figure should
not have been included; there is only one data panel in this
figure. The corrected version of Fig. 8 is shown opposite. Secondly, the authors have realized that, in the main title and
in the running title, exendin‑4 was incorrectly spelt as
‘extendin‑4’. The correct version of the title, as it should have
appeared in this paper, is shown above. Figure 8. Effects of exendin‑4 on GLUT‑1 and GLUT‑4 translocation in
H/R‑treated H9c2 cells. H9c2 cells were pre‑treated with exendin‑4 (200 nM)
for 30 min prior to H/R (12/4 h). Two inhibitors of p38 mitogen‑activated pro‑
tein kinase, BIRB796 (1 μM) and SB203580 (5 μM), were added to the culture
medium and kept for 10 min before the cells were treated with exendin‑4. GLUT‑1 and GLUT‑4 translocation was investigated by measuring GLUT
expression on the cytomembrane following H/R treatment using western
blot analysis. Relative expression levels of GLUT‑1/GLUT4 were analyzed. Values are expressed as the mean ± standard deviation (n=4). *P<0.05 vs. control group; #P<0.05 vs. H/R group; &P<0.05 vs. exendin‑4 + HR group. H/R, hypoxia/reoxygenation; GLUT, glucose transporter. Note that these errors do not alter the interpretation of the
results and conclusions, and all the authors agree to this
corrigendum. The authors apologize to the readership of the
Journal for any confusion these errors have caused.
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Empowering Non-Terrestrial Networks with Artificial Intelligence: A Survey
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cc-by
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This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE Empowering Non-Terrestrial Networks with Artificial
Intelligence: A Survey This paper was downloaded from TechRxiv (https://www.techrxiv.org). CC BY 4.0 SUBMISSION DATE / POSTED DATE
31-05-2023 / 07-06-2023 AMJAD IQBAL1,2, MAU-LUEN THAM1, YI JIE WONG1, ALA’A AL-HABASHNA2, GABRIEL
WAINER2, Yong Xu Zhu3, AND TASOS DAGIUKLAS4
1Department of Electrical and Electronic Engineering, Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Sungai
Long Campus, Selangor 43000, Malaysia. ,
g
,
of Electrical and Electronic Engineering, Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Sungai
us, Selangor 43000, Malaysia. g
g
y
4Cognitive Systems Research Centre, School of Engineering/Computer Science, London South Bank University, 103 Borough Road, London SE1
0AA, UK. ve Systems Research Centre, School of Engineering/Computer Science, London South Bank University, 103 Borough Road, London SE1
K 4Cognitive Systems Research Centre, School of Engineering/Computer Science, London South Bank University, 103 Borough Road, L
0AA, UK. Corresponding authors: Mau-Luen Tham (e-mail: thamml@utar.edu.my). Corresponding authors: Mau-Luen Tham (e-mail: thamml@utar.edu.my). Corresponding authors: Mau-Luen Tham (e-mail: thamml@utar.edu.my). This work was supported in part by the Universiti Tunku Abdul Rahman (UTAR), Malaysia, under UTAR Research Fund (UTARRF)
(IPSR/RMC/UTARRF/2021C1/T05) and in part by the British Council under UK-ASEAN Institutional Links Early Career Researchers Scheme with
project number 913030644 and partially supported by NSERC Canada).” ABSTRACT The fifth generation (5G) of wireless communication technology has revolutionized the way
we connect with each other; enabling faster data transfer rates, lower latency, and higher reliability. However, the demand for even more efficient and ubiquitous connectivity is ever-growing. As such,
researchers have already been exploring the potential of the sixth generation (6G) of wireless
communications. The 6G networks are expected to provide unprecedented connectivity and reliability. An
important component of 6G networks is the use of non-terrestrial networks (NTNs) that have the potential
to extend the coverage of 6G networks to areas where connectivity with traditional terrestrial networks is
not feasible or cost-effective. NTNs include technologies with low earth orbit (LEO) satellites, high-altitude
platforms (HAPs), and unmanned aircraft systems (UASs). However, the deployment and management of
NTNs face numerous technical and operational challenges, such as network planning, resource allocation
(RA), and interference management. Recent advances in artificial intelligence (AI) offer new opportunities
for optimizing the performance of NTNs. By leveraging AI techniques, such as machine learning (ML),
deep learning (DL), and reinforcement learning (RL), network operators can enhance the efficiency,
reliability, and security of NTNs in 6G wireless communications. This survey paper comprehensively
reviews the state-of-the-art research on AI-powered NTNs for 6G wireless communications. It covers key
NTN technologies, AI techniques for network optimization, and recent advances in AI-powered NTNs, as
well as challenges and opportunities for future research. The paper also discusses the potential impact of
advancing AI on the development of 6G networks and beyond. CITATION Iqbal, Amjad (2023). Empowering Non-Terrestrial Networks with Artificial Intelligence: A Survey. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.23269337.v1 10.36227/techrxiv.23269337.v1 10.36227/techrxiv.23269337.v1 Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Digital Object Identifier 10.1109/ACCESS.2017.Doi Number Digital Object Identifier 10.1109/ACCESS.2017.Doi Number AMJAD IQBAL1,2, MAU-LUEN THAM1, YI JIE WONG1, ALA’A AL-HABASHNA2, GABRIEL
WAINER2, Yong Xu Zhu3, AND TASOS DAGIUKLAS4
1Department of Electrical and Electronic Engineering, Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Sungai
Long Campus, Selangor 43000, Malaysia. The findings of this survey paper deliver
valuable insights for researchers, practitioners, and policy-makers in the field of 6G networks and AI. INDEX TERMS Non-Terrestrial Networks (NTNs), Artificial Intelligence (AI), 5G/6G, Unmanned
Aircraft System (UAS), Resource Allocation (RA), Reinforcement Learning (RL), Deep Learning (DL). INDEX TERMS Non-Terrestrial Networks (NTNs), Artificial Intelligence (AI), 5G/6G, Unmanned
Aircraft System (UAS), Resource Allocation (RA), Reinforcement Learning (RL), Deep Learning (DL). I. INTRODUCTION to guarantee ubiquitous 5G-Advanced coverage everywhere
with existing network infrastructures. In times of natural
disasters, connectivity outages are widespread, which can
prove detrimental to critical actions necessary for saving
lives and properties. To address these challenges, research
focus
for
5G-Advanced
and
sixth-generation
(6G)
communication networks is shifting towards non-terrestrial
networks (NTNs), which can include low earth orbit (LEO)
satellites,
high
altitude
platform
stations
(HAPS),
unmanned aircraft systems (UASs), or a combination of The evolution of wireless communication technology has
been rapid in recent years, with mobile network operators
deploying the fifth-generation (5G) technology worldwide. The third Generation Partnership Project (3GPP) has also
begun the standardization of 5G-Advanced, which is
expected to offer higher data rates, lower latency, increased
capacity, and more efficient spectrum utilization than any
of its predecessors [1]-[2]. However, due to limited
coverage areas and geographic constraints, it is challenging 1 1 VOLUME XX, 2017 and [15] explore the potential impact of 6G networks on
various industries, including agriculture, transportation, and
healthcare. The work in [16]-[17] focuses on the technical
aspects of NTNs, such as the different radio access
technologies available, the requirements and challenges of
integrating 6G with existing technologies, and the potential
security threats of NTNs. Overall, these provide valuable
insights into the potential of NTNs for 6G and the various
challenges to consider for their successful implementation. The authors in [18]-[19] provide an overview of the vision,
requirements, and challenges of 6G wireless networks. In
[20]-[21], the authors review the current status and future
directions of 6G wireless networks. Furthermore, the works
in [22] and [23] discuss the key enabling technologies for
6G networks, such as terahertz communication, visible light
communication,
and
wireless
power
transfer. The
applications, technologies, and challenges of 6G wireless
networks are explored in [24]-[25]. The challenges and
opportunities in 6G networks, including ultra-reliable, low-
latency, and massive machine-type communication, are
discussed in [26] and [27]-[28]. A comprehensive 6G
wireless communication survey is presented in [29]-[30]. The concept and standardization of 6G networks, including
new spectrum bands and wireless technologies, are
explained in [31]-[32]. In [33]-[34], the authors explore the
opportunities and challenges of 6G networks, including the
use of satellite communication and energy-efficient design. A comprehensive survey of 6G networks, including new
radio access technologies and security challenges, is
presented in [35] and [36]. I. INTRODUCTION In [37], the authors present a
comprehensive survey of 6G wireless networks, including
new antenna technologies and network slicing. Similarly,
the authors in [38] explain the five facets of the new
wireless generation, along with its research challenges, and
different opportunities for the new wireless generation. these technologies [3]. NTNs can provide uninterrupted
communication and high data transfer rates in remote,
disaster-stricken, and rural areas where terrestrial networks
are not available. Recent studies have shown that NTNs can
offer high availability and low latency, making them an
ideal candidate for 6G communication systems [4],[5]. Despite these advantages, NTNs present significant
challenges related to their deployment and management. One of the primary challenges is how to deploy and manage
efficiently, including the physical and ground-based
infrastructure, such as antennas, base stations (BSs), and
backhaul networks. Furthermore, the dynamic nature of
NTNs, with platforms moving at high speeds and in
different directions, introduces additional challenges related
to
signal
propagation,
interference,
and
handover
management [6]-[7]. Researchers are turning to artificial intelligence (AI) as a
promising solution to address these challenges. AI can help
to optimize the performance of NTNs by analyzing data
from various sources, predicting network behavior, and
adapting to changing conditions [8]. For example, AI can
enable intelligent power management and beamforming to
maximize the use of available resources while minimizing
interference and maintaining the quality of service (QoS)
[9]. Furthermore, AI can provide advanced analytics and
automated decision-making to enhance the efficiency and
reliability of NTNs. The integration of AI in cellular networks is still in its early
stages compared to other fields, primarily due to wireless
networks’ complexity and time-varying nature [10]. The
multi-dimensional topology of the next-generation wireless
networks adds an additional layer of complexity to the
existing communication networks, making it challenging to
solve problems that arise in real networks. Nevertheless, AI
techniques can be exploited to overcome these challenges
and provide efficient solutions. NTNs, being an integral
part of next-generation wireless networks, holds great
potential for the application of AI. However, implementing
these algorithms in real-world environments while ensuring
reliable vertical connectivity between ground and space
networks can present practical challenges. To achieve
optimal performance in future networks, proper AI
solutions must complement theoretical advancements in
communication systems design. The related work just discussed shows a variety of surveys
on NTNs for 6G wireless communications that do not
incorporate the use of AI in their analysis. I. INTRODUCTION As such, this
survey focuses on the potential of AI-powered NTNs for
enhancing 6G wireless communications networks. Through
a comprehensive literature review, summarized in Table 1,
we identify the gaps in the existing literature and suggest
potential areas for future research. Unlike existing survey papers, this survey paper analyzes
AI's utilization in NTNs to enhance 6G wireless
communications networks. Firstly, we discuss the potential
benefits and challenges of integrating AI in NTNs,
including its impact on network performance, reliability,
and security. A. RELATED WORKS AND PAPER CONTRIBUTION The main focus
is on the transition from 5G to B5G and on the advanced features that will be required for 6G. [40]
2022
The role of wireless backhauls in 5G networks and its integration with new technologies like UAV, HAPS, mmWave, mMIMO, and
beamforming are presented. This article focuses mainly on rural connectivity, mobile edge computing, and security issues related to
wireless backhaul in 5G and B5G. [34]
2023
The design of an energy-efficient resource allocation system for NTNs is explained. The main focus is to maximize system energy
efficiency by collaboratively optimizing user equipment association, power control, and UAV deployment. [38]
2023
Researchers are exploring five Facets of 6G to develop next-generation solutions, i.e., next-generation architectures, networking, IoT,
wireless positioning and sensing, and deep learning applications. The main focus is to review promising techniques and architectures,
address vulnerabilities, and advocate for multi-component Pareto optimization for optimal solutions. [41]
2023
Distributed edge learning (EL) techniques and their integration with advanced communication optimization designs for B5G wireless
networks are explored. The main focus is to present the open problems and emerging application opportunities for the B5G network. This work
We discuss the use of AI in NTNs for 6G communication networks. The main focus of the survey is:
1. NTNs role in 6G networks and unique security/privacy concerns. 2. Applicable AI approaches for NTN problems and proper technique selection. 3. AI-enabled NTN research avenues and superiority over traditional methods. 4. AI-based NTN resource allocation case studies and research. 5. Future open issues in AI for NTNs, considering constraints for its maximum potential. Table 1. Summary of the related work on 6G the fundamental aspects of AI techniques used in the
NTNs. As a result, we can select appropriate AI
approaches for dealing with various NTN issues. analyze the use cases for AI-powered NTNs in these
industries, including how they can improve efficiency and
reduce costs. The main contributions of this survey paper
can be summarized as follows: 3) We provide a detailed overview of AI-enabled NTN
research
and
a
motivating
argument
for
their
implementation. We explore the challenges of NTNs,
outline the issue associated with traditional methods,
and provide ideas for the superiority of AI techniques. 1) We provide a brief overview of NTNs, including their
introduction in the context of 6G networks, their role in
enhancing network performance, and their key features
and requirements. A. RELATED WORKS AND PAPER CONTRIBUTION Several studies and surveys have been conducted to explore
the possibility of NTNs for 6G wireless communications. It
is very rare to explore the concept of AI for NTNs. There is
numerous research on network architectures, standards,
regulations, and use cases. For instance, [11]-[12] gives an
overview the use of different NTN, including satellite
communication, aerial drones, and terrestrial devices. The
work in [13] examines the challenges associated with
satellite communication in 6G, including power constraints,
latency issues, and frequency allocation. The work in [14] Additionally, we show different AI techniques that can be
applied to NTNs, such as machine learning (ML), deep
learning (DL), and deep reinforcement learning (DRL). Furthermore, this survey paper also discusses the potential
applications of AI-powered NTNs in various industries,
including healthcare, transportation, and smart cities. We Additionally, we show different AI techniques that can be
applied to NTNs, such as machine learning (ML), deep
learning (DL), and deep reinforcement learning (DRL). Furthermore, this survey paper also discusses the potential
applications of AI-powered NTNs in various industries,
including healthcare, transportation, and smart cities. We 1 VOLUME XX, 2017 Table 1. Summary of the related work on 6G
Ref
Year
Contribution and Main Focus
[22]
2019
A vision for 6G networks in 2030 for superior performance and enabling emerging services and applications is discussed. The focus is to
propose a large-dimensional, autonomous network architecture integrating various networks and advanced technologies. [17]
2020
The security and privacy issues associated with 6G networks are being explored as next-generation solutions due to the limitations of 5G
networks. The main focus is to discuss four key aspects of 6G networks and their associated security and privacy issues. [19]
2020
Vision, technology trends, and challenges for 6G are discussed. The main focus is providing the key enabler of a ubiquitous intelligent
mobile society and suggesting a roadmap for the 6G standards. [21]
2020
Recent advances in 6G wireless systems are discussed. The main focus is to present a taxonomy of key technologies and open research
challenges and propose practical guidelines such as neural networks and blockchain-based secure business models. [31]
2020
A discussion of 6G wireless communication technologies is provided, emphasizing fundamental breakthroughs at the physical layer. The
main focus is to provide an overview of these technologies, including holographic radio, terahertz communication, large, intelligent
surface, and orbital angular momentum. A. RELATED WORKS AND PAPER CONTRIBUTION [32]
2020
The limitations of 5G and the need to develop the 6G wireless system are discussed. The main focus is to provide the vision for 6G and
outline a research agenda for enabling the new services and technologies required. [13]
2021
The potential of UAVs in beyond 5G and 6G wireless networks are discussed. The main focus is to highlight the use of cellular networks,
advanced technologies, machine learning, and non-terrestrial networks to support UAVs in 6G. [23]
2021
A fiber-wireless network architecture is presented based on full spectrum, fully adaptive, and coordinated radio access networks (RANs). The main focus is to offer promising scenarios such as NR-free space optical backhauling and indoor systems via visible light
communication for high-speed data link and VLC-aided positioning systems. [24]
2021
The need for 6G to overcome the limitations of current cellular networks and support high-bandwidth applications are discussed. The
main focus is to provide an overview of system requirements, potential technologies, and recent research progress. [25]
2021
The overviews of reconfigurable intelligent surfaces (RISs) for 6G wireless networks are explained. The main focus is to provide the use
case of RISs to create a favorable propagation channel and improve performance gains. [26]
2021
A comparison of 5G and 6G technologies, including terahertz communication, RIS, and blockchain, are presented. The main focus is to
illustrate how IRS can enhance signal quality by controlling passive reflecting elements and how blockchain can enhance system
security. [16]
2022
The possibility of integrating terrestrial and NTNs is discussed as a means of improving user experience and connecting unconnected
devices. The main focus is identifying the opportunities and challenges for defining and orchestrating a new 3D wireless network
architecture. [28]
2022
The challenges of 5G technology and the potential benefits of 6G technology for edge networks in processing real-time applications are
examined. The focus is on integrating ultra-reliable 6G technology into edge computing networks. [33]
2022
The potential of IoT devices and energy-efficient 6G wireless communication in transforming smart cities into super-smart cities is
conferred. The main focus is to review key technologies and applications, including quantum communication, blockchain, and VLC and
identifies promising trends for using 6G through IoT devices in smart cities. [39]
2022
An overview of 6G mobile networks, including motivations, use cases, requirements, and research projects, are reviewed. A. RELATED WORKS AND PAPER CONTRIBUTION Additionally, we analyze the unique
security and privacy concerns associated with NTNs,
providing valuable insights into their intrinsic nature. 4) We summarize relevant case studies and existing
research used to solve the resource allocation problem
associated with AI in NTNs. 2) We briefly discuss the fAI approaches by explaining the 1 VOLUME XX, 2017 VII. Conclusions
I. Introduction
II. Non-terrestrial
Networks
III. Security
and Privacy in
NTN
IV. AI-techniques
for
NTN
optimization
V. Recent
advances in AI-
power NTN for
6G network
VI. Challenges and
opportunities in ai-
powered NTN for 6G
networks
Related works
and paper
contribution
Paper
Organization
Overview of NTN
Technologies
Role of NTN in 6G
Networks
Opportunities for
Future Research
Key NTN Features
and Requirements
Possible AI-
Applications in NTN
for 6G Networks
Network
Optimization and
Management
Case Studies and
Experimental
Results
Key Research
Directions for AI-
Powered NTN
Challenges and
Open Issues in
Deploying AI-
Power NTN
AI-Driven Network
Planning and
Optimization
AI-Powered reconfigurable
intelligent surface (RIS) with
NTNs VII. Conclusions
I. Introduction
II. Non-terrestrial
Networks
III. Security
and Privacy in
NTN
IV. AI-techniques
for
NTN
optimization
V. Recent
advances in AI-
power NTN for
6G network
VI. Challenges and
opportunities in ai-
powered NTN for 6G
networks
Related works
and paper
contribution
Paper
Organization
Overview of NTN
Technologies
Role of NTN in 6G
Networks
Opportunities for
Future Research
Key NTN Features
and Requirements
Possible AI-
Applications in NTN
for 6G Networks
Network
Optimization and
Management
Case Studies and
Experimental
Results
Key Research
Directions for AI-
Powered NTN
Challenges and
Open Issues in
Deploying AI-
Power NTN
AI-Driven Network
Planning and
Optimization
AI-Powered reconfigurable
intelligent surface (RIS) with
NTNs VI. Challenges and
opportunities in ai-
powered NTN for 6G
networks III. Security
and Privacy in
NTN VII. Conclusions Paper
Organization Key NTN Features
and Requirements AI-Powered reconfigurable
intelligent surface (RIS) with
NTNs Figure 1. Article Structure A. OVERVIEW OF NTNs TECHNOLOGIES 5) We finally summarize the main challenges and
opportunities of using AI for NTNs. In addition, we
provide a set of recommendations for future research
directions in this area. Furthermore, we also identify
potential applications of AI in areas such as network
optimization and resource allocation. I. Low Earth Orbit (LEO) Satellites: LEO satellites are
satellites that orbit the Earth at altitudes between 200 and
2000 kilometers. They have emerged as a promising
platform for providing connectivity to remote and rural
areas. Recent developments in satellite technology have
reduced the cost of launching and operating LEO
satellites, making it a viable option for commercial use. Some specific technologies for LEO include the
International Space Station (ISS), the Hubble Space
Telescope, and the Global Positioning System (GPS)
[42]-[43]. B. PAPER ORGANIZATION This paper is organized as follows. Section II provides an
overview of NTNs and their potential role in 6G
communication systems. Different security and privacy
concerns of NTNs are discussed in Section III. In Section
IV, we introduce an overview of AI techniques that can be
used to optimize the performance of NTNs. Section V
discusses the recent research in AI-powered NTNs for 6G
communication systems. Section VI identifies the key
research directions and challenges in this field, and Section
VII concludes the paper. The structure of the paper can be
shown in Fig. 1. II. High Altitude Platform Stations (HAPS): HAPS are
unmanned platforms that operate in the stratosphere at
altitudes between 17 to 22 kilometers. They have the
potential to provide connectivity to areas that are difficult
to access or where terrestrial infrastructure is not
feasible. HAPS can be used for a range of applications,
including
communication,
surveillance,
and
environmental monitoring. HAPS includes balloons
and airships. Some specific examples might include
Google’s Project Loon, Facebook’s Aquila, and the
Stratobus airship [44]-[45]. II. NON-TERRESTRIAL NETWORKS NTNs have emerged as a promising solution for extending
coverage and improving connectivity in 6G networks. NTNs are a class of wireless networks that operate using
non-earth-based
platforms
to
provide
connectivity
[3],[10],[11]. Unlike terrestrial networks, NTNs can cover
large
areas,
operate
independently
of
terrestrial
infrastructure, and be quickly deployed in areas without
adequate terrestrial infrastructure [8]. In the following, we
present an overview of the existing NTN technologies. III. Unmanned Aircraft Systems (UASs): UASs are aircrafts
that are operated without a pilot on board. UASs can be
quickly deployed to provide connectivity in areas
affected by natural disasters or emergencies. They can be
used
for
a
variety
of
applications,
including
communication,
surveillance,
and
delivery. Some
specific examples that use the UASs are DJI Mavic, the
Parrot Bebop 2, and the Lockheed Martin Indago3 drone
[46], [47],[48],[49]. 9 VOLUME XX, 2017 NTN Features
Latency
Energy
Efficiency
Capacity
Scalability
Flexibility
Cost-
Effective
Reliability
Modulation
Schemes
Global
Coverage
Security and
Privacy
Resilience
Altitude
Support
Link
Reliability
Figure 2. Basic Features of NTN Latency Energy
Efficiency Link
Reliability Altitude
Support Resilience NTN Features Security and
Privacy Global
Coverage Cost-
Effective Modulation
Schemes Figure 2. Basic Features of NTN VOLUME XX, 2017 B. ROLE OF NTNs IN 6G NETWORKS routing
algorithms,
and
interference
management
techniques [50]. Similarly, the use of HAPs in 6G networks
has been investigated, focusing on developing efficient
communication protocols, beamforming techniques, and
energy-efficient power management schemes [51]. The use
of UASs in 6G networks has also been explored, with a
focus on developing aerial base stations and efficient
trajectory planning algorithms [52]. 6G wireless communications networks will require a
variety of new technologies to meet the high data rate, low
latency, and mobility requirements of the future. One
possible solution is using NTNs that offer a viable option to
provide these services cost-effectively and efficiently. They
are expected to play a vital role in enabling the full
potential of 6G networks. The terrestrial network infrastructure, including 5G
networks, has limitations in terms of coverage (e.g.,
remote/rural areas, sea/air, etc.). One of the key roles of
NTNs in 6G networks is to complement the terrestrial
network infrastructure and overcome its limitations. NTNs
can provide broader coverage, higher capacity, and mobility
support in areas where terrestrial networks are unavailable
or impractical. Moreover, NTNs can also serve as a backup
or redundant network in case of network failures or
disasters, ensuring high reliability and availability of
communication services. Overall, the role of NTNs in 6G networks is crucial for
providing ubiquitous, reliable, and high-capacity wireless
communication services. The unique characteristics of
NTNs, such as high altitude, 3D mobility support, and
broad coverage, make them a promising solution for
meeting the requirements of future wireless networks. Ongoing research efforts are expected to further improve
the performance and efficiency of NTNs in 6G networks
and pave the way for the realization of the full potential of
these networks. To date, several research efforts have been undertaken to
explore the potential of NTNs for 6G networks. For
instance, the integration of LEO satellites into 6G
communications networks has been studied extensively,
with a focus on optimizing the satellite constellation design, C. KEY NTNs FEATURES AND REQUIREMENTS This is done by detecting errors, isolating
them, and recovering from them without impacting the
system. Fault tolerance is a key element in network security
and helps prevent unauthorized access or misuse of the
network. It also helps to protect against data loss. Fault Tolerance: Identifying and mitigating faults in the
network is essential for maintaining the network’s security
and privacy. This is done by detecting errors, isolating
them, and recovering from them without impacting the
system. Fault tolerance is a key element in network security
and helps prevent unauthorized access or misuse of the
network. It also helps to protect against data loss. Fault Tolerance: Identifying and mitigating faults in the
network is essential for maintaining the network’s security
and privacy. This is done by detecting errors, isolating
them, and recovering from them without impacting the
system. Fault tolerance is a key element in network security
and helps prevent unauthorized access or misuse of the
network. It also helps to protect against data loss. Trust Management: As the network becomes more
complex, managing trust becomes more challenging, and
the risk of trust-related attacks increases. AI can play a crucial role in mitigating these security and
privacy concerns. AI can be leveraged to provide enhanced
security and privacy features that go beyond traditional
security measures. The following are some of the ways AI
can help resolve these concerns. III. SECURITY AND PRIVACY IN NTN Security and privacy are critical considerations in designing
and implementing NTNs for 6G. The increasing reliance on
advanced technologies in the telecommunications industry
means that network security threats are becoming more
sophisticated and complex. As the next generation of
wireless networks, 6G networks are expected to bring a
revolutionary
transformation
to
the
world
of
communication. In the context of 6G, security and privacy
concerns are magnified due to the large-scale deployment
of networked devices and the increasing complexity of
network architectures. These concerns include data leakage,
identity theft, and unauthorized access to sensitive
information. Developing secure and privacy-preserving
technologies is essential to ensure the secure operation of
6G networks. The following is a list of security and privacy
concerns for the NTNs in 6G networks. Trust Management: As the network becomes more
complex, managing trust becomes more challenging, and
the risk of trust-related attacks increases. AI can play a crucial role in mitigating these security and
privacy concerns. C. KEY NTNs FEATURES AND REQUIREMENTS C. KEY NTNs FEATURES AND REQUIREMENTS
NTNs are critical for ensuring their success in 6G
communication systems. NTNs architecture must be
designed to address key challenges such as 3D mobility link
reliability, latency, energy efficiency, and capacity. The 9 VOLUME XX, 2017 data from unauthorized access becomes more challenging,
especially as it is transmitted across multiple network
segments. NTNs system must be able to support a large number of
devices with high data rates and low latency while also
being scalable, flexible, and cost-effective. One key feature of NTNs is the use of advanced antenna
technologies, such as beamforming, which can enhance
signal strength and reduce interference. Other key features
include the use of multi-frequency bands, efficient power
management
techniques,
and
advanced
modulation
schemes. To ensure security and privacy, NTNs must have
strong authentication, encryption, and access control
mechanisms. Network Congestion: The 6G network’s high-speed
capabilities can cause network congestion, leading to a
higher risk of network attacks. With more devices
connected to the network, the chances of network
congestion increase, creating a larger attack surface. Network Congestion: The 6G network’s high-speed
capabilities can cause network congestion, leading to a
higher risk of network attacks. With more devices
connected to the network, the chances of network
congestion increase, creating a larger attack surface. Malware and Hacking: As 6G networks become more
complex, the risk of malware and hacking increases, putting
the security and privacy of the network at risk. It is
essential to have robust, secure technologies in place, such
as encryption, authentication, and firewalls, to protect the
network. Additionally, users should follow best practices
for online security, such as using strong passwords and
being aware of potential phishing scams. NTNs requirements for 6G communication systems include
their ability to provide ubiquitous connectivity, reliability,
and high data rates with low latency. The system must
support seamless integration with terrestrial networks and
enable global coverage with minimal delay. Additionally,
NTNs must be designed to meet the specific needs of
various applications, such as the Internet of Things (IoT),
smart cities, and connected vehicles. It must be able to
support high-altitude platforms, LEO satellites, and
geostationary orbit satellites. The system should also be
resilient to natural disasters and cyber-attacks. Fig. 2 summarizes the key features and requirements of
NTNs for 6G communication systems. Fault Tolerance: Identifying and mitigating faults in the
network is essential for maintaining the network’s security
and privacy. C. KEY NTNs FEATURES AND REQUIREMENTS AI can be leveraged to provide enhanced
security and privacy features that go beyond traditional
security measures. The following are some of the ways AI
can help resolve these concerns. Threat Detection and Prevention: AI can be used to
detect and prevent network threats in real-time, thereby
minimizing the risk of attacks. Anomaly Detection: AI can be used to identify and flag
unusual behavior on the network, which may be an
indicator of a potential security breach. Predictive Analysis: AI can be leveraged to predict
potential security threats, allowing for proactive measures
to be taken before an attack occurs. Anomaly Detection: AI can be used to identify and flag
unusual behavior on the network, which may be an
indicator of a potential security breach. Predictive Analysis: AI can be leveraged to predict
potential security threats, allowing for proactive measures
to be taken before an attack occurs. Anomaly Detection: AI can be used to identify and flag
unusual behavior on the network, which may be an
indicator of a potential security breach. Anomaly Detection: AI can be used to identify and flag
unusual behavior on the network, which may be an
indicator of a potential security breach. Authentication and Authorization: Authentication and
authorization are critical for securing the network and
ensuring that only authorized devices are allowed to access
the network. To address this challenge, 6G networks must
provide strong authentication mechanisms that can detect
and prevent unauthorized access from malicious actors. Furthermore, authentication and authorization policies must
be updated regularly to prevent any potential security
breaches. Predictive Analysis: AI can be leveraged to predict
potential security threats, allowing for proactive measures
to be taken before an attack occurs. Predictive Analysis: AI can be leveraged to predict
potential security threats, allowing for proactive measures
to be taken before an attack occurs. Identity Management: AI can be used to manage
identities and access control more effectively, ensuring that
only authorized devices and users can access the network. Privacy and Confidentiality: The massive amount of data
that will be generated by 6G networks, coupled with the
increasing use of AI-powered devices, poses a significant
privacy and confidentiality concern. Protecting sensitive Encryption: AI can be used to improve encryption
techniques, making it more challenging for attackers to 9 VOLUME XX, 2017 Figure 3 Artificial Intelligence Architecture Figure 3. Artificial Intelligence Architecture optimization. C. KEY NTNs FEATURES AND REQUIREMENTS First, they require prior knowledge of the
network parameters and environment, which may not be
accurate or up-to-date, especially in dynamic and uncertain
environments. Second, they are not adaptive enough to
handle the changing conditions and demands of the
network, as they rely on fixed and predefined models and
rules. Third, they may not be able to handle the
heterogeneity and complexity of the network, as they
assume a uniform and idealized network topology and
behavior. started exploring new approaches based on AI
techniques, which can overcome these limitations and
provide more accurate and adaptive solutions for NTN
optimization [58]. These AI techniques can learn from the
data and adapt to changing environments. The AI
techniques can be broadly classified into three categories:
supervised learning [59], unsupervised learning [60], and
RL [61]. The basic architecture of AI can be shown in Fig. 3. gain access to sensitive data. Behavioral Analysis: AI can be used to analyze user and
device behavior, enabling the network to detect and prevent
abnormal behavior that may indicate a potential security
threat. In conclusion, the 6G network’s security and privacy
concerns are significant and must be addressed to ensure
the network’s safety and secure operation. AI can provide
enhanced security and privacy features, enabling the
network to mitigate these concerns. IV. AI TECHNIQUES FOR NTNs OPTIMIZATION In addition,
unsupervised learning may suffer from the problem of
overfitting, where the model memorizes the data instead of
learning the underlying structure. The basic structure of
unsupervised learning is seen in Fig. 5. DRL has been able to achieve state-of-the-art performance
in various domains, such as robotics, games, wireless
communication, and natural language processing. These
successes have motivated researchers to explore the use of
DRL for NTN optimization, where the state and action
spaces are often high-dimensional and continuous. By using
DRL, it is possible to learn an optimal policy for resource
allocation [71], [72], [73],[74], routing [75], and scheduling
[76] that operate in dynamic and uncertain conditions in
NTN. RL involves training an agent to interact with an
environment and learn an optimal policy to maximize a
cumulative reward [69]. In NTN optimization, RL can be
used for various tasks, such as beamforming, routing, and
scheduling. For example, a Q-learning algorithm can be
used to find the optimal beamforming angles and power
levels for a given set of antennas and users [70]. Another advantage of DRL is that it can handle the
heterogeneity and complexity of the network, which is
difficult to model using traditional approaches. For
example, in a satellite network, the number and position of
the satellites may change over time, the users may move in
different directions, and the signal quality may vary
depending on the atmospheric conditions. DRL can adapt to
these changes by continuously updating the policy and
value functions based on the new observations, which can
result in more robust and efficient operations. One of the advantages of RL is that it can handle dynamic
environments with large state and action spaces. However,
it is important to note that RL is typically used for discrete
state and action spaces and is not well-suited for problems
with continuous state and action spaces. Additionally, while
RL can handle dynamic environments but may struggle
with large and complex ones. Overall, while RL has shown promising results in a range ,
p
g
g
Figure 5. Unsupervised Learning In summary, DRL offers a powerful approach to solving the
challenges of NTN optimization. It can handle complex and
dynamic environments, high-dimensional and continuous
state and action spaces, and heterogeneity and uncertainty
of the network. As shown in Table 2. IV. AI TECHNIQUES FOR NTNs OPTIMIZATION The optimization process plays an important role in
improving the performance and efficiency of wireless
networks deployed in non-terrestrial environments such as
satellites, drones, and balloons. Optimization can be used to
identify the best settings for the network, such as the power
level, frequency, and type of antenna, as well as the optimal
placement of devices. This can lead to improved coverage,
increased bandwidth, and reduced latency. Additionally,
optimization can save energy by reducing the amount of
power required to operate the network. Supervised learning involves training a model using
labelled data, where the input-output pairs are known in
advance. The model can then be used to make predictions
on new inputs. In NTN optimization, supervised learning
can be used for various tasks, such as channel estimation
[62], interference mitigation [63], and resource allocation
[64]. For example, a deep neural network can be trained to
predict the best channel and power allocation for a given set Numerous approaches were defined for NTN optimization. These traditional methods are based on mathematical
models and simulations that use prior knowledge of the
network parameters and environment to make predictions
and decisions. For example, network planning involves
designing the network architecture, coverage area, and
capacity based on the expected traffic and user
requirements
[53]. Link
budget
analysis
involves
calculating the power budget and signal-to-noise ratio for
each link in the network to ensure reliable and high-quality
communication [54]. Antenna design involves selecting the
antenna’s type, size, and orientation based on the
frequency, gain, and radiation pattern [55]-[56]. Signal
processing involves filtering, equalizing, and modulating
the signal to optimize transmission and reception [57]. Th
di i
l
h
h
b
d f
l Figure 4. Supervised Learning These traditional approaches have been used for a long
time, but they have some limitations when it comes to NTN Figure 4. Supervised Learning VOLUME XX, 2017 Figure 6. Reinforcement Learning of users and resources. The advantage of supervised
learning is that it can produce accurate results with high
precision. Supervised learning requires labelled data, where
the input-output pairs are known in advance. However, in
NTN optimization, it may be difficult to obtain labelled
data, as the network operates in dynamic and uncertain
conditions, and the ground truth may not be available. In
addition, the labelled data may not be representative of the
entire network, leading to biased or inaccurate models. Supervised learning is shown in Fig. 4. IV. AI TECHNIQUES FOR NTNs OPTIMIZATION Figure 6. Reinforcement Learning p
g
g
Unsupervised learning involves training a model using
unlabeled data, where the input-output pairs are not known
in advance. The model can then discover patterns and
structures in the data and group them into clusters or
categories. In NTN optimization, unsupervised learning can
be used for various tasks, such as anomaly detection [65],
network clustering [66], and traffic analysis [67]. For
example, a self-organizing map can be used to cluster the
satellites or drones based on their location and connectivity
[68]. The advantage of unsupervised learning is that it can
find hidden patterns in data that may not be easily visible. Unsupervised learning does not require labelled data, but it
relies on the assumption that the data has some inherent
structure or pattern that can be discovered. However, in
NTN optimization, the network may be too complex or
heterogeneous, and the data may not have clear patterns or
clusters that can be easily identified. In addition,
unsupervised learning may suffer from the problem of
overfitting, where the model memorizes the data instead of
learning the underlying structure. The basic structure of
unsupervised learning is seen in Fig. 5. of domains, it is crucial to consider its applicability to the
specific problem. In the context of NTN optimization, RL of domains, it is crucial to consider its applicability to the
specific problem. In the context of NTN optimization, RL
can be a powerful tool, but it may not always be the best
choice depending on the complexity and nature of the
problem. A basic representation of RL is shown in Fig. 6. DRL is an extension of RL that has been shown to be more
effective in handling complex and high-dimensional
problems. In RL, the policy and value functions are
represented using simple linear or nonlinear models,
whereas, in DRL, deep neural networks are used to
represent these functions. This allows the DRL models to
learn more complex and abstract representations of the state
and action spaces, which can result in better performance
and more efficient exploration of the environment. Unsupervised learning does not require labelled data, but it
relies on the assumption that the data has some inherent
structure or pattern that can be discovered. However, in
NTN optimization, the network may be too complex or
heterogeneous, and the data may not have clear patterns or
clusters that can be easily identified. A. POSSIBLE APPLICATIONS OF AI IN NTNs AI has the potential to revolutionize NTN optimization in
6G networks by enabling faster and more efficient
communication. There are several possible applications of
AI in NTNs for 6G adoption, including Terahertz
communications, Optical wireless communication, IoT, and
remote sensing. AI-powered
network
optimization
and
management
involves the application of AI techniques to improve the
performance,
efficiency,
and
reliability
of
6G
communication networks. These techniques include ML,
DL, and RL, which can be used to learn from network data,
predict network behavior, and make automated decisions. One of the key benefits of AI-powered network
optimization is its ability to handle the complexity and
heterogeneity of modern communication networks. With
the rise of 5G and the advent of 6G, networks are becoming
more diverse and dynamic, with a wide range of devices,
protocols, and services. AI can help manage this
complexity by providing automated monitoring, analysis,
and optimization tools. AI-powered
network
optimization
and
management
involves the application of AI techniques to improve the
performance,
efficiency,
and
reliability
of
6G
communication networks. These techniques include ML,
DL, and RL, which can be used to learn from network data,
predict network behavior, and make automated decisions. One possible application is satellite communication. AI can
be used to optimize communication between satellites,
ground stations, and users. It can also help reduce latency
and interference, thereby improving the system's overall
performance. For instance, AI can be used to optimize the
scheduling and routing of satellite links and predict and
mitigate the effects of atmospheric attenuation and weather
conditions. One of the key benefits of AI-powered network
optimization is its ability to handle the complexity and
heterogeneity of modern communication networks. With
the rise of 5G and the advent of 6G, networks are becoming
more diverse and dynamic, with a wide range of devices,
protocols, and services. AI can help manage this
complexity by providing automated monitoring, analysis,
and optimization tools. Another application is drone-based connectivity. AI can be
used to optimize the coverage and capacity of drone-based
networks, which can be used for various applications, such
as search and rescue, environmental monitoring, and
precision agriculture. For example, AI can be used to
optimize the trajectory and positioning of drones and
manage the handover between drones and ground stations. AI can also be used to optimize the altitude, trajectory, and
communication of balloon-based networks. Table 2. Dynamics of Supervised Learning, Unsupervised Learning, and RL
Issues
Supervised
Learning
Unsupervised
Learning
Reinforcement
Learning
Dynamic and uncertain environment
❌
❌
✔️
Heterogeneity and complexity of the network
❌
✔️
✔️
High-dimensional and continuous state and action spaces
❌
❌
✔️
Anomaly detection
✔️
✔️
❌
Channel estimation
✔️
❌
❌
Interference mitigation
✔️
❌
❌
Resource allocation
✔️
❌
❌
Network clustering
❌
✔️
❌
Traffic analysis
❌
✔️
❌
Beamforming
❌
❌
✔️
Routing
❌
❌
✔️
Scheduling
❌
❌
✔️ Table 2. Dynamics of Supervised Learning, Unsupervised Learning, and RL them can address the dynamic and uncertain environment
of NTN optimization, which is a critical factor for NTNs. them can address the dynamic and uncertain environment
of NTN optimization, which is a critical factor for NTNs. DRL, on the other hand, can address all of the above issues,
as it can learn the optimal policy for complex and dynamic
environments where the state and action spaces are high-
dimensional and continuous. DRL can be used for
beamforming, routing, and scheduling tasks requiring real-
time decision-making and optimization. Therefore, RL,
especially DRL, is preferred for NTN optimization in 6G
networks, where the goal is to provide reliable and high-
speed connectivity to remote and underserved areas. predict and mitigate the effects of atmospheric conditions
and other environmental factors. predict and mitigate the effects of atmospheric conditions
and other environmental factors. Finally, AI can be used to optimize energy consumption,
data processing, and transmission of IoT and remote
sensing devices. This can be particularly important in
limited energy applications, such as remote and off-grid
locations. AI can be used to optimize the routing and
processing of data and predict and mitigate the effects of
interference and other environmental factors. Finally, AI can be used to optimize energy consumption,
data processing, and transmission of IoT and remote
sensing devices. This can be particularly important in
limited energy applications, such as remote and off-grid
locations. AI can be used to optimize the routing and
processing of data and predict and mitigate the effects of
interference and other environmental factors. Table 3 below summarizes the possible applications of AI
in NTNs for 6G networks. IV. AI TECHNIQUES FOR NTNs OPTIMIZATION supervised learning can address some
issues of NTN optimization, such as anomaly detection,
channel estimation, interference mitigation, and resource
allocation. Unsupervised learning can address issues like
network clustering and traffic analysis. However, neither of Figure 5. Unsupervised Learning 9 VOLUME XX, 2017 VOLUME XX, 2017 A. POSSIBLE APPLICATIONS OF AI IN NTNs For example, AI-powered
RIS can intelligently direct signals to avoid obstacles and
optimize coverage, which can be especially useful in deep-
space communications where distance can severely
attenuate signal strength [83]. In summary, AI-powered network optimization and
management have the potential to transform the way we
design, operate, and maintain communication networks. By
providing automated monitoring, analysis, and decision-
making tools, AI can help improve network performance,
efficiency, and reliability and enable new applications and
services. Moreover, AI-powered RIS can enable more efficient use
of the available spectrum, which is a precious resource in
NTNs [84]. By intelligently managing signal strength and
direction, RIS can increase the capacity of the existing
spectrum, reduce the risk of interference, and enable more
reliable data transmission. A. POSSIBLE APPLICATIONS OF AI IN NTNs AI can help
optimize the positioning and deployment of balloons and There are several areas in which AI-powered network
optimization can be applied. One of these is network
planning and design, where AI can be used to optimize the
placement and configuration of network elements such as
antennas, base stations, and routers. This can help to
improve coverage, capacity, and QoS while minimizing
costs and energy consumption. Another area is resource allocation, where AI can be used
to optimize the allocation of network resources, such as 9 VOLUME XX, 2017 Table 3. AI Possible Applications
Application
Description
Satellite
communication
Optimize communication between satellites, ground stations, and users; reduce latency and
interference; optimize scheduling and routing of satellite links. Drone-based
connectivity
Optimize coverage and capacity of drone-based networks; manage handover between drones and
ground stations; enable search and rescue, environmental monitoring, and precision agriculture. Balloon-based
networks
Optimize altitude, trajectory, and communication of balloon-based networks; provide connectivity to
remote and underserved areas; predict and mitigate effects of atmospheric conditions and
environmental factors. IoT and remote
sensing
Optimize energy consumption, data processing, and transmission of IoT and remote sensing devices;
optimize data routing and processing; predict and mitigate effects of interference and environmental
factors. Table 3. AI Possible Applications bandwidth, power, and spectrum. This can help to improve
network efficiency and capacity while minimizing
interference and congestion. and large networks [78]. Recent research has shown that
integrating AI with RIS technology can improve the
performance and reliability of NTNs, such as those used for
satellite communications, deep-space exploration, and
interplanetary networks. These networks face unique
challenges, such as limited bandwidth, high latency, and
harsh environmental conditions that can impede data
transmission and connectivity [79]-[80]. AI can also be used for fault detection and diagnosis,
automatically identifying and diagnosing network faults
and anomalies and providing recommendations for
remedial action. This can help to reduce downtime and
improve network availability and reliability. Finally, AI can be used for network security, automatically
detecting and preventing security threats such as intrusion,
malware, and denial-of-service attacks. This can help to
protect network assets and data and maintain user privacy
and trust. AI-powered RIS can help to overcome some of these
challenges by optimizing the wireless channel in real-time. By leveraging AI algorithms, RIS can adapt to changing
network conditions and dynamically adjust its configuration
to improve signal quality, increase network capacity, and
reduce interference [81]-[82]. VOLUME XX, 2017 V. RECENT ADVANCES IN AI-POWER NTN FOR 6G
NETWORK In recent years, significant progress has been made in
developing AI-powered NTNs. The use of AI in NTNs
provides numerous benefits, such as intelligent network
management,
automatic
network
optimization,
and
predictive maintenance. Researchers have explored various
AI-based approaches, such as DL, DRL, and Convolutional
Neural Networks (CNNs), to improve network performance
in the past few years [58],[87]. These approaches have been
used for various applications such as intelligent network
slicing, load balancing, and resource allocation, enabling
the network to operate optimally in dynamic and
unpredictable environments. DRL and Genetic Algorithms (GA) have also been applied
to improve the network architecture and adapt to changing
environmental conditions, enhancing the network's overall
performance. DRL is an AI technique that involves training
an agent to act in an environment to maximize a cumulative
reward. This approach has been used to optimize network
routing, enabling the network to adapt to changing traffic
patterns and reduce latency. Conversely, GA has been used
to optimize network parameters such as the number of BSs
and the frequency allocation, enabling the network to
operate
optimally
in
dynamic
and
unpredictable
environments. The integration of AI into NTNs has also led to the
development of new and innovative network architectures
that can efficiently handle the vast amounts of data
generated by 6G networks. One such architecture is the AI-
powered Open Radio Access Network (ORAN) [88], which
utilizes AI to optimize network coverage and capacity and
minimize interference and latency. The AI-powered ORAN
also allows for dynamic network configuration, enabling
the network to adapt to changing network conditions in
real-time. Furthermore, researchers have also explored the
use of AI for network security and privacy in 6G networks. AI-based security solutions such as Intrusion Detection
Systems (IDS) and Intrusion Prevention Systems (IPS) can
identify and prevent security threats in real-time, improving
the network's overall security [89]. AI-powered privacy
solutions such as differential privacy can also protect user
data while enabling network operators to collect valuable
data for network optimization and management. In addition,
AI is being used to address energy efficiency challenges in
6G networks [58]. With the increasing demand for high-
speed connectivity, the energy consumption of 6G networks
is expected to increase significantly. However, AI-based
approaches such as energy-efficient resource allocation,
dynamic sleep mode management, and intelligent power
control can help to reduce energy consumption and improve
the overall energy efficiency of the network. C. AI-POWERED RECONFIGURABLE INTELLIGENT
SURFACE WITH NTNs SVMs can be used to classify network traffic based on
various criteria, such as the source of the traffic, the type of
data being transmitted, and the destination of the traffic. This enables service providers to allocate network resources
more efficiently and reduce congestion. On the other hand,
Random Forests can be used to predict network traffic
based on historical data and network behavior, enabling
service providers to allocate network resources more
effectively. network
management,
automatic
optimization,
and
intelligent decision-making. AI-driven network planning
can help ensure optimal resource allocation, reduce the cost
of network deployment, and enhance network performance. With the ability to analyze vast amounts of data from
multiple sources, such as user behavior, network traffic, and
resource utilization, AI can enable efficient network
management and improve users' QoS. Although the integration of AI and RIS technology has
great promise for enhancing the performance and reliability
of NTNs for future advancements in space exploration,
satellite communications, and interplanetary networks,
however, the technical challenges still need to be addressed;
ongoing research and development in this field suggests
that AI-powered RIS will play an increasingly important
role in shaping the future of wireless technology in non-
terrestrial environments. ML algorithms, such as Support Vector Machines (SVMs)
and Random forests, have been utilized to predict network
traffic, optimize routing, and allocate resources effectively. SVMs can be used to classify network traffic based on
various criteria, such as the source of the traffic, the type of
data being transmitted, and the destination of the traffic. This enables service providers to allocate network resources
more efficiently and reduce congestion. On the other hand,
Random Forests can be used to predict network traffic
based on historical data and network behavior, enabling
service providers to allocate network resources more
effectively. C. AI-POWERED RECONFIGURABLE INTELLIGENT
SURFACE WITH NTNs Reconfigurable intelligent surfaces (RIS) is a new
technology that can manipulate electromagnetic waves by
dynamically changing their surface properties [77]. They
consist of a planar array of small, passive, and tunable
elements that can manipulate electromagnetic waves to
improve the performance of wireless networks. By
adjusting
the
phase,
amplitude,
and
direction
of
electromagnetic waves, RIS technology can enhance
wireless networks' coverage, capacity, and quality of
service. One of the key benefits of AI-powered RIS is its ability to
create a more dynamic and adaptive wireless environment. By constantly analyzing and adjusting the wireless channel,
RIS can ensure that the network remains resilient and
reliable, even in the face of equipment failure or
environmental disturbances. Additionally, RIS can be used
to create more secure and resilient NTNs by controlling and
manipulating the propagation of radio waves to prevent
unauthorized access and mitigate the risk of cyber-attacks
[85]. One of the key benefits of AI-powered RIS is its ability to
create a more dynamic and adaptive wireless environment. RIS can be used to optimize the signal strength and quality
of NTN, improving its coverage and capacity. When RIS
technology is combined with NTNs, the resulting system
can improve wireless communication performance even
more. This performance improvement is especially
beneficial in areas with poor network coverage. RIS is also
more cost-effective than other methods of improving NTN
signal strength, making it a great solution for both small AI-powered RIS also has the potential to revolutionize the
field of space exploration. By enabling more advanced and
sophisticated communications systems, RIS can improve
the accuracy and reliability of spacecraft navigation
systems, enabling more precise targeting and maneuvering. Furthermore, RIS can create more efficient and robust data
transmission systems for deep-space exploration, enabling VOLUME XX, 2017 9 more rapid and reliable transmission of scientific data and
images [86]. network
management,
automatic
optimization,
and
intelligent decision-making. AI-driven network planning
can help ensure optimal resource allocation, reduce the cost
of network deployment, and enhance network performance. With the ability to analyze vast amounts of data from
multiple sources, such as user behavior, network traffic, and
resource utilization, AI can enable efficient network
management and improve users' QoS. ML algorithms, such as Support Vector Machines (SVMs)
and Random forests, have been utilized to predict network
traffic, optimize routing, and allocate resources effectively. V. RECENT ADVANCES IN AI-POWER NTN FOR 6G
NETWORK The study
proposed an AI-based approach for the placement of virtual
network functions (VNFs) in the network, which utilized a
DRL algorithm. The results showed that the proposed
approach can significantly improve network performance
and reduce the resource utilization of the network. B. CASE STUDIES AND EXPERIMENTAL RESULTS
With the rapid development of 6G networks, AI-powered
NTNs have emerged as critical area of research to improve
network management, optimization, and performance. Several recent case studies have been conducted to evaluate
the effectiveness of AI-based approaches in 6G networks. One such study focused on using AI in network slicing,
which allows network operators to partition the network
into virtual slices with customized functionalities and
resources. The study demonstrated that the use of AI-based
approaches such as DL and CNNs could significantly
improve network slicing performance, enabling better
resource utilization and enhancing the QoS for end-users
[90]. In a different study [96] the authors proposed an AI-based
approach for network traffic prediction in 6G networks. The
proposed approach utilized a Deep Belief Network (DBN)
algorithm to predict network traffic, which can enable
better resource allocation and network management. The
experimental results showed that the proposed approach can
achieve higher prediction accuracy compared to traditional
approaches. In [97], the authors focused on the use of AI in network
function placement for edge computing in 6G networks. The study proposed an AI-based approach for the
placement of network functions in the network, which
utilized a Q-learning algorithm. The results showed that the
proposed approach could significantly improve network
performance. In [91], the authors focused on using RL to optimize
network resource allocation in dynamic and unpredictable
environments. The study proposed a new approach to
resource allocation, which combined RL with the GA to
enable the network to adapt to changing environmental
conditions. The experimental results demonstrated that the
proposed approach can significantly improve network
performance by optimizing the allocation of resources to
different network functions. In [92], an AI-based approach
for dynamic network slicing is proposed, which allows the
network to adapt to changes in network traffic and user
demands. The proposed approach utilized a Multi-Agent
RL (MARL) algorithm to enable the network to learn and
adapt to changes in network conditions. The results showed
that the proposed approach can significantly improve
network performance by optimizing the allocation of
resources to different network functions. V. RECENT ADVANCES IN AI-POWER NTN FOR 6G
NETWORK AI can analyze data from multiple sources, such as user
behavior, network traffic, and resource utilization to
identify areas where network resources are being
underutilized or overutilized. This can help service
providers optimize their network architecture and allocate
resources more efficiently, reducing the overall cost of
network deployment. Furthermore, AI-driven network
planning can ensure efficient resource allocation and reduce
the cost of network deployment, thereby enabling service
providers to deliver high-quality services while reducing
operational costs. The application of AI in network planning and optimization
is expected to become more widespread with the
advancement of technology and the increasing demand for
high-speed and reliable connectivity in 6G networks. However, there are several challenges that must be
addressed to ensure the effective implementation of AI in
network planning and optimization. These challenges
include the need for standardized data formats, the
integration of AI algorithms with existing network
infrastructure, and the development of ethical guidelines for
the use of AI in network management. A. NETWORK PLANNING AND OPTIMIZATION
The use of AI algorithms in network planning and
optimization offers numerous benefits, including efficient In conclusion, AI-driven network planning and optimizati- 9 VOLUME XX, 2017 -on have emerged as critical research areas in developing
AI-powered NTNs for 6G networks. AI algorithms offer
numerous benefits, such as efficient network management,
automatic optimization, and intelligent decision-making. ML algorithms such as SVMs and Random Forests have
been utilized to predict network traffic and allocate
resources effectively, while DRL and GA have been
applied to improve the network architecture and adapt to
changing environmental conditions. AI-driven network
planning can ensure efficient resource allocation and reduce
the cost of network deployment, thereby enabling service
providers to deliver high-quality services while reducing
operational costs. However, there are several challenges
that
must
be
addressed
to
ensure
the
effective
implementation
of
AI
in
network
planning
and
optimization. In [94], an AI-based approach for energy-efficient resource
allocation in 6G networks is proposed. The proposed
approach utilized a Genetic Algorithm to optimize the
allocation of network resources, considering each network
function's energy consumption. The results demonstrated
that the proposed approach can significantly reduce the
network's energy consumption while maintaining the
desired level of network performance. Another study conducted by [95] focused on the use of AI
in network function virtualization (NFV) to improve
network efficiency and resource utilization. V. RECENT ADVANCES IN AI-POWER NTN FOR 6G
NETWORK The authors in [87] propose a DRL approach to maximize
the worst-case secrecy energy efficiency of a reconfigurable
intelligent surface (RIS)-aided millimeter-wave rotary-wing
unmanned aerial vehicle (UAV) communication system. The simulation results show that the proposed approach
outperforms the traditional method in terms of secrecy
energy savings. The above-explained case studies are
summarized in Table. 4. VI. CHALLENGES
AND
OPPORTUNITIES
IN
AI-
POWERED NTNs FOR 6G NETWORKS VI. CHALLENGES
AND
OPPORTUNITIES
POWERED NTNs FOR 6G NETWORKS The development of the 6G networks is rapidly advancing,
and AI has emerged as a key technology in realizing the
vision of intelligent and efficient 6G networks. AI-powered
network management is expected to provide numerous
benefits,
including
improved
network
performance,
enhanced user experience, and reduced operational costs. However, achieving these benefits comes with significant
challenges and requires overcoming various technical and
practical obstacles. Therefore, this survey paper discusses
the challenges and opportunities in AI-powered network
management for 6G networks. In [93], an AI-based approach for dynamic network slicing
is proposed, allowing the network to adapt to changes in
traffic and user demands. The proposed approach utilized a
MARL algorithm to enable the network to learn and adapt
to changes in network conditions. The results showed that
the proposed approach can achieve 27% better network
performance and utilization compared to traditional 9 VOLUME XX, 2017 VOLUME XX, 2017 Table 4. Case Studies Summary
Case
Study
Approach
Key Findings
[90]
DL and CNN
The AI-based approach can significantly improve network slicing performance,
enabling better resource utilization and enhancing the QoS for end-users
[91]
RL and GA
The proposed approach can significantly improve network performance by
optimizing the allocation of resources to different network functions. [93]
MARL algorithm
The proposed approach can achieve 27% better network performance and
utilization compared to traditional approaches. [94]
GA
The proposed approach can significantly reduce the network's energy
consumption while maintaining the desired level of network performance
[95]
DRL algorithm
The proposed approach can significantly improve network performance and
reduce the resource utilization of the network. [96]
Deep Belief Network
algorithm
The proposed approach can achieve higher prediction accuracy for network
traffic prediction compared to traditional approaches. [97]
Q-learning algorithm
The proposed approach can significantly improve network performance for
network function placement in edge computing. [98]
DRL approach
The proposed approach outperforms the traditional method in terms of secrecy
energy
savings
for
RIS-aided
millimeter-wave
rotary-wing
UAV
communication system. A. KEY RESEARCH DIRECTIONS FOR AI-POWERED
NTNs A. KEY RESEARCH DIRECTIONS FOR AI-POWERED
NTNs This can be used to train AI models for NTNs in a privacy-
preserving and energy-efficient manner. As AI technologies continue to evolve, they offer
tremendous potential to revolutionize the way future 6G
networks are designed, deployed, and managed. AI-
powered NTNs is an emerging research area that aims to
leverage AI techniques to enhance the performance and
efficiency of 6G networks. However, realizing the full
potential of AI-powered NTNs requires identifying key
research directions that can overcome the challenges posed
by the highly dynamic and complex nature of 6G networks. This section discusses some of the most promising research
directions for AI-powered NTNs in 6G networks. Network slicing for AI-powered NTNs: Network slicing
allows multiple logical networks to be created on top of a
shared physical infrastructure. This can help to meet the
diverse requirements of different applications in AI-
powered NTNs, such as low latency, high bandwidth, and
high reliability. Edge intelligence for AI-powered NTNs: Edge computing
can perform AI computations at the network edge, reducing
latency and improving energy efficiency. This includes
using edge devices, such as BSs and user equipment, to
perform AI computations and make real-time decisions. Edge intelligence for AI-powered NTNs: Edge computing
can perform AI computations at the network edge, reducing
latency and improving energy efficiency. This includes
using edge devices, such as BSs and user equipment, to
perform AI computations and make real-time decisions. Multi-objective optimization for AI-powered NTNs: 6G
networks are expected to support diverse applications with
different quality-of-service requirements. Multi-objective
optimization can help balance conflicting objectives, such
as energy efficiency, spectral efficiency, and reliability, in
AI-powered NTNs. Explainable AI for NTNs: The development of
explainable AI can enhance the transparency and
interpretability of NTNs powered by AI. This includes
developing algorithms that can explain how AI models
make decisions and identify potential biases in the data. AI-driven security for NTNs: The use of AI can help to
improve the security of NTNs, including detecting and
mitigating
attacks,
identifying
vulnerabilities,
and
enhancing privacy and data protection. Intelligent network planning and deployment: AI-
powered NTNs can leverage intelligent algorithms to
automate the network planning and deployment process. This includes using ML to identify the optimal network
topology, antenna placement, and resource allocation to
minimize interference and maximize coverage. B. CHALLENGES AND OPEN ISSUES IN DEPLOYING
AI-POWERED NTNs While AI-powered NTNs have significant potential, there
are also several challenges and open issues that must be
addressed. These include but are not limited: Data privacy and security: AI techniques require large
amounts of data to train models, but ensuring data privacy
and security is a significant concern. Organizations must
ensure that sensitive data is protected and not accessible to
unauthorized personnel. Additionally, organizations must
be transparent about the data they are collecting, how it will
be used, and who has access to it. Resource limitations: AI-powered networks may require
significant computational resources, which can be a
challenge
in
resource-constrained
environments. Organizations must ensure that AI-powered networks are
designed to operate within the resource constraints of the
environment. Additionally, organizations must consider AI-
powered networks' power consumption and environmental
impact. Trustworthiness:
AI-powered
networks
must
be
trustworthy
and
reliable,
with
clear
accountability
mechanisms in place to ensure the network functions as
intended. Organizations must ensure that AI-powered
networks are transparent and explainable so that users can
understand how the network is making decisions. Additionally, organizations must be transparent about the
limitations of AI-powered networks so that users
understand when the network may not be appropriate for
certain use cases. Lack
of
standardization:
There
is
a
lack
of
standardization in AI-powered network technologies and
protocols, which can hinder interoperability and scalability. Organizations must work together to develop standards for
AI-powered networks that promote interoperability and
scalability. Bias and fairness: AI-powered networks must address bias
and fairness issues in decision-making, particularly in areas
such as resource allocation and network management. Organizations must ensure that AI-powered networks are
designed to be fair and unbiased and that decisions made by
the network are transparent and explainable. Ethical concerns: AI-powered networks must consider
ethical concerns related to the use of AI, including
transparency, accountability, and potential unintended
consequences. Organizations must ensure that AI-powered
networks are designed to be transparent and accountable
and
that
they
do
not
have
unintended
negative
consequences. Bias and fairness: AI-powered networks must address bias
and fairness issues in decision-making, particularly in areas
such as resource allocation and network management. Organizations must ensure that AI-powered networks are
designed to be fair and unbiased and that decisions made by
the network are transparent and explainable. Integration and interoperability: AI-powered networks
must be able to integrate and interoperate with existing
networks
and
devices,
including
legacy
systems. A. KEY RESEARCH DIRECTIONS FOR AI-POWERED
NTNs AI-driven resource allocation and management: The use
of AI can help to efficiently allocate and manage network
resources, including frequency bands, power levels, and
computing
resources. This
can
improve
network
performance and reduce energy consumption. Federated learning for distributed NTNs: Federated
learning is an ML technique allowing devices to
collaboratively learn a shared model without sharing data Intelligent network planning and deployment: AI-
powered NTNs can leverage intelligent algorithms to
automate the network planning and deployment process. This includes using ML to identify the optimal network
topology, antenna placement, and resource allocation to
minimize interference and maximize coverage. Intelligent network planning and deployment: AI-
powered NTNs can leverage intelligent algorithms to
automate the network planning and deployment process. This includes using ML to identify the optimal network
topology, antenna placement, and resource allocation to
minimize interference and maximize coverage. AI for network optimization and self-healing: AI can be
used to continuously monitor and optimize network
performance and detect and repair faults automatically. This includes using AI to predict and prevent network
failures, optimize routing and traffic flow, and manage
network congestion. AI-driven resource allocation and management: The use
of AI can help to efficiently allocate and manage network
resources, including frequency bands, power levels, and
computing
resources. This
can
improve
network
performance and reduce energy consumption. AI for user experience and behavior analysis: AI can be
used to analyze user behavior and preferences and predict
and personalize services and content. This includes using
AI to optimize user engagement, enhance content delivery,
and improve overall user experience. The key research dir- Federated learning for distributed NTNs: Federated
learning is an ML technique allowing devices to
collaboratively learn a shared model without sharing data. Federated learning for distributed NTNs: Federated
learning is an ML technique allowing devices to
collaboratively learn a shared model without sharing data. 9 VOLUME XX, 2017 Table 5. Key Research Directions Summary
Key Research Directions
Description
Multi-objective optimization for AI-powered
NTNs
Balancing conflicting objectives in network design
Intelligent network planning and deployment
Automating network planning and deployment
AI-driven
resource
allocation
and
management
Efficiently allocating and managing network resources. Federated learning for distributed NTNs
Collaboratively learning a shared model without sharing data. Network slicing for AI-powered NTNs
Creating multiple logical networks on a shared physical infrastructure
Edge intelligence for AI-powered NTNs
Performing AI computations at the network edge
Explainable AI for NTNs
Enhancing the transparency and interpretability of AI models
AI-driven security for NTNs
Improving the security of NTNs
AI for network optimization and self-healing
Continuously monitoring and optimizing network performance. AI for user experience and behavior analysis
Analyzing user behavior and preferences -ections for AI-Powered NTNs are summarized in Table 5. human factors involved in network management, including
user
behavior,
network
administrator
skills,
and
organizational structures. Organizations must ensure that
AI-powered networks are designed to meet the needs of the
users and the organization. This requires understanding the
skills and knowledge of the network administrators and
users and the organizational structure and culture. Resource limitations: AI-powered networks may require
significant computational resources, which can be a
challenge
in
resource-constrained
environments. Organizations must ensure that AI-powered networks are
designed to operate within the resource constraints of the
environment. Additionally, organizations must consider AI-
powered networks' power consumption and environmental
impact. human factors involved in network management, including
user
behavior,
network
administrator
skills,
and
organizational structures. Organizations must ensure that
AI-powered networks are designed to meet the needs of the
users and the organization. This requires understanding the
skills and knowledge of the network administrators and
users and the organizational structure and culture. B. CHALLENGES AND OPEN ISSUES IN DEPLOYING
AI-POWERED NTNs Organizations must ensure that AI-powered networks are
designed to work with existing infrastructure and can
operate
within
existing
networks. Additionally,
organizations must ensure that AI-powered networks can
communicate with other devices and networks using
standard protocols. Ethical concerns: AI-powered networks must consider
ethical concerns related to the use of AI, including
transparency, accountability, and potential unintended
consequences. Organizations must ensure that AI-powered
networks are designed to be transparent and accountable
and
that
they
do
not
have
unintended
negative
consequences. Ethical concerns: AI-powered networks must consider
ethical concerns related to the use of AI, including
transparency, accountability, and potential unintended
consequences. Organizations must ensure that AI-powered
networks are designed to be transparent and accountable
and
that
they
do
not
have
unintended
negative
consequences. Human factors: AI-powered networks must consider the 9 VOLUME XX, 2017 Table 6. Summary of Challenges and Open Issues
Challenge/Open Issue
Description
Data privacy and security
Ensuring data privacy and security is a significant concern when using AI techniques
that require large amounts of data to train models. Trustworthiness
AI-powered networks must be trustworthy and reliable, with clear accountability
mechanisms to ensure the network functions as intended. Integration
and
interoperability
AI-powered networks must be able to integrate and interoperate with existing
networks and devices, including legacy systems. Human factors
AI-powered networks must consider the human factors involved in network
management,
including
user
behavior,
network
administrator
skills,
and
organizational structures. Resource limitations
AI-powered networks may require significant computational resources, which can be
a challenge in resource-constrained environments. Lack of standardization
A lack of standardization in AI-powered network technologies and protocols can
hinder interoperability and scalability. Bias and fairness
AI-powered networks must address bias and fairness issues in decision-making,
particularly in areas such as resource allocation and network management. Ethical concerns
AI-powered networks must consider ethical concerns related to the use of AI,
including transparency, accountability, and potential unintended consequences. Regulatory
and
legal
frameworks
AI-powered networks must comply with regulatory and legal frameworks related to
privacy, security, and other issues. Lack of domain expertise
Developing AI-powered networks requires domain expertise in networking and AI,
which can be challenging to find in a single individual or organization. Table 6. Summary of Challenges and Open Issues Regulatory and legal frameworks: AI-powered networks
must comply with regulatory and legal frameworks related
to privacy, security, and other issues. B. CHALLENGES AND OPEN ISSUES IN DEPLOYING
AI-POWERED NTNs Organizations must
ensure that AI-powered networks are designed to meet
regulatory and legal requirements and that they are
transparent and accountable to regulators and other
stakeholders. of AI-powered network management with emerging
technologies such as edge computing, blockchain, and the
IoT can create intelligent and efficient networks. Research
in this area can lead to the development of new
architectures and protocols that can leverage the benefits of
these emerging technologies. For example, edge computing
can reduce latency and improve network response time,
while blockchain can provide secure and decentralized
network management. Lack of domain expertise: Developing AI-powered
networks requires domain expertise in networking and AI,
which can be challenging to find in a single individual or
organization. Organizations must ensure that they have
access to the necessary expertise to develop and deploy AI-
powered networks and that they are able to collaborate with
other organizations to share expertise and resources. We
have summarized these challenges and open issues in Table
6 for better and easier understanding. Adoption of new network architecture: AI-powered
NTNs for 6G networks requires adopting new network
architectures
optimized
for
AI-powered
network
management. Research in this area can lead to the
development of new architectures and protocols that can
provide better scalability, reliability, and performance in
network management. For example, software-defined
networks (SDNs) and network function virtualization
(NFV) can provide greater flexibility and programmability
in network management, which AI-powered techniques can
leverage. C. OPPORTUNITIES FOR FUTURE RESEARCH C. OPPORTUNITIES FOR FUTURE RESEARCH
Despite the challenges and open issues, AI-powered NTNs
for 6G networks present numerous future research
opportunities. In the following, we list such opportunities:
Development of new AI techniques: AI-powered NTNs
for 6G networks require the development of new AI
techniques that can address the unique challenges of 6G
networks. These challenges include the need for real-time
processing, large-scale data analysis, and dynamic network
management. Research in this area can lead to the
development of new algorithms and models that can
provide better performance, accuracy, and efficiency in
network management. Exploration of new applications: AI-powered NTNs for
6G networks can enable a wide range of new applications,
such as immersive virtual reality, connected vehicles, and
smart cities. Research in this area can lead to the
development of new use cases and applications that can
leverage the benefits of AI-powered network management. For example, AI-powered network management can
optimize network performance and resource allocation for
virtual reality applications or enable intelligent traffic
management for connected vehicles. Standardization: This is an important area for future Integration with emerging technologies: The integration 9 VOLUME XX, 2017 research
in
AI-powered
NTNs
for
6G
networks. Standardization involves the development of consistent and
uniform technology and protocols across the industry. This
is important to ensure the interoperability and scalability of
AI-powered network technologies. In other words,
standardization ensures that different AI-powered network
management systems can work together seamlessly and that
they can be easily scaled up or down depending on the
network size and complexity. To achieve standardization,
researchers can work on developing new standards and
protocols that can facilitate the adoption and deployment of
AI-powered NTNs for 6G networks. This can involve
creating standardized communication protocols, data
formats, and interfaces that can be used across different
network management systems. Researchers can also work
with industry stakeholders and regulatory bodies to create
industry-wide standards and guidelines that can promote the
development and adoption of AI-powered NTNs for 6G
networks. By achieving standardization, AI-powered NTNs
for 6G networks can become more efficient, effective, and
reliable, ultimately leading to a better user experience and
improved network performance. In conclusion, AI-powered NTNs for 6G networks present
significant challenges and opportunities. Addressing the
challenges and leveraging the opportunities will require
significant research and development efforts, but the
potential benefits of intelligent and efficient networks make
are worthy of these efforts. VII. CONCLUSIONS In his paper, we provided a comprehensive survey of AI-
powered NTNs for 6G wireless communications networks. We first discussed the background and motivation for using
NTNs in 6G networks and their deployment and
management challenges. We then provide an overview of
the key features and requirements of NTNs, including the
role of LEO satellites, HAPS, and UAVs in 6G networks. Afterwards, we discussed various AI techniques for
optimizing NTNs, including ML, DL, RL and DRL, and
highlighted recent advances in AI-powered NTNs for 6G
networks. We also identified key research directions,
challenges, and opportunities in AI-powered NTNs for 6G
networks. Based on our survey, we conclude that AI-powered NTNs
can provide significant benefits in terms of network
management, optimization, and performance. Recent
advances in AI-based approaches have shown that
exploiting AI techniques could help the network to operate
optimally in dynamic and unpredictable environments. AI-
driven network planning and optimization have also
emerged as key areas of research, enabling the network to
adapt to changing environmental conditions and improve
network performance. Experimental results from various
case studies have demonstrated the effectiveness of AI-
based approaches in improving network performance and
efficiency. However, deploying AI-powered NTNs in 6G
networks poses several challenges, including the need for
massive
data
processing
and
storage,
reliable
communication links, and security and privacy concerns. Sustainability: AI-powered NTNs for 6G networks must
also consider the sustainability of network management,
including the use of renewable energy sources, the
reduction of energy consumption, and the optimization of
network resources. Researchers can work on developing
new techniques and protocols that can ensure the
sustainability of AI-powered network management. Explainability and transparency: AI-powered network
management should be explainable and transparent to
ensure trust and accountability. Researchers can work on
developing new techniques and models that can provide
insights into how AI-powered network management makes
decisions and provide explanations for these decisions. Resilience: AI-powered NTNs for 6G networks must be
resilient to cyber-attacks and other security threats. Researchers can work on developing new techniques and
protocols that can ensure the resilience of AI-powered
network management and protect against these threats. In summary, our survey provides a clear vision for the
potential of AI-powered NTNs for 6G networks and
highlights the need for further research and development in
this field. [1]
X. Lin, “An Overview of 5G Advanced Evolution in 3GPP
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83, 2022, doi: 10.1109/MCOMSTD.0001.2200001. VII. CONCLUSIONS We believe that our survey can serve as a
valuable reference for industry and academic researchers
interested in deploying and managing AI-powered NTNs
for 6G networks. Collaboration and partnerships: AI-powered NTNs for
6G networks require collaboration and partnerships
between academia, industry, and government to address the
complex challenges involved in network management. Research in this area can lead to the development of new
collaborative models and frameworks that can facilitate
sharing of knowledge, resources, and expertise. C S
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from the University of Engineering and
Technology (UET), Peshawar, in 2017, and the
Ph.D. degree in Wireless Communication from
University Tunku Abdul Rahman (UTAR),
Malaysia, in 2022. He is currently a Postdoc
research fellow in the Department of Systems
and
Computer
Engineering
at
Carleton
University, Canada. His research interests
include resource allocation optimization, machine learning, deep
reinforcement learning, 5G/B5G cellular networks, and Reflecting
Intelligent Surface (RIS). [89] [89]
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2020, doi: 10.1109/TWC.2020.3003719. THAM MAU LUEN received the Bachelor of
Engineering and Doctor of Philosophy degrees in
telecommunication
engineering
from
the
University of Malaya. He is currently an
Assistant Professor with Universiti Tunku Abdul
Rahman. He has been a principal investigator (PI)
and a co-investigator of more than 15 research
and development projects. This includes five
international
grants,
two
of
which
are
simultaneously led by him as the PI/Co-PI under
the support of ASEAN IVO and the British Council. He has published two
IEEE Transactions papers as a principal author. His research interests
include the IoT, machine learning/deep learning/deep reinforcement
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Function Virtualization in C-RAN,” IEEE Trans. Cogn. Commun. Netw., vol. 6, no. 2, pp. 428–435, 2020, doi:
10.1109/TCCN.2020.2982886. YI JIE WONG received the B.Eng. degree (Hons.)
in biomedical engineering from the Universiti
Tunku Abdul Rahman, Sungai Long, Malaysia,
in 2022, where he is currently pursuing the Ph.D. degree in digital technology with specialization
of reinforcement learning-based federated learning. His research interests include the Internet of
Things (IoT), machine learning, federated learning,
deep reinforcement. YI JIE WONG received the B.Eng. degree (Hons.)
in biomedical engineering from the Universiti
Tunku Abdul Rahman, Sungai Long, Malaysia,
in 2022, where he is currently pursuing the Ph.D. degree in digital technology with specialization
of reinforcement learning-based federated learning. His research interests include the Internet of
Things (IoT), machine learning, federated learning,
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Y. Fu and X. Wang, “Traffic Prediction-Enabled Energy-
Efficient Dynamic Computing Resource Allocation in CRAN
Based on Deep Learning,” IEEE Open J. Commun. Soc., vol. 3,
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pp. 159–175,
2022,
doi: Y. Fu and X. Wang, “Traffic Prediction-Enabled Energy-
Efficient Dynamic Computing Resource Allocation in CRAN
Based on Deep Learning,” IEEE Open J. Commun. Soc., vol. 3, no. February,
pp. 159–175,
2022,
doi: 9 VOLUME XX, 2017 TASOS
DAGIUKLAS
received
the
engineering degree from the University of
Patras, Greece, in 1989, the M.Sc. degree
from the University of Manchester, U.K., in
1991, and the Ph.D. degree from the
University of Essex, U.K., in 1995, all in
electrical engineering. He is a Leading
Researcher and Expert in the fields of smart
internet technologies. He is the Leader of the
Smart Internet Technologies (SuITE) Research Group, London South
Bank University, where he also acts as the Head of the Cognitive Systems
Research Centre. He has been a principal investigator, a co-investigator,
the project and technical manager, a coordinator, and a focal person of
more than 25 internationally research and development and capacity
training projects in the areas of fixed- mobile convergence, 4G/5G
networking technologies, VoIP, and multimedia networking. His research
interests lie in the field of systems beyond 5G/6G networking
technologies, programmable networks, UAVs, V2X communications, and
cyber security for smart internet systems. TASOS
DAGIUKLAS ALA’A AL-HABSHANA received his Master of
Engineering degree from Memorial University of
Newfoundland in 2010, and his PhD in Electrical
and
Computer
Engineering
from
Carleton
University in 2018. Currently, Dr. Al-Habashna is
an Adjunct Research Professor at Carleton
University, Ottawa, Canada. Dr. Al-Habashna is
also a Senior Researcher at Statistics Canada,
Ottawa, Canada. He has won multiple awards
including excellence and best-paper awards. He
worked as a reviewer for many conferences and journals and as a
Technical Program Committee Member of multiple conferences. He has
also been the co-chair of the Communications and Networking Simulation
(CNS) track of the Annual Modeling and Simulation Conference. His
current research interests include 5G wireless networks, machine learning,
computer
vision,
IoT
applications,
localization,
multimedia
communication over wireless networks, signal detection and classification,
cognitive radio systems, and discrete-event modeling and simulation. GABRIEL WAINER received the PhD degree
(Highest Hons.) from UBA/Université d'Aix-
Marseille III, Marseille, France, in 1998. He held
visiting positions with the University of Arizona;
LSIS
(CNRS),
Université
Paul
Cézanne,
University of Nice, INRIA Sophia-Antipolis,
Université de Bordeaux (France); UCM, UPC
(Spain), University of Buenos Aires, National
University of Rosario (Argentina) and others. He
is currently a full professor with Carleton
University, Ottawa, ON, Canada, where he is also the head with the
Advanced Real-Time Simulation Laboratory, Centre for advanced
Simulation and Visualization (V-Sim). He is Editor in Chief of
SIMULATION (Sage) and a member of the editorial board of the IEEE
Computing in Science & Engineering, Wireless Networks (Elsevier), and
The Journal of Defense Modeling and Simulation (SCS). He is a fellow of
the Society for Modeling and Simulation International (SCS). YONGXU ZHU received the Ph.D. degree in
electrical engineering from University College
London in 2017. From 2017 to 2019, she was a
Research
Associate
with
Loughborough
University. She is currently a Senior Lecturer
with the Division of Computer Science and
Informatics, London South Bank University. Her
research interests include B5G/6G, heterogeneous
networks, UAV communications, and physical-layer security. She also
serves as an Editor for IEEE Wireless Communications Letters and IEEE
Transactions on Wireless Communications. VOLUME XX, 2017
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Underpinning Rural Bumiputera Mompreneurs' Work-Life Balance Framework Towards Sustainable Entrepreneurship
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To Cite this Article: Hazudin, S. F., Sabri, M. F., Sharif, T. I. S. T., Aziz, N. N. A., & Ridzuan, M. R. (2021).
Underpinning Rural Bumiputera Mompreneurs’ Work-Life Balance Framework Towards Sustainable
Entrepreneurship. International Journal of Academic Research in Business and Social Sciences, 11(19), 169–
179. International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS
Underpinning Rural Bumiputera Mompreneurs' Work-Life
Balance Framework Towards Sustainable Entrepreneurship
Siti Fahazarina Hazudin, Mohamad Fazli Sabri, Tengku Intan Suzila Tengku
Sharif, Nurul Nadia Abd Aziz, Mohd Rozaimy Ridzuan
To Link this Article: http://dx.doi.org/10.6007/IJARBSS/v11-i19/11725 DOI:10.6007/IJARBSS/v11-i19/11725
Received: 18 October 2021, Revised: 20 November 2021, Accepted: 30 November 2021
Published Online: 11 December 2021 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS
Underpinning Rural Bumiputera Mompreneurs' Work-Life
Balance Framework Towards Sustainable Entrepreneurship
Siti Fahazarina Hazudin, Mohamad Fazli Sabri, Tengku Intan Suzila Tengku
Sharif, Nurul Nadia Abd Aziz, Mohd Rozaimy Ridzuan
To Link this Article: http://dx.doi.org/10.6007/IJARBSS/v11-i19/11725 DOI:10.6007/IJARBSS/v11-i19/11725
Received: 18 October 2021, Revised: 20 November 2021, Accepted: 30 November 2021
Published Online: 11 December 2021 Interna
Vol. 11, No Underpinning Rural Bumiputera Mompreneurs' Work-Life
Balance Framework Towards Sustainable Entrepreneurship Siti Fahazarina Hazudin, Mohamad Fazli Sabri, Tengku Intan Suzila Tengku
Sharif, Nurul Nadia Abd Aziz, Mohd Rozaimy Ridzuan
To Link this Article: http://dx.doi.org/10.6007/IJARBSS/v11-i19/11725 DOI:10.6007/IJARBSS/v11-i19/11725
Received: 18 October 2021, Revised: 20 November 2021, Accepted: 30 November 2021 Published Online: 11 December 2021 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS Underpinning Rural Bumiputera Mompreneurs' Work-Life
Balance Framework Towards Sustainable Entrepreneurship Abstract This paper aims to underpin the Bumiputera women entrepreneurs' (BWEs) sustainable work-
life balance framework based on business-family relationships. Aiming to propel holistic
success among BWEs in rural society, the study focuses on marital and parenthood balances. The Conversion of Resource Theory (COR) has been adopted to identify the factors
contributing to the level of women’s entrepreneurship activities in the context of family
relationships from the past empirical studies. This study provides a basis for the starting point
in filling the existing gap by discovering BWEs and their families' views on factors that can
cause work-life imbalances within a business family relationship. The study's baseline should
be directed towards meeting the inclusive socio-economics agenda for an equitable share of
the economic resources of disadvantaged minority women and families. The life balances and
support needed to be addressed by policymakers so that the economic potential of this group
can be fully utilised especially when entrepreneurship is considered a reliable economic
weapon to combat poverty for the rural society. Keywords:
Family
Relationships,
Women
Entrepreneurs,
Work-Life
Balance,
Entrepreneurship Activities Siti Fahazarina Hazudin2,3, Mohamad Fazli Sabri1,2, Tengku
Intan Suzila Tengku Sharif4, Nurul Nadia Abd Aziz5, Mohd
Rozaimy Ridzuan6 1Institute for Social Science Studies, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, 2Department of Resource Management & Consumer Studies, Faculty of Human
Ecology, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, 3Faculty of Business and
Management, Universiti Teknologi MARA Pahang, 26400 Bandar Jengka, Pahang, 4Academy
of Language Studies, Universiti Teknologi MARA Pahang, 26400 Bandar Jengka, Pahang,
5Faculty of Business and Management, Universiti Teknologi MARA Pahang, 27600 Raub,
Pahang, 5Faculty of Science Administration and Policy Studies, Universiti Teknologi MARA
Pahang, 27600 Raub, Pahang, Malaysia
Email: fazli@upm.edu.my Copyright: © 2021 The Author(s) Published by Human Resource Management Academic Research Society (www.hrmars.com)
This article is published under the Creative Commons Attribution (CC BY 4.0) license. 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 license may be seen
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Special Issue Title: Youth and Community Wellness, 2021, Pg. 169 - 179
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http://hrmars.com/index.php/pages/detail/publication-ethics 169 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS
Underpinning Rural Bumiputera Mompreneurs'
Work-Life Balance Framework Towards
Sustainable Entrepreneurship
Siti Fahazarina Hazudin2,3, Mohamad Fazli Sabri1,2, Tengku
Intan Suzila Tengku Sharif4, Nurul Nadia Abd Aziz5, Mohd
Rozaimy Ridzuan6
1Institute for Social Science Studies, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, 2Department of Resource Management & Consumer Studies, Faculty of Human
Ecology, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, 3Faculty of Business and
Management, Universiti Teknologi MARA Pahang, 26400 Bandar Jengka, Pahang, 4Academy
of Language Studies, Universiti Teknologi MARA Pahang, 26400 Bandar Jengka, Pahang,
5Faculty of Business and Management, Universiti Teknologi MARA Pahang, 27600 Raub,
Pahang, 5Faculty of Science Administration and Policy Studies, Universiti Teknologi MARA
Pahang, 27600 Raub, Pahang, Malaysia
Email: fazli@upm.edu.my Underpinning Rural Bumiputera Mompreneurs'
Work-Life Balance Framework Towards
Sustainable Entrepreneurship Underpinning Rural Bumiputera Mompreneurs
Work-Life Balance Framework Towards
Sustainable Entrepreneurship International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS without acknowledging challenges and demands on Bumiputera Women Entrepreneurs
(BWEs) self-family relationships, thus shattering the BWEs entrepreneurial sustainability and
growth prospect. Many perennial issues and challenges grounded women entrepreneurs
from dominating entrepreneurial activities (Filzah et al., 2019). The stresses and ability to
contain strain shall positively and negatively affect the well-being of family members,
relationships, and happiness. As families begin to understand the work demands of BWEs,
family business lifestyles will be built (Abdullahi and Zainol, 2016). Women entrepreneurs in rural areas have reportedly had long-standing problems due
to a lack of institutional support, economic and social resources, which are evidence of
insufficient protective factors in dealing with vulnerable events (Roberts et al., 2017). Apart
from being homemakers, women in Malaysia have a vital role in Malaysia's economy, yet
many women entrepreneurs are still reviving their businesses, and some are still suffering
due to the pandemic (Adib, 2020). Women-owned businesses in rural society, especially
Federal Land Development Authority (FELDA) for domestic eco-sustainability is significant to
support the family income. FELDA BWEs create many homogenous problems that demand
attention. Not only FELDA participants are ranked poor, FELDA venture has been at loss since
2013 led by an unsustainable agriculturally based economy (Ooi, 2019) where agriculturally
based products may suffer price fluctuation tremendously. European boycott on palm oil exaggerates the problems (Klepper, 2018) along with
agricultural mismanagement and income lobbying (Che Wan, 2017) have driven BWEs to
route their resources to business for life sustenance, driven by presently low returns earned
in agriculturally based income, and a high number of loaners in FELDA co-operation scheme
with low pay-ability prospect among FELDA settlers force BWEs to act. Prominent poverty and
unruly income seeking behaviour may accelerate social issues involving FELDA families,
especially children. This chain of reaction is prosperously affecting the government on a larger
scale (Selamat et al., 2015). The lack of a reliable picture in understanding women entrepreneurial phenomena in
family relationships and family well-being results in a wastage of attention given by
policymakers, NGOs, and government bodies on this important topic. This makes crucial
interventions difficult to assist the BWEs successfully. International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS The women entrepreneurship sector is
highly valued in the country as continuous big spending from national budget allocation
clearly shows the significant role women entrepreneurs play in the economy, especially in
tackling issues of poverty and life-work balance for women and families. There are several questions concerning BWEs, such as (1) how would BWEs describe the
factors that cause work-life imbalances in business-family relationships? (2) How do their
family describe the factors that cause work-life imbalances in business-family relationships? And (3) what are the elements of BWEs' sustainable work-life balance model based on
business-family relationships? Therefore, this study aims to explore BWEs' and BWEs family
members' views on factors that can cause work-life imbalances within the context of business
family relationships. In addition, this study also aims to develop BWEs' sustainable work-life
balance model based on business-family relationships. Introduction Existing government policies may not capture all of the impediments to women's
business growth, suggesting that role, growth, and space issues are vital (Selamat et al., 2015). Narrowing the problem on the role of women - family work life balance may not be achieved 170 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS economics since their population is equal to men (Kanapathipillai and Azam, 2019). The
rampant policy executions towards harnessing women in entrepreneurial activities are
evident for their strong presence needed in the economic development plan throughout the
world. Empowering women in the economy through entrepreneurship is a way up to tighten
gender gaps and is key to achieving the 2030 Agenda for Sustainable Development Goals. Despite their undeniable roles in helping society generate employment and domestic
income, women entrepreneurs struggle a lot in juggling the work-life balance between family
and businesses. Consequently, equal opportunity between men and women to excel from the
perspective of entrepreneurship is still rhetoric rather than a reality. Worsen, cultural bias
discriminating against women's possibility to become successful entrepreneurs if not properly
managed may lead to business market failure. Likewise, if they do not function according to
their expected traditional roles, the price for such sacrifice to succeed in business could only
be culturally shameful. Policy makers need to address these market and cultural biases so
that the economic potentially induced of this group can be fully utilised, especially when
entrepreneurship is considered a reliable economic weapon to combat poverty for rural
society. The COVID-19 pandemic is particularly hard-hit on all yet, even more, the already
marginalised women entrepreneurs. The pandemic caused the jobs and livelihoods of many
sections of society at risk, a lower average of women's incomes, and potential over-exposure
to job losses (Organisation for Economic Co-operation and Development (OECD), 2020). Despite a tendency to pursue more risk-averse business strategies, women-owned businesses
may be at greater risk of closure over extended periods with substantially reduced or no
revenues. Social distancing measures, lockdowns and distorted supply chains and markets
have exacerbated structural inequities and challenges that typically hinder the performance
and growth of women-owned enterprises and have resulted in many temporary or
permanent closures of operations (International Labour Organization, 2020). More than 190,000 women entrepreneurs, mostly small and medium-sized
enterprises, expose themselves to economic and social risk (Alzahrin, 2020). The negative
consequence of the Covid-19 pandemic not only hurt BWEs businesses, but concurrently it
spills over to a family relationship, livelihood, and well-being. Women and Entrepreneurship Women are a basic unit in society. They traditionally prioritise greatly on preserving
family-relationship above any other endeavours. Women are capable of producing a great
family, turning the great family into a good society, and subsequently contributing to a great
nation. Nonetheless, women are still behind and considered an underdeveloped source of 171 Development of Rural Bumiputera Mompreneurs' Work-Life Balance Framework
Underpinning Theory As women entrepreneurs have relatively fixed and limited amounts of time and
energy, they have the motive and motivation to acquire, foster and assimilate more resources
from their environment according to the Conservation of Resource Theory (COR) (Hobfoll,
1989; 1998; 2002; 2012). This theory is well known in ecology literature but interestingly, only
a handful of prior studies have identified its impacts on entrepreneurship. Therefore, based
on the COR theory, we posit that women entrepreneurs can receive, maintain, and then
expand resources from their family members, the current nuclear family. Therefore, it is
theoretically consequential to investigate entrepreneurship from the familial perspective and
to understand the underlying influences better. The COR theory defines resources as matters that individuals' value and need, such as
objects, states, and conditions, which can help them to reduce stress and pursue life and
business activities. In contrast, the loss of resources will cause them to feel stress. A career as an entrepreneur consumes an individual's resources significantly, even
severely if one is a mother and wife. Thus, it is of critical value for individuals to seek, acquire,
and spend the resources available from their families. To summarise, a harmonious family
encourages entrepreneurial support, serving as critical resources to facilitate entrepreneurial
activities. This study makes an important contribution to the literature on entrepreneurship. By drawing basis on the COR theory (Hobfoll, 1989), we examine the relationships between
supports from immediate family members and entrepreneurship activities, thus casting light
on how support from the family spheres can help women entrepreneurs to manage
entrepreneurial activities, which contributes to the development of entrepreneurship theory
within family-relationship context. Secondly, this study further contributes to the development of entrepreneurship
theory in identifying the factors that mediate the effect of immediate family members on
women's likelihood of success in managing entrepreneurial activities. Specifically, we
postulate that family support expedites the effects of immediate family relationships on
entrepreneurship activities but reduces conflicts that facilitate the current family's effect on
entrepreneurship. Thirdly, from a broader perspective, this study contributes to the COR theory in that
we identified that the supports from current family serve as important resources that
individuals can resort to and apply into such life ventures and expeditions, such as business
entrepreneurship, expanding the meaning and nature of the concept of resource and thus
contributing to developing the COR theory. Figure 1 illustrates the conceptual model of this
study. International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS In this case, severe domestic
vulnerabilities such as domestic violence, depression, divorce filing, income deficits, savings
depletion and even bankruptcy forces could not be controlled (OECD, 2020). Thus, all-
inclusive policy planning that advocates BWEs may narrow the poverty gap between urban
and rural society. The president of the Bumiputera Women's Association in Business and Profession
Malaysia (Peniagawati) said that helping women entrepreneurs also means helping families
because women are known to contribute around 70% of their income to family well-being
(Alzahrin, 2020). As women entrepreneurs are generally less resilient to the impact of adverse
economic shocks, ensuring their access to critical support services will be essential to ensure
their post-COVID-19 stabilisation and recovery (International Labour Organization, 2020). In
Malaysia, small and medium-sized enterprises are one of the sectors most directly affected
by the movement of control orders. This severely inhibits the income earned by small traders,
particularly in rural areas (Abdul Rashid et al., 2020). Intending to improve the settlers' quality of life and socio-economic status, FELDA, as
one of Malaysia's government agencies, is taking the initiative to transform the FELDA
community into an entrepreneurial community (Selamat et al., 2015). Deo et al (2016) found
a positive and robust relationship between women entrepreneurs and family socio-economic 172 Development of Rural Bumiputera Mompreneurs' Work-Life Balance Framework
Underpinning Theory Specifically, we hypothesise that a positive relationship with one's spouse and a
positive relationship with one's children within the immediate family members will lead to
reduced conflicts, which, in turn, can help women entrepreneurs to facilitate entrepreneurial
activities. Family Relationship and Entrepreneurial Activities
Family relationships represent an important source of resources in social and human
capital (Granovetter 1985; Matzek et al., 2010; Michel et al., 2011). Abdullahi and Zainol Family Relationship and Entrepreneurial Activities International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS development. Therefore, the present study is worthy and holds merit. It may suggest
measures for the sustainability of family income and better understand the well-being of
family members involved and society. development. Therefore, the present study is worthy and holds merit. It may suggest
measures for the sustainability of family income and better understand the well-being of
family members involved and society. International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS (2016) agrees as families begin to understand the work demands of BWEs, family business
lifestyles will be built. This creates a path for BWEs entrepreneurial success. (2016) agrees as families begin to understand the work demands of BWEs, family business
lifestyles will be built. This creates a path for BWEs entrepreneurial success. Family is generally recognised as one crucial resource reservoir that fuels an
individual's passion, whereas work-family conflicts negatively impact or even destroy their
performance (Michel et al., 2011). This kind of family relationship has the potential to induce
either competitive advantages or disadvantages (Chrisman et al., 2005). Entrepreneurship
requires individuals with much more physical, emotional, and psychological resources to deal
with relevant stresses and challenges. The regional learning literature argues that family relationships may stimulate learning
processes due to the level of trust and commitment embedded in the relationship (Maskell &
Malmberg, 1999). Rimsha et al (2020) raised Stigma, Time, Growth, Family Balance (STGFB)
as challenges for Pakistani Mompreneurs. They found there is a positive relation between
Stigma and Mumpreneurship. Domestic and global economic stability will be increased due
to the empowerment in gender as all political, legal, and social factors ultimately affect
Pakistan's entrepreneurial activities. Similar information must unfold for the more
remarkable achievement of Malaysian BWEs. Family relationships are also highly beneficial because they lower agency and
transaction costs by offering bundles of inimitable, rare, and valuable resources via the
interactions between individual family members and the business (Habbershon and
Williams, 1999). For instance, women may experience work-life conflict and such conflicts
undermine business success. However, the effects of conflicts on entrepreneurial activities
and the role of family conflict/support as an important contextual contingency in initiating
entrepreneurial activities have been under-discussed (Zhu et al., 2020). Thus, we believe that
it is necessary better to integrate the literature in these two seemingly separate spheres to
better understand the family environment factor. Additionally, because women entrepreneurs need to assume multiple roles, their
time, resources, and energy will be shared and expanded to different spheres. Therefore,
resource depletion will eventually influence entrepreneurs' well-being and venture success. In this view, the family relationship is a vital source of support to women entrepreneurs
(Zachary, 2011). International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS Therefore, it is of theoretical value and necessary to consider one's family
environment as a predictor of their entrepreneurial abilities to cope with work-life balance. Thus, this study puts the following propositions: Proposition 1: There is a Positive Relationship between Family Relationships and BWEs
Entrepreneurship Activities. Family Relationship and Entrepreneurial Activities 173 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS (Van Auken and Werbel, 2006). In fact, women entrepreneurs will get even more inspired to
excel in business to gratify their spouse by sharing responsibility and burden to increase
domestic income in a way it shows how they appreciate the husband from that heart-winning
relationship (Roy and Manna, 2014). The role of the husband in helping women entrepreneurs
prosper in their entrepreneurial activities were not only for emotional reasons, but they
provide instant access to internal funding and business mentoring which critical to survival
for small size business (Maden, 2015; Wolf and Frese, 2018). It is notable that a supportive
husband in a marital relationship is far more important than other types of social support as
Brüderl and Preisendörfer (1998) emphasise that this marital bonding is regarded as strong
ties than weak ties, which is undoubtedly a pivotal resource to sustain the work-life balance
of women entrepreneurs. Therefore, in view of the above points, this study puts the following
propositions: Proposition 2: There is a positive relationship between marital relationships and BWEs
entrepreneurship activities. Marital Relationships and Entrepreneurship Activities The marital relationship has a considerable influence on women entrepreneurial
activities because it can facilitate direct and indirect spouse involvement in any aspects of the
family business (Kurniawan and Sanjaya, 2016). Many research in consensus on the
prominent role of husband plays in women entrepreneurship and serve as a critical enabling
factor to positive venture performance (Matzek et al., 2010; Van Auken and Werbel, 2006). Furthermore, as married women typically had cultural barriers limiting their business growth
prospect (Khan et al., 2021), having a good relationship with their spouse offers a form of
emotional and instrumental advantage, which is very helpful to encounter business
challenges (Chay, 1993). The way that emotional comfort influences women entrepreneurs is
pretty apparent in stimulating positive behaviours such as self-confidence and self-efficacy 174 Marital Relationship Mediates the Relationship between Family Relationship and
Entrepreneurship Activities Figure 1: Conceptual Model of the Study
As Figure 1 illustrates, the arrangement of the model suggests that the role of the
marital relationship and family relationships towards entrepreneurship activities of BWEs. These factors are underpinned by the conservation of resource theory. This framework
emphasises marital relationships as an intermediary between family relationships and BWEs
entrepreneurship activities. Family
Relationship
Marital
Relationship
Entrepreneurship
Activities Figure 1: Conceptual Model of the Study
Family
Relationship
Marital
Relationship
Entrepreneurship
Activities Marital
Relationship Family
Relationship Entrepreneurship
Activities Figure 1: Conceptual Model of the Study As Figure 1 illustrates, the arrangement of the model suggests that the role of the
marital relationship and family relationships towards entrepreneurship activities of BWEs. These factors are underpinned by the conservation of resource theory. This framework
emphasises marital relationships as an intermediary between family relationships and BWEs
entrepreneurship activities. Marital Relationship Mediates the Relationship between Family Relationship and
Entrepreneurship Activities Marital Relationship Mediates the Relationship between Family Relationship and
Entrepreneurship Activities In most developing countries, women are still bounded by culture and tradition, and
in such cases, men obviously dominate most of the family state of affairs. In marital context,
it was found that even family relationship can be a strong predictor to positive
entrepreneurial activities, including growth and success destiny of an enterprise, it is
contingence to how supportive the husband is in maintaining work-life balance relationships
(Wolf and Frese, 2018). With house chores and parenting responsibilities where women are
expecting to be good at them, it is very challenging to juggle multitasking roles without
husband support, even with the presence of help from other family members. As previously
discussed, the wife-husband relationship is capable of achieving a state of mental comfort or
feeling that "being supported", which often works primarily during the critical period
compared to other tangible family supports (Carels and Baucom, 1999). To some extent, where family resources are available, spouses' commitment to the
business can facilitate synergy alignment between business objectives and family goals (Van
Auken and Werbel, 2006). Eddleston and Powell (2012) provides this justification, suggesting
that a husband can act as invisible leader in directing the right business path through his
supportive behaviours (for example sharing domestic responsibilities to enable their partners
to spend more time on business development). On the other hand, potential conflict among
family members who get involved in the business can also be mitigated effectively by the
functional role of the husband-wife relationship in reducing business-related conflicts (Owen
et al., 2013). The vital notion in literature has made it clear that spouse involvements are able to
create a positive influence towards women entrepreneurs' success, which makes their life
partners ranked highest among social ties, including the other family members (Jianakoplos
and Bernasek, 2008; Garcia et al., 2007). Therefore, strengthening the outcome of women
entrepreneurship need full support from their partners, and thus this study puts the following
propositions: 175 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS Proposition 3: Marital relationship mediates the relationship between family relationship and
BWEs entrepreneurship activities. This study identifies the factors of work-life balance in business-family relationships among
BWEs. The conceptual model proposed by this paper is presented below as Figure 1. International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS "economic" and a reliable solution to ensure families continue earning before a tragic
phenomenon Covid-19 can be solved. The existing theoretical is still unexplored and cannot
explain women entrepreneurship's phenomena, especially single mother entrepreneurs. Therefore, this discussion offers some direction for future research in women
entrepreneurship, particularly on the model of successful single mother entrepreneurs. Conclusion Issues and challenges pertaining to the quality of life and well-being within domestics
or family context during this pandemic Covid-19 challenges are inevitable. The work-life
balance between family and entrepreneurial commitment became extreme; thus, positive
interventions demanded its urgency. The chances of women entrepreneurs to succeed in
business are in determinant on their family's relationships, happiness, and well-being. Thus,
it is vital to underpin the impact of this challenging time on the women entrepreneurs,
families, and communities so that assistance and support can be aggressively offered to the
most in need relationships. The findings of this research shall proclaim supportive networks, resources and policy
undertaken needed to be undertaken to remedy the hindrances faced by BWEs and their
families in developing a holistic work-life self-family relationship thus, ensuring sustainable
entrepreneurial ventures among BWEs. This study can be replicated by other prominent
bodies like Federal Land Consolidation and Rehabilitation Authority (FELCRA) and Rubber
Industry Smallholders Development Authority (RISDA). This shall benefit their participants as
the research findings offer a set of modelling to rectify any similar issues. Women entrepreneurs in rural society mainly are known to be emotionally fragile. They need tangible and intangible support especially following the negative consequences of
the Covid-19 pandemic. The research aims to collectively assemble the life experiences of the
Mompreneurs in managing the challenging situation to continue business for the sake of
earning sufficient income to pay basic expenses. Their views matter most to be heard through
a research initiative. The research findings can be communicated effectively and directly from
a trusted research entity, the university, to responsible agencies. Movement Control Order
(MCO) had witnessed massive temporary job discontinuance and job termination, which
drastically impact family economic well-being where many of them are men-beheaded
breadwinners. Therefore, empowerment of women entrepreneurship is considered 176 International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS International Journal of Academic Research in Business and Social Sciences
Vol. 11, No. 19, Youth and Community Wellness, 2021, E-ISSN: 2222-6990 © 2021 HRMARS Hobfoll, S. E. (2002). Social and psychological resources and adaptation. Review of General
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English
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PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration
|
PloS one
| 2,012
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cc-by
| 10,459
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ETH Library ETH Library PhOTO Zebrafish: A Transgenic
Resource for in vivo Lineage
Tracing During Development and
Regeneration Journal Article
Author(s):
Dempsey, William P.; Fraser, Scott E.; Pantazis, Periklis
Publication date:
2012-03-14
Permanent link:
https://doi.org/10.3929/ethz-b-000062531
Rights / license:
Creative Commons Attribution 3.0 Unported
Originally published in:
PLoS ONE 7(3), https://doi.org/10.1371/journal.pone.0032888 Abstract * E-mail: periklis.pantazis@bsse.ethz.ch * E-mail: periklis.pantazis@bsse.ethz.ch lineage tracing analyses involving early stage dye injection (e.g. fluorescein, etc.), followed by fixed sample analysis or live cell
tracking after sufficient developmental time [3]. More recent
sparse labeling modalities take advantage of genetically encoded
means for generating random transgenic mosaics expressing cell-
targeted fluorescent proteins (FPs) – for example, using random
gene insertion events [4,5], drug induced recombination [6,7] or
heat shock promoters [8]. Other partial cell labeling techniques
revolve around tissue specific promoter driven FP expression to
isolate only cells in a particular tissue of interest [9,10,11,12,13]. The second variety, global cell labeling, is most often achieved by
ubiquitous nuclear FP expression driven by constitutive promoter
sequences [14,15]. William P. Dempsey1,2, Scott E. Fraser1, Periklis Pantazis2* Institute (139-74), California Institute of Technology, Pasadena, California, United States of America, 2 ETH Zurich, Department o
ring, Basel, Switzerland 1 Division of Biology, Beckman Institute (139-74), California Institute of Technology, Pasadena, California, United States of America, 2 ETH Zurich, Department of
Biosystems Science and Engineering, Basel, Switzerland Author(s): This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. PLoS ONE | www.plosone.org March 2012 | Volume 7 | Issue 3 | e32888 Abstract Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying
embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space
and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible
optical tracking of nuclear and membrane dynamics in vivo. Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible
FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2
photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through
adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and
photoconversion of a ,100mm stripe along the cut area, marked differences seen in how cells contribute to the new tissue
give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate
were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a
subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly
regenerating fin. Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early
embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and
regeneration, cancer progression, and stem cell behavior. Citation: Dempsey WP, Fraser SE, Pantazis P (2012) PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and
Regeneration. PLoS ONE 7(3): e32888. doi:10.1371/journal.pone.0032888 Editor: Shree Ram Singh, National Cancer Institute, United States of America Received December 18, 2011; Accepted February 1, 2012; Published March 14, 2012 Copyright: 2012 Dempsey 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. orted by a Caltech Rosen Scholar Graduate fellowship. The funders had no role in study design, data collection and analysis, decision to
f the manuscript. Funding: WPD was supported by a Caltech Rosen Scholar Graduate fellowship. The funders had no role in study design, data co
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. PhOTO Zebrafish for Development and Regeneration PhOTO Zebrafish for Development and Regeneration terminus of the protein [19]. Self-cleavage of a highly efficient
Thosea asigna virus (TaV) 2A sequence between the two FPs at the
ribosome separates the two proteins to their respective target
locations within the cell at a 1:1 stoichiometric ratio [20]. Two
stable
transgenic
lines
(Tg(bactin2:memb-Cerulean-2A-H2B-Den-
dra2)pw1 and Tg(bactin2:memb-Dendra2-2A-H2B-Cerulean)pw2) were
established
after
Tol2
mediated
[21]
genome
integration. PhOTO-N zebrafish constitutively express H2B-Dendra2 to label
all nuclei and memb-Cerulean to label all membranes, and the
complementary PhOTO-M zebrafish line expresses H2B-Cerule-
an and memb-Dendra2. Efficient cleavage and proper FP
localization were verified by western blot (Figure S1) and
confocal microscopy (Figure 1B). Founders were established for
each line (7 for PhOTO-N; 5 for PhOTO-M), and only offspring
with ubiquitous, bright FP expression in all cells were considered
for this analysis. lineage tracing tool should combine the strengths of both and
should be directly applicable for studies of early development as
well as adult tissue regeneration. Here, we report a set of transgenic zebrafish lines that we
named PhOTO (photoconvertible optical tracking of…), which
meets the challenges of long-term lineage tracing in both early
development as well as adult epimorphic tissue regeneration. These lines combine the benefits of global and sparse imaging
approaches for lineage analysis. Whole organism imaging and
tracking is enhanced by the multicompartment (nuclear and
membrane) expression of spectrally distinct FPs from a single
coding sequence, providing better accuracy for segmentation and
tracking of cells in vivo. Additionally, with the aid of optically
induced photoconversion, long-lasting sparse cell labeling is easily
achieved, allowing high signal-to-noise tracking of a subset of cells
in space and time at any point during the lifetime of the zebrafish. Results Establishing the PhOTO-N and PhOTO-M transgenic lines
In order to best address the requirements for long-term imaging
of
complex
vertebrate
development
and
regeneration,
we
constructed the PhOTO vector (Figure 1A), which allows for
constitutive b-actin2 [16] driven expression of two FPs – green-to-
red photoconvertible Dendra2 [17] and the blue FP Cerulean [18]
– targeted to either (1) the nucleus of the cell by means of an H2B
fusion or (2) the membrane (‘‘memb’’) via a palmitoylation and
myristoylation fatty acid substrate sequence included at the N- Since nuclear tracking has become standard practice for
developmental lineage tracing [14,15], we illustrated the simplicity
of targeting down to the level of a single cell during the dynamic
period of gastrulation in the PhOTO-N line (Figure S2 and
Videos S1, S2). Our approach only requires semi-automated
image analysis (see methods), and we segmented and tracked the
photoconverted H2B-Dendra2 nuclei in time through ,6 hours of
development. Simultaneous collection of memb-Cerulean and
unconverted nuclear H2B-Dendra2 fluorescence data from all Figure 1. Description of PhOTO Vector and PhOTO Transgenics. (A) Depicted is a schematic of the general PhOTO vector. The major
components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on
the outside of the plasmid circle. Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily
switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note
that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–
19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts
unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells
in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100mm. doi:10.1371/journal.pone.0032888.g001 Figure 1. Description of PhOTO Vector and PhOTO Transgenics. (A) Depicted is a schematic of the general PhOTO vector. The major
components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on
the outside of the plasmid circle. Introduction Extensive migratory events, morphological changes, and cell
divisions coordinated by cell signaling drive complex vertebrate
patterning events during embryonic development [1] as well as
during epimorphic tissue regeneration [2]. Lineage tracing – the
process of tracking a cell and its progeny in space and time as it
moves from an early to a specified state – provides an overall
picture of the coordinated events required to set up or repair a
proper body plan. Two major considerations are required for this
tracking strategy: first, a precise, indelible mark must be placed
within cells of interest at an early stage of development or
regeneration; and second, after sufficient time, the final location of
labeled cells and their progeny must be scored accurately [3]. Thus, techniques capable of monitoring long-term behavior (i.e. movements as well as morphology) in precise cell locations are key
to tease out the timing and cellular events controlling general cell
fate decisions in vivo. These two techniques have opposing benefits and disadvantages
that limit their effectiveness as lineage tracing tools. Sparse/partial
methods simplify the tracking problem by focusing solely on cells
of interest, though the data lacks context with other cell types in a
given tissue environment. In contrast, global methods supply cell
behavior data in context to every cell in the organism at the
expense
of
requiring
sophisticated
volumetric
imaging
and
algorithms for cell segmentation and tracking. An attractive Most techniques for lineage tracing can be split into two general
categories, (1) sparse/partial and (2) global cell labeling and
tracking. Sparse cell labeling methodologies include traditional 1 March 2012 | Volume 7 | Issue 3 | e32888 Results Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily
switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note
that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–
19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts
unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells
in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100mm. doi:10.1371/journal.pone.0032888.g001 March 2012 | Volume 7 | Issue 3 | e32888 PLoS ONE | www.plosone.org 2 PhOTO Zebrafish for Development and Regeneration Figure 2. Monitoring Dynamic Membrane Movements and Tracking Nuclei in a PhOTO-M Zebrafish During Gastrulation. Animal pole
view maximum intensity projection (MIP) images of the first 21mm (in depth) of an ,5 hour time-lapse of a heterozygote F1 PhOTO-M zebrafish from
late gastrulation (.80% epiboly) until early segmentation. (A) Merged MIP of the first frame of the time-lapse, showing unconverted (green) memb-
Dendra2, segmented photoconverted (red) memb-Dendra2, and H2B-Cerulean (blue) labeled nuclei as well as segmented H2B-Cerulean nuclei (multi-
colored surfaces in the photoconverted cells). (B) Merged MIP image of the final frame of the time-lapse. (C) Zoomed MIP images of segmented,
photoconverted, and migrating memb-Dendra2 intensity data (red) and segmented H2B-Cerulean nuclei (multi-colored surfaces) at 8 different time-
points. Note that H2B-Cerulean was segmented using the surrounding membranes as a guide in three dimensions. Cells are migrating in front of the
developing head and move apart laterally. Arrowheads depict a cell division event and the arrow depicts a cell moving into the field of view from
below. Note that a few overlying enveloping layer (EVL) cells were photoconverted in addition to the tracked cells, but since these remained
stationary throughout the time-lapse, they were neglected when performing semi-automated membrane segmentation of the data. Scale bars are
50mm. doi:10 1371/journal pone 0032888 g002 Figure 2. Monitoring Dynamic Membrane Movements and Tracking Nuclei in a PhOTO-M Zebrafish During Gastrulation. Results Animal pole
view maximum intensity projection (MIP) images of the first 21mm (in depth) of an ,5 hour time-lapse of a heterozygote F1 PhOTO-M zebrafish from
late gastrulation (.80% epiboly) until early segmentation. (A) Merged MIP of the first frame of the time-lapse, showing unconverted (green) memb-
Dendra2, segmented photoconverted (red) memb-Dendra2, and H2B-Cerulean (blue) labeled nuclei as well as segmented H2B-Cerulean nuclei (multi-
colored surfaces in the photoconverted cells). (B) Merged MIP image of the final frame of the time-lapse. (C) Zoomed MIP images of segmented,
photoconverted, and migrating memb-Dendra2 intensity data (red) and segmented H2B-Cerulean nuclei (multi-colored surfaces) at 8 different time-
points. Note that H2B-Cerulean was segmented using the surrounding membranes as a guide in three dimensions. Cells are migrating in front of the
developing head and move apart laterally. Arrowheads depict a cell division event and the arrow depicts a cell moving into the field of view from
below. Note that a few overlying enveloping layer (EVL) cells were photoconverted in addition to the tracked cells, but since these remained
stationary throughout the time-lapse, they were neglected when performing semi-automated membrane segmentation of the data. Scale bars are
50mm. d i 10 1371/j
l
0032888 002 doi:10.1371/journal.pone.0032888.g002 Figure 2C) with high precision. Photoconverted membranes are
still
visible above
background
greater
than
20 hours after
photoconversion in different cell types (Figure 3), ensuring the
documentation of membrane behavior throughout early embryo-
genesis as well as during major tissue specification events later in
development. surrounding cells gives context to the behavior of the photo-
converted nuclei. PhOTO-M highlights the dynamics of cell membranes
and nuclei during early development To extend lineage tracing beyond the level of nuclei alone, we
utilized the PhOTO-M line to capture a condensed, high-
resolution
view
of
cell
dynamics
at
the
membrane
level
(Figure 2 and Videos S3, S4). Although gross membrane
morphological changes (e.g. total cell volume changes) in all cells
are visible in the unconverted memb-Dendra2 channel, small-scale
membrane
dynamics
of
cells
within
the
embryo
can
be
appreciated only in a sparsely labeled environment such as in
the segmented (see methods) photoconverted memb-Dendra2
channel, where short membrane extensions can be seen to reach
out as the cells crawl across the surface of the embryo. We also
used the photoconverted membranes as visible boundaries to
segment the H2B-Cerulean labeled nuclei within these cells,
allowing us to distinguish cell division events (arrowheads in
Figure 2C) and cell migration into the field of view (arrow in PhOTO-N enables identification of cell contributions to
zebrafish epimorphic tail fin regeneration Since distinct, constitutive FP expression continues throughout
the lifetime of PhOTO zebrafish, these lines can be extended to
approaching problems such as live imaging of epimorphic tissue
regeneration. Building upon previous studies of heart [10,11] and
tail fin [12] regeneration, we used the PhOTO-N line to probe cell
contributions to the regenerating tail fin (Figure 4A). We
amputated the caudal portion of an adult (,7 months old)
PhOTO-N zebrafish tail fin (Figure S3), photoconverted all
nuclei within an ,100mm stripe along the amputation plane (see
methods) (Figure S4), and allowed the fin to regenerate for
7 days. Ubiquitous memb-Cerulean and unconverted H2B- PLoS ONE | www.plosone.org March 2012 | Volume 7 | Issue 3 | e32888 3 PhOTO Zebrafish for Development and Regeneration Figure 3. PhOTO-M Photoconversion Persists in Multiple Cell Types. Zebrafish were photoconverted in a ,100mm circular region of interest
near the animal pole during gastrulation and were imaged the following day to visualize cell membranes at high resolution in different cell
compartments. Even after multiple rounds of division, photoconverted membranes are still clearly visible above background. (A) Photoconverted
membranes are clearly visible in the epithelium a day after photoconversion. Maximum intensity projections of a ventrally mounted zebrafish
(anterior top) embryo 1 day post fertilization. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted memb-
Dendra2 (red) and a merged image of the three channels. (B) Photoconverted membranes are visible and are separated by unconverted membranes
in the eye a day after photoconversion. Single focal plane images of the developing eye in a ventrally mounted zebrafish (anterior top) embryo 1 day
post fertilization. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted memb-Dendra2 (red) and a merged image
of the three channels. (C) Photoconverted membranes are visible in the developing forebrain a day after photoconversion. Single focal plane images
of the developing forebrain in a ventrally mounted zebrafish (anterior top) embryo 1 day post fertilization. The star indicates an individual
photoconverted cell in mitosis during prometaphase. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted
memb-Dendra2 (red) and a merged image of the three channels. Scale Bar in (A) is ,150mm. Scale Bar in (B) and (C) is ,30mm. doi:10.1371/journal.pone.0032888.g003 Figure 3. PhOTO-M Photoconversion Persists in Multiple Cell Types. PhOTO-N enables identification of cell contributions to
zebrafish epimorphic tail fin regeneration Zebrafish were photoconverted in a ,100mm circular region of interest
near the animal pole during gastrulation and were imaged the following day to visualize cell membranes at high resolution in different cell
compartments. Even after multiple rounds of division, photoconverted membranes are still clearly visible above background. (A) Photoconverted
membranes are clearly visible in the epithelium a day after photoconversion. Maximum intensity projections of a ventrally mounted zebrafish
(anterior top) embryo 1 day post fertilization. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted memb-
Dendra2 (red) and a merged image of the three channels. (B) Photoconverted membranes are visible and are separated by unconverted membranes
in the eye a day after photoconversion. Single focal plane images of the developing eye in a ventrally mounted zebrafish (anterior top) embryo 1 day
post fertilization. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted memb-Dendra2 (red) and a merged image
of the three channels. (C) Photoconverted membranes are visible in the developing forebrain a day after photoconversion. Single focal plane images
of the developing forebrain in a ventrally mounted zebrafish (anterior top) embryo 1 day post fertilization. The star indicates an individual
photoconverted cell in mitosis during prometaphase. Left to Right: Unconverted memb-Dendra2 (green), H2B-Cerulean (blue), photoconverted
memb-Dendra2 (red) and a merged image of the three channels. Scale Bar in (A) is ,150mm. Scale Bar in (B) and (C) is ,30mm. doi:10.1371/journal.pone.0032888.g003 Notably, a subset of these cells was aligned and evenly spaced
along the growth axis of the regenerating tissue (Figure 4I and
arrowheads in Figure 4E). Dendra2 expression was re-established in the regenerating tissue
(Figure 4C, D), indicating little, if any, down-regulation of the b-
actin2 promoter driving FP expression. Using the photoconverted
nuclei (Figure 4E), we obtained localization and intensity data
that act as a readout for the extent of cell migration and division,
respectively. We observed a large subset of cells with bright
photoconverted nuclei that sit just rostral to the amputation plane
(Figure 4F), presumably maintaining structural integrity of the fin
during regeneration. Signal from these bright photoconverted
nuclei does not colocalize with unconverted H2B-Dendra2
(Figure 4F9), indicating that these cells did not divide, since the
H2B protein has a long half-life [22] in non-dividing cells. Discussion The PhOTO lines enable non-invasive, non-random mosaic
labeling of the membranes or nuclei of any subset of cells at any
time during development by use of the photoconvertible property
of Dendra2, combining the strengths of global and sparse/partial
lineage tracing techniques and enabling selective lineage tracing
during adult regeneration. The photoconversion is immediate and
nontoxic, circumventing experimental shortcomings seen with
heat shock or drug inducible promoters for mosaic labeling, which
require significant incubation time before expression and have
possible cytotoxic effects during temperature or drug treatment. An additional feature is that the multi-compartment labeling in the
PhOTO lines eliminates the need for exogenous counterstains. p
g
[
]
g
Within the regenerated portion of the tail fin, we segmented all
of the photoconverted H2B-Dendra2 nuclei that remained above
background
using
an
automated
script
(see
methods)
and
discovered three distinct intensity populations (Figure 5). Cells
with no red signal over background – presumably from the
contribution of unconverted cells more rostral to the amputation
plane [12] – and those with slight increases over background made
up the majority of cells throughout the newly regenerating region,
indicating that most of the cells in the new territory result from
extensive proliferation as the fin structure is re-established. As
expected, a subset of bright cells were located at the distal edge of
the tail fin, consistent with previous studies [23] (Figure 4H). However, we also see bright cells in the middle of the regenerate
(Figure 4G) that continue to stay visible for over 14 days (data not
shown). The lack of colocalized unconverted H2B-Dendra2
(green) signal with the brightest photoconverted (red) nuclei
(Figure 4G9, H9) suggests that these particular photoconverted
cells managed to populate the regenerate without dividing at all. PhOTO zebrafish and recent lineage tracing methods (i) An adult zebrafish is
anesthetized and (ii) the tail is amputated using a razor blade (dotted
line refers to amputation plane). (iii) Photoconversion is achieved by
fluorescent illumination along the amputation plane. (iv) In addition to
the H2B-Dendra2 (green) and memb-Cerulean (blue) signal, there is
now a line of photoconverted H2B-Dendra2 nuclei (red). (B-I) Lateral
view (anterior left, ventral down) MIP images of the regenerating tail fin photoconverted H2B-Dendra2 (red) alone. (F9-H9) Merged unconverted
H2B-Dendra2 (green) and photoconverted H2B-Dendra2 (red) within
the zoomed boxed regions from (D), (E). (F, F9) Many photoconverted
cells in the ventral and dorsal portions of the amputated fin stayed
behind the amputation plane during the regeneration process. (G, G9)
Surprisingly, a subset of brightly photoconverted cells were found in
the central portion in addition to (H, H9) other bright cells at the distal
edge of the regenerate. (I) Zoomed binary image of a subset of the
segmented nuclei from the photoconverted H2B-Dendra2 channel in
panel (E). Among the scattered cells with photoconverted signal over
background (gray), certain cells (red) seem aligned and evenly spaced
along the anterior-posterior axis of the tail fin within the regenerating
region. Scale bar for (B-E) is 300mm. Scale bar for (F-H) is ,10mm. Scale
bar for (I) is ,100mm. photoconverted H2B-Dendra2 (red) alone. (F9-H9) Merged unconverted
H2B-Dendra2 (green) and photoconverted H2B-Dendra2 (red) within
the zoomed boxed regions from (D), (E). (F, F9) Many photoconverted
cells in the ventral and dorsal portions of the amputated fin stayed
behind the amputation plane during the regeneration process. (G, G9)
Surprisingly, a subset of brightly photoconverted cells were found in
the central portion in addition to (H, H9) other bright cells at the distal
edge of the regenerate. (I) Zoomed binary image of a subset of the
segmented nuclei from the photoconverted H2B-Dendra2 channel in
panel (E). Among the scattered cells with photoconverted signal over
background (gray), certain cells (red) seem aligned and evenly spaced
along the anterior-posterior axis of the tail fin within the regenerating
region. Scale bar for (B-E) is 300mm. Scale bar for (F-H) is ,10mm. Scale
bar for (I) is ,100mm. doi:10.1371/journal.pone.0032888.g004 within labeled tissues for lineage analysis is desired, either PhOTO
transgenic line may be incrossed with the tissue specific line of
interest. PhOTO zebrafish and recent lineage tracing methods (C) Memb-Cerulean (blue) and (D) unconverted H2B-
Dendra2 (green) channels indicate that expression is still consistent as
the tail fin regenerates. (E) Many of cells with fluorescence above
background appear both before and after the plane of amputation in
the photoconverted H2B-Dendra2 (red) channel. Cells in the central
region of the tail show the least expression, suggesting extensive cell
division contributing to regeneration in this area. However, certain cells
to the right of the amputation plane are bright, aligned, and evenly
spaced (arrowheads). (F-H) Zoomed boxed regions from (D) and (E) of Figure 4. Photoconverted Cell Organization After Epimorphic
Tail Fin Regeneration in a Living Adult PhOTO-N Zebrafish. (A)
Overview of fin regeneration protocol. (i) An adult zebrafish is
anesthetized and (ii) the tail is amputated using a razor blade (dotted
line refers to amputation plane). (iii) Photoconversion is achieved by
fluorescent illumination along the amputation plane. (iv) In addition to
the H2B-Dendra2 (green) and memb-Cerulean (blue) signal, there is
now a line of photoconverted H2B-Dendra2 nuclei (red). (B-I) Lateral
view (anterior left, ventral down) MIP images of the regenerating tail fin
of a live, anesthetized adult zebrafish 7 days post amputation. The
dotted yellow line indicates the approximate amputation plane. (B)
Bright field channel. Bright signal to the left of the dotted line arises
from tissue birefringence. The terminal end of this signal indicates the
amputation plane. (C) Memb-Cerulean (blue) and (D) unconverted H2B-
Dendra2 (green) channels indicate that expression is still consistent as
the tail fin regenerates. (E) Many of cells with fluorescence above
background appear both before and after the plane of amputation in
the photoconverted H2B-Dendra2 (red) channel. Cells in the central
region of the tail show the least expression, suggesting extensive cell
division contributing to regeneration in this area. However, certain cells
to the right of the amputation plane are bright, aligned, and evenly
spaced (arrowheads). (F-H) Zoomed boxed regions from (D) and (E) of Figure 5. Histogram Readout for the Extent of Cell Division
After 7 Days of Regeneration. Intensity data from individual nuclei
within the regenerated region (posterior to the amputation plane) of
the tail fin in the photoconverted H2B-Dendra2 channel (Figure 4E)
was segmented. The average intensity of each segmented nucleus was
recorded and this data was plotted as a histogram. PhOTO zebrafish and recent lineage tracing methods The sole constraint for applying this PhOTO incross
method for lineage analysis is that the expressed FP from the tissue
specific transgenic should not have significant spectral overlap with
the photoconverted Dendra2 channel. Additionally, a useful non-transgenic labeling approach for
zebrafish lineage tracing has been described recently, the so-called
SNAP-Tag [26]. Like the PhOTO lines, SNAP-Tags can be
targeted to different cell compartments (e.g. nuclei and mem-
branes) and may be designed for photouncaging experiments. It is
unclear, however, how long injected SNAP-Tag fluorescent
conjugates will remain visible within the zebrafish, which may
preclude lineage tracing past early developmental time windows. Thus, SNAP-Tags and PhOTO zebrafish are both valuable tools
for early developmental lineage tracing, while the PhOTO Figure 5. Histogram Readout for the Extent of Cell Division
After 7 Days of Regeneration. Intensity data from individual nuclei
within the regenerated region (posterior to the amputation plane) of
the tail fin in the photoconverted H2B-Dendra2 channel (Figure 4E)
was segmented. The average intensity of each segmented nucleus was
recorded and this data was plotted as a histogram. The data was fit to a
sum of 3 Gaussian curves, suggesting at least three distinct levels of
fluorescence in the photoconverted cells above background. These
three levels are indicative of the extent of divisions undergone during
the re-establishment of the tail fin: the population with the highest
average intensity underwent the fewest divisions, while the other two
populations divided more often during the 7 day regeneration period. doi:10.1371/journal.pone.0032888.g005 Figure 4. Photoconverted Cell Organization After Epimorphic
Tail Fin Regeneration in a Living Adult PhOTO-N Zebrafish. (A) Figure 4. Photoconverted Cell Organization After Epimorphic
Tail Fin Regeneration in a Living Adult PhOTO-N Zebrafish. (A)
Overview of fin regeneration protocol. (i) An adult zebrafish is
anesthetized and (ii) the tail is amputated using a razor blade (dotted
line refers to amputation plane). (iii) Photoconversion is achieved by
fluorescent illumination along the amputation plane. (iv) In addition to
the H2B-Dendra2 (green) and memb-Cerulean (blue) signal, there is
now a line of photoconverted H2B-Dendra2 nuclei (red). (B-I) Lateral
view (anterior left, ventral down) MIP images of the regenerating tail fin
of a live, anesthetized adult zebrafish 7 days post amputation. The
dotted yellow line indicates the approximate amputation plane. (B)
Bright field channel. Bright signal to the left of the dotted line arises
from tissue birefringence. The terminal end of this signal indicates the
amputation plane. PhOTO zebrafish and recent lineage tracing methods PhOTO zebrafish and recent lineage tracing methods
Current analyses of early tissue specification are often facilitated
by the use of tissue specific promoters driving FP expression [24]. In many situations, tissue specific transgenic lines expressing FP
reporters exist and represent attractive tools to quickly identify a
region of interest to focus on for lineage analysis. To target
subpopulations of tissue specific promoter driven sequences, clever
methodologies have been devised, including combining tissue
specific FP expression with clonal mosaic analysis [5] or with
mutant analysis [25]. As a complementary approach, when tissue
specificity and instantaneous targeting of subpopulations of cells PLoS ONE | www.plosone.org March 2012 | Volume 7 | Issue 3 | e32888 4 PhOTO Zebrafish for Development and Regeneration PhOTO Zebrafish for Development and Regeneration within labeled tissues for lineage analysis is desired, either PhOT
transgenic line may be incrossed with the tissue specific line
interest. The sole constraint for applying this PhOTO incr
method for lineage analysis is that the expressed FP from the tiss
specific transgenic should not have significant spectral overlap w
the photoconverted Dendra2 channel. Additionally, a useful non-transgenic labeling approach
zebrafish lineage tracing has been described recently, the so-call
SNAP-Tag [26]. Like the PhOTO lines, SNAP-Tags can
targeted to different cell compartments (e.g. nuclei and me
branes) and may be designed for photouncaging experiments. I
unclear, however, how long injected SNAP-Tag fluoresce
conjugates will remain visible within the zebrafish, which m
preclude lineage tracing past early developmental time window
Thus, SNAP-Tags and PhOTO zebrafish are both valuable to
for early developmental lineage tracing, while the PhOT
Figure 4. Photoconverted Cell Organization After Epimorphic
Tail Fin Regeneration in a Living Adult PhOTO-N Zebrafish. (A)
Overview of fin regeneration protocol. (i) An adult zebrafish is
anesthetized and (ii) the tail is amputated using a razor blade (dotted
line refers to amputation plane). (iii) Photoconversion is achieved by
fluorescent illumination along the amputation plane. (iv) In addition to
the H2B-Dendra2 (green) and memb-Cerulean (blue) signal, there is
now a line of photoconverted H2B-Dendra2 nuclei (red). (B-I) Lateral
view (anterior left, ventral down) MIP images of the regenerating tail fin
of a live, anesthetized adult zebrafish 7 days post amputation. The
dotted yellow line indicates the approximate amputation plane. (B)
Bright field channel. Bright signal to the left of the dotted line arises
from tissue birefringence. The terminal end of this signal indicates the
amputation plane. PhOTO zebrafish and recent lineage tracing methods (C) Memb-Cerulean (blue) and (D) unconverted H2B-
Dendra2 (green) channels indicate that expression is still consistent as
the tail fin regenerates. (E) Many of cells with fluorescence above
background appear both before and after the plane of amputation in
the photoconverted H2B-Dendra2 (red) channel. Cells in the central
region of the tail show the least expression, suggesting extensive cell
division contributing to regeneration in this area. However, certain cells
to the right of the amputation plane are bright, aligned, and evenly
spaced (arrowheads). (F-H) Zoomed boxed regions from (D) and (E) of
photoconverted H2B-Dendra2 (red) alone. (F9-H9) Merged unconvert
H2B-Dendra2 (green) and photoconverted H2B-Dendra2 (red) wit
the zoomed boxed regions from (D), (E). (F, F9) Many photoconvert
cells in the ventral and dorsal portions of the amputated fin stay
behind the amputation plane during the regeneration process. (G, G
Surprisingly, a subset of brightly photoconverted cells were found
the central portion in addition to (H, H9) other bright cells at the dis
edge of the regenerate. (I) Zoomed binary image of a subset of t
segmented nuclei from the photoconverted H2B-Dendra2 channel
panel (E). Among the scattered cells with photoconverted signal o
background (gray), certain cells (red) seem aligned and evenly spac
along the anterior-posterior axis of the tail fin within the regenerat
region. Scale bar for (B-E) is 300mm. Scale bar for (F-H) is ,10mm. Sc
bar for (I) is ,100mm. doi:10.1371/journal.pone.0032888.g004
Figure 5. Histogram Readout for the Extent of Cell Divisi
After 7 Days of Regeneration. Intensity data from individual nuc
within the regenerated region (posterior to the amputation plane)
the tail fin in the photoconverted H2B-Dendra2 channel (Figure 4
was segmented. The average intensity of each segmented nucleus w
recorded and this data was plotted as a histogram. The data was fit t
sum of 3 Gaussian curves, suggesting at least three distinct levels
fluorescence in the photoconverted cells above background. The
three levels are indicative of the extent of divisions undergone dur
the re-establishment of the tail fin: the population with the high
average intensity underwent the fewest divisions, while the other t
populations divided more often during the 7 day regeneration peri
doi:10.1371/journal.pone.0032888.g005 Figure 4. Photoconverted Cell Organization After Epimorphic
Tail Fin Regeneration in a Living Adult PhOTO-N Zebrafish. (A)
Overview of fin regeneration protocol. PhOTO Zebrafish for Development and Regeneration zebrafish also has the advantage of lineage tracing during any
stage in the lifetime of the zebrafish. PhOTO-N zebrafish proved to be especially suited to identify cells
that did not divide during the regeneration period. As a potential
extension, slowly dividing stem cells and possibly cancer stem cells
may be selected from the general population of cells in a similar
manner in the adult PhOTO zebrafish; photoconversion of a
region of interest within a tissue followed by a several day recovery
period should allow the identification and subsequent time-lapse
imaging of cells within the photoconverted tissue that have divided
the least. PhOTO Constructs Additionally, technological advances in imaging may be applied
to improve resolution in both time and space for cell tracking (e.g. a recent lateral line lineage tracing study [30]). As mentioned, the
PhOTO zebrafish have the advantage of targeted regional
specificity when a specific transgenic line is unavailable, and they
may also be used in conjunction with sophisticated imaging
modalities – such as with modified selective plane illumination
microscopy techniques [31,32] – to enhance cell tracking. The DNA constructs were generated based on a zebrafish
expression vector containing a minimal b-actin2 promoter [16],
flanking Tol2 transposable elements [21], and a polyadenylation
sequence. The TaV 2A sequence was generated using polymerase
chain reaction primer extension and further subcloned with PacI and
SnaBI restriction sites. A glycine-serine-glycine spacer, which has
been demonstrated to ensure high cleavage efficiency of the 2A [39],
was inserted immediately upstream of the 2A sequence. Restriction
sites were subcloned into the original zebrafish expression vector to
facilitate easy modification of any element. The bicistronic template
vector was generated using this vector by first incorporating a TaV
2A sequence between the promoter and polyA sequences by
subcloning. The FP Dendra2 [17] or Cerulean [18] was localized
to the membrane using two repeats of a myristoylated and
palmitoylated N-terminal MGCIKSKRKDNLNDDE signal se-
quence from Lyn kinase [19]. For nuclear localization, the C-
terminus of the H2B protein was fused with either Dendra2 or
Cerulean as previously described [40]. The fused Dendra2 and
Cerulean constructs were subcloned with restriction sites for
insertion into the PhOTO vector (sites shown in Figure 1A). Revealing the extent of cell division using PhOTO-N
zebrafish Segmentable signal from a subset of photoconverted nuclei in
the PhOTO-N line persists for at least 14 days, which makes the
line an optimal tool for tracking of cells during organ formation as
well as major regenerative processes in multiple tissue types (e.g. tail fin [12], heart [10,11] and maxillary barbel [33]). Thus, any
cell and its progeny can be traced from its origin before
amputation to newly established regions within the regenerate,
giving insight into the contributions of individual cells when
repopulating a damaged area. For instance, having non-dividing
cells within the regenerating region of the tail fin may suggest that
cells important for guidance during the tissue regeneration process
are maintained at some frequency throughout the re-established
region and not solely at the leading edge. This observation seems
even more plausible when considering the alignment of cells in the
regenerating tissue. Such an organization seems reminiscent of the
evenly spaced arrangement of cells during posterior lateral line
development, where the establishment and maintenance of certain
signaling cues – some of which are integral to fin regeneration [34]
– guide cell migration and deposition [35]. It will be interesting to
explore whether similar spatially restricted signaling contributes to
the establishment of the regenerating fin. Zebrafish Transgenics To establish transgenic zebrafish lines (Tg(bactin2:memb-Cerulean-
2A-H2B-Dendra2)pw1 and
Tg(bactin2:memb-Dendra2-2A-H2B-Ceru-
lean)pw2), WT zebrafish were first injected with either 20ng
PhOTO-N
or
PhOTO-M
plasmid
DNA
and
80ng
Tol2
transposase
mRNA
at
the
zygote
stage. Translated
Tol2
transposase proteins recognize Tol2 elements flanking the coding
region of the construct during the early developmental phases of
the zebrafish, and the coding region may be inserted randomly
into a cell’s genome by one of these transposase proteins [21]. Injected fish were raised at 28uC until 7 days post fertilization,
when they were screened for fluorescence. Healthy-looking,
brightly expressing mosaic embryos/larvae from the injected
population were selected and raised to adulthood. Fish that Implications of PhOTO zebrafish for large-scale, systems-
level analyses of cell behavior Considerable efforts have been made to segment and track
dense cell populations during development using nuclear labeling
[14,15,27], though density and spacing of segmentable features as
well as time resolution between frames increase uncertainty of
these cell tracking experiments [28]. Regional reduction in
segmentation and tracking complexity, which is possible with the
PhOTO lines, can decrease computational effort required for
large-scale embryo reconstruction, especially when selecting out
areas of high cell density and mobility, as in the shield of early
developing embryos. Since both the membrane and nuclear
compartments are labeled, segmentation errors that occur when
nuclei are in close proximity with each other can be avoided [29]. Finally, data taken within separate photoconverted regions from
different embryos – especially in the case of the PhOTO-M line
where small scale membrane dynamics are visible in photocon-
verted subsets – can ultimately be superimposed to get a sum-of-
parts view of cellular dynamics during particular stages of
development or regeneration, effectively eliminating uncertainty
from any given region in a whole-embryo context. With the combination of targeted cell tracking and global cell
monitoring, the effects of therapeutics on cell behavior may also be
monitored in real time. For example, since cancer stem cells have
been suggested to resist traditional chemotherapies [36], photo-
conversion of a zebrafish tumor followed by chemotherapeutic
treatment may enable time-lapse visualization of tumor regrowth
from a subset of tumor cells unaffected by the treatment. Similarly,
tracking cells of interest after any drug treatment that results in cell
behavior changes at the single cell level (e.g. loss-of-function
studies using retinoic acid [37] or morpholino oligonucleo-
tides [38] for manipulating zebrafish regeneration) is also possible
using the PhOTO zebrafish lines. Zebrafish Husbandry Zebrafish were raised, hatched, injected, and maintained in a
house colony as previously described [41]. PLoS ONE | www.plosone.org PhOTO zebrafish and recent lineage tracing methods The data was fit to a
sum of 3 Gaussian curves, suggesting at least three distinct levels of
fluorescence in the photoconverted cells above background. These
three levels are indicative of the extent of divisions undergone during
the re-establishment of the tail fin: the population with the highest
average intensity underwent the fewest divisions, while the other two
populations divided more often during the 7 day regeneration period. doi:10.1371/journal.pone.0032888.g005 Figure 5. Histogram Readout for the Extent of Cell Division
After 7 Days of Regeneration. Intensity data from individual nuclei
within the regenerated region (posterior to the amputation plane) of
the tail fin in the photoconverted H2B-Dendra2 channel (Figure 4E)
was segmented. The average intensity of each segmented nucleus was
recorded and this data was plotted as a histogram. The data was fit to a
sum of 3 Gaussian curves, suggesting at least three distinct levels of
fluorescence in the photoconverted cells above background. These
three levels are indicative of the extent of divisions undergone during
the re-establishment of the tail fin: the population with the highest
average intensity underwent the fewest divisions, while the other two
populations divided more often during the 7 day regeneration period. doi:10.1371/journal.pone.0032888.g005 March 2012 | Volume 7 | Issue 3 | e32888 PLoS ONE | www.plosone.org 5 Imaging and Dendra2 Photoconversion After crossing founder PhOTO zebrafish with WT adults,
embryos were raised in egg water [41] at 28uC until they were
ready to be imaged. Prior to imaging, embryos were screened for
fluorescence using an Olympus MVX10 fluorescence microscope. Positive embryos were embedded in 1% low melting point agarose
(Invitrogen) in 30% Danieau’s solution [42] within Lab-Tek 2-well
imaging chambers with #1 coverslip bottoms (Nalge Nunc
International). Embryos past 16 hours post fertilization were
anesthetized using 0.015%–0.03% Tricaine methanesulfonate
(Finquel/MS-222, Argent Labs) and were maintained in anesthet-
ic at the same concentration when embedded in the 1% agarose/
30% Danieau’s solution for imaging. We obtained live images in
space and time using a 20x/0.8NA Plan-Apochromat air objective
(Zeiss) using a Zeiss LSM 710 confocal microscope. Embryos were
maintained at a temperature between 28uC and 32uC during time-
lapse experiments. The tiled image in Figure 1 was taken with a
Leica True Confocal Scanner SP5 Spectral High-Speed Confocal
System with AOBS (Acoustical Optical Beam Splitter) (Leica
Microsystems Inc., Deerfield, IL) using a 20x/0.7NA objective
(Leica). Photoconversion was achieved by prolonged (.30sec)
illumination of a region of interest within the zebrafish sample with
a scanned 405nm laser on the Leica confocal microscope. Images
and time-lapse data from embryos were processed and cell
segmentation and tracking was performed in a semi-automated
manner using the spot tracking tool (for PhOTO-N nuclei) and the
surface segmentation tool (PhOTO-N membranes and PhOTO-
M membranes and nuclei) within Imaris software (Bitplane AG). Ad lt
b
fi h
(
7
th
ld)
th ti
d Western Blotting Screened larval zebrafish from each PhOTO line as well as WT
control fish were collected and homogenized in ice-cold lysis buffer
(150mM NaCl, 10mM Tris-HCl at ,pH 7.55, 1mM EDTA, 1%
Triton X-100) with a protease inhibitor (Complete Protease
Inhibitor Cocktail Tablets, Roche). Extracts from ,2 fish per lane
underwent SDS-PAGE (5% stacking gel/12% running gel)
electrophoresis using a Mini-Protean Gel Doc (BioRad). The
resulting bands were transferred to a PVDF membrane (Im-
mobilon P, Millipore) after wet electroblotting (transfer buffer: 25
mMTris, 192mM glycine, 20% Methanol, 0.1% SDS, pH 8.1–8.5)
using the Mini-Protean Gel Doc (BioRad). Rabbit anti-Dendra2
polyclonal antibodies (1:5000, Evrogen) and mouse anti-a-Tubulin
monoclonal antibodies (1:10000, Sigma Aldrich, loading control)
were used to probe the western blots. Horseradish peroxidase
linked goat anti-rabbit was used as a secondary antibody (1:10000,
Jackson ImmunoResearch Laboratories, Inc.). Western blots were
visualized
using
an ECL
Plus chemiluminescence
kit (GE
Healthcare). PhOTO zebrafish – further applications The complexity of cell behaviors during other cell process may
be ultimately unraveled due to the high signal-to-noise ratio of
photoconverted fluorescence in the context of global labeling when
using the PhOTO lines. Recall that three distinct cell populations
could be distinguished in the regenerate, and, surprisingly, the March 2012 | Volume 7 | Issue 3 | e32888 6 PhOTO Zebrafish for Development and Regeneration of amputation [43]. Images in Figures S3 and S4 were taken
with
an
Olympus
MVX10
fluorescence
microscope. After
imaging, the fish was immediately revived using a method
reported previously [44] and was then put back on the fish
husbandry system (Aquaneering). For confocal imaging of the tail
after 7 days, the tail was restrained under anesthesia using 2% low
melting point agarose (Invitrogen) in 30% Danieau’s solution. To
keep the fish alive during imaging, either system water or 30%
Danieau’s solution containing 0.03% Tricaine methanesulfonate
was flowed across the gills of the anesthetized fish, and gill motion
was monitored constantly. Confocal images were taken using the
same Zeiss LSM 710 confocal microscope. All images and videos
were processed and compiled using Adobe Photoshop CS3 (Adobe
Systems). survived to adulthood were crossed to WT adults, and the
resulting embryos were screened for fluorescence. Founders
positive for germline transmission and for strong, ubiquitous
expression were crossed with WT zebrafish to continue to
propagate the line. Embryos from these founder crosses were also
used for imaging. of amputation [43]. Images in Figures S3 and S4 were taken
with
an
Olympus
MVX10
fluorescence
microscope. After
imaging, the fish was immediately revived using a method
reported previously [44] and was then put back on the fish
husbandry system (Aquaneering). For confocal imaging of the tail
after 7 days, the tail was restrained under anesthesia using 2% low
melting point agarose (Invitrogen) in 30% Danieau’s solution. To
keep the fish alive during imaging, either system water or 30%
Danieau’s solution containing 0.03% Tricaine methanesulfonate
was flowed across the gills of the anesthetized fish, and gill motion
was monitored constantly. Confocal images were taken using the
same Zeiss LSM 710 confocal microscope. All images and videos
were processed and compiled using Adobe Photoshop CS3 (Adobe
Systems). PhOTO-N Nuclei Segmentation and Cell Division Analysis
After 7 Days of Regeneration PhOTO-N Nuclei Segmentation and Cell Division Analysis
After 7 Days of Regeneration Photoconverted H2B-Dendra2 MIP data from Figure 4E was
first processed in Photoshop CS3 (Adobe Systems) to isolate the
regenerated tail fin portion from the tail fin region rostral to the
amputation plane using the magnetic lasso tool. Using a custom
Matlab (Mathworks) script, nuclei were segmented that had
intensity above background, and the segmented mean intensity
data from each nucleus was fit to a sum of three Gaussians in a
manner similar to one described previously [45] to generate
Figure 5. Note that the segmented data was converted to a binary
image and modified in Photoshop CS3 (i.e. changing color of the
binary nuclei from white to red and gray) in order to generate
Figure 4I. PLoS ONE | www.plosone.org (MOV) Figure S4
Amputated Adult PhOTO-N Zebrafish Tail
Fin After Photoconversion. A fluorescent stereomicroscope
image of the photoconverted amputated tail fin (anterior left,
ventral down) showing (A) bright field, (B) memb-Cerulean (blue),
(C) unconverted H2B-Dendra2 (green), (D) photoconverted H2B-
Dendra2 (red), and (E) a merged image of (C) and (D). Note that
almost all of the H2B-Dendra2 fluorescence has been photo-
converted in the ,50–100mm region along the amputation plane,
as is indicated by the lack of green signal in the photoconverted
stripe in (C). Scale bar is ,300mm. (TIF) Video S4
Segmented Time-Lapse Data for PhOTO-M
Membrane Dynamics Visualization During Late Gas-
trulation. This video shows segmented data for each frame of the
time-lapse summarized in Figure 2 in the text. First, the
photoconverted membranes were segmented in 3D. Then, using
the segmented data, nuclei within the surrounding photoconverted
membranes were identified and represented as colored surfaces. The
nuclei were tracked starting with the final frame, moving backward
in time. (A) Segmented and photoconverted memb-Dendra2 (red) as
well as the segmented H2B-Cerulean channel (multi-colored
surfaces) along with the bright field (grayscale) from the time-lapse
video. Note that the developing head and optic primordia can be
seen during the time-lapse. (B) Segmented and photoconverted
memb-Dendra2 (red) as well as the segmented H2B-Cerulean (multi-
colored surfaces) along with the merged animal pole images as seen
in Video S3D. (C) Isolated zoomed images of the segmented nuclei
and membranes during the time-lapse. Scale bars are 50mm. (MOV) Video S1
Time-Lapse Data for PhOTO-N Development
During Late Gastrulation. This video shows each frame of the
time-lapse summarized in Figure S2. (A) Memb-Cerulean inten-
sity data (blue), (B) unconverted H2B-Dendra2 intensity data
(green), (C) and segmented photoconverted H2B-Dendra2 intensity
data (red). Initially, 3 cells were photoconverted (2 EVL cells and
one epiblast). Both EVL cells divide between the first and second
time point, and the epiblast cell divides between 1 hour, 19minutes
and 2 hours. (D) Merged image of panels (A–C). Scale bar is 50mm. (MOV) 9. Caron SJ, Prober D, Choy M, Schier AF (2008) In vivo birthdating by
BAPTISM reveals that trigeminal sensory neuron diversity depends on early
neurogenesis. Development 135: 3259–3269. Ethics Statement The animal experiments conducted were covered under the animal
license protocol number 1227 ‘Zebrafish Develomental Biology’ approved
by Caltech’s Institutional Animal Care and Use Committee (IACUC). The animal experiments conducted were covered under the animal
license protocol number 1227 ‘Zebrafish Develomental Biology’ approved
by Caltech’s Institutional Animal Care and Use Committee (IACUC). Author Contributions Conceived and designed the experiments: WPD SEF PP. Performed the
experiments: WPD PP. Analyzed the data: WPD PP. Wrote the paper:
WPD SEF PP. Conceived and designed the experiments: WPD SEF PP. Performed the
experiments: WPD PP. Analyzed the data: WPD PP. Wrote the paper:
WPD SEF PP. 7. Ramachandran R, Reifler A, Parent JM, Goldman D (2010) Conditional
gene expression and lineage tracing of tuba1a expressing cells during
zebrafish development and retina regeneration. J Comp Neurol 518:
4196–4212. 8. Collins RT, Linker C, Lewis J (2010) MAZe: a tool for mosaic analysis of gene
function in zebrafish. Nat Methods 7: 219–223. Supporting Information Figure S1
Efficient TaV2A-Mediated Protein Cleavage
in the PhOTO Zebrafish Lines. Western blot analysis of each
PhOTO line: lane (1) WT (2) PhOTO-M (3) PhOTO-N. The
membrane was probed for Dendra2 as well as a-Tubulin (loading
control, upper blot). No Dendra2 was seen in the WT (lane 1), and
each PhOTO lane had the Dendra2 protein at approximately the
molecular weight expected from the particular fusion protein. Note that for lane 2, two bands are present, reflecting the post-
translational palmitoylation/myristoylation additions. Efficient
protein cleavage as a result of the TaV 2A sequence was confirmed
by the lack of an uncleaved product within the two PhOTO lanes. (absence of a band above 50kDa in the lower blot). (TIF) Figure S2
Nuclear Photoconversion and Segmentation
in a PhOTO-N Zebrafish During Gastrulation. Animal
pole view maximum intensity projection (MIP) images of the first
10.5mm (in depth from the animal pole) of an ,6 hour time-lapse
of a heterozygote F1 PhOTO-N zebrafish from late gastrulation
(.80% epiboly) until early segmentation. (A) Merged MIP of the
first frame of the time-lapse, showing memb-Cerulean (blue) and
both unconverted (green) and segmented photoconverted (red)
H2B-Dendra2. (B) Merged MIP image of the final time frame of
the time-lapse. (C) Zoomed in MIP areas at four different time-
points of the merged fluorescence images as a reference for the
photoconverted images seen in panels (D) and (E). (D) Intensity
images from segmented photoconverted nuclei (red) for the same 4
timepoints as the merged image in panel (C). (E) Segmented nuclei
from the intensity images from panel (D). Two enveloping layer
(EVL) cells are photoconverted (the orange and red spheres) as
well as an epiblast cell (the white sphere). Each cell undergoes a
single cell division during the course of the time-lapse. Note that
the epiblast cells move beneath the field of view in the last frame, March 2012 | Volume 7 | Issue 3 | e32888 March 2012 | Volume 7 | Issue 3 | e32888 7 PLoS ONE | www.plosone.org PhOTO Zebrafish for Development and Regeneration PhOTO Zebrafish for Development and Regeneration due to the development of the head during early segmentation. Scale bars are 50mm. (TIF) animal pole images as seen in Video S1D. (C) Isolated zoomed
images of the segmented nuclei and membranes during the time-
lapse. Scale bars are 50mm. (MOV) animal pole images as seen in Video S1D. 1. Stern CD, Charite J, Deschamps J, Duboule D, Durston AJ, et al. (2006) Head-
tail patterning of the vertebrate embryo: one, two or many unresolved problems?
Int J Dev Biol 50: 3–15. Acknowledgments Video S2
Segmented Time-Lapse of PhOTO-N Devel-
opment
During
Late
Gastrulation. This
video
shows
segmented data for each frame of the time-lapse summarized in
Figure S2. The nuclei were segmented: the red and orange
spheres represent the EVL nuclei and the white spheres represent
the migrating epiblast nuclei. The membrane of the orange-
labeled EVL nucleus was segmented and is highlighted in all three
panels of the image in enhanced contrast blue. (A) Segmented and
photoconverted H2B-Dendra2 data (red) as well as the segmented
EVL membrane data (blue) with the bright field data (grayscale)
from the time-lapse video. The developing head and optic
primordia can be seen during the time-lapse. (B) Segmented and
photoconverted H2B-Dendra2 data as well as the enhanced
contrast segmented EVL membrane data along with the merged We would like to thank Le Trinh and Sean Megason for sharing reagents
and Le Trinh, Cambrian Liu, and Tatiana Hochgreb for assistance with
the western blotting. We would like to thank Andres Collazo for access to
the Confocal Microscopy Core at the House Research Institute, and Le
Trinh and Christopher Dempsey for comments on the manuscript. 2. Poss KD (2010) Advances in understanding tissue regenerative capacity and
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Amputation of Adult PhOTO-N Zebrafish
Before Photoconversion. (A) A digital camera photo of the
anesthetized PhOTO-N zebrafish after amputating a small portion
of the upper half of the tail fin. (B–E) A fluorescent stereomicro-
scope image of the amputated tail fin (anterior left, ventral down)
showing (B) bright field, (C) memb-Cerulean (blue), (D) uncon-
verted H2B-Dendra2 (green), and (E) background signal when
imaging with the same fluorescence emission filter as photo-
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detritus in the water that is picked up in the red fluorescence
channel. Scale bar for (B–E) is ,300mm. (TIF) Video S3
Time-Lapse Data for PhOTO-M Membrane
Dynamics Visualization During Late Gastrulation. This
video shows each frame of the time-lapse summarized in Figure 2
in the text. (A) H2B-Cerulean intensity data (blue), (B) unconvert-
ed memb-Dendra2 intensity data (green), (C) segmented photo-
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dynamic throughout the time-lapse, starting as a tight cluster of
cells and eventually moving apart laterally and away from the
developing head toward the ventral side of the embryo as the time-
lapse progresses. (D) Merged image of panels (A–C), which also
includes segmented nuclear data (multi-colored surfaces) within
each of the photoconverted membranes. Scale bar is 50mm. (MOV) Video S3
Time-Lapse Data for PhOTO-M Membrane
Dynamics Visualization During Late Gastrulation. This
video shows each frame of the time-lapse summarized in Figure 2
in the text. (A) H2B-Cerulean intensity data (blue), (B) unconvert-
ed memb-Dendra2 intensity data (green), (C) segmented photo-
converted memb-Dendra2 intensity data (red). Membranes are
dynamic throughout the time-lapse, starting as a tight cluster of
cells and eventually moving apart laterally and away from the
developing head toward the ventral side of the embryo as the time-
lapse progresses. (D) Merged image of panels (A–C), which also
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Marine pests in paradise: capacity building, awareness raising and preliminary introduced species port survey results in the Republic of Palau
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Author Campbell, ML, Hewitt, CL, Miles, J Title Title
Marine pests in paradise: capacity building, awareness raising and preliminary introduced species port survey
results in the Republic of Palau Copyright information This version of work is made accessible in the repository with the permission of the copyright holder/s under
the following, This version of work is made accessible in the repository with the permission of the copyright holder/s und
the following,
Licence. Rights statement: Copyright 2016 The Author(s)
If you believe that this work infringes copyright, please email details to: oa.repository@utas.edu.au
Downloaded from University of Tasmania Open Access Repository
Please do not remove this coversheet as it contains citation and copyright information. University of Tasmania Open Access Repository
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utas.edu.au Bibliographic citation Bibliographic citation
Campbell, ML; Hewitt, CL; Miles, J (2016). Marine pests in paradise: capacity building, awareness raising and
preliminary introduced species port survey results in the Republic of Palau. University Of Tasmania. Journal
t ib ti Campbell, ML; Hewitt, CL; Miles, J (2016). Marine pests in paradise: capacity building, awareness raising and
preliminary introduced species port survey results in the Republic of Palau. University Of Tasmania. Journal
contribution. https://figshare.utas.edu.au/articles/journal_contribution/Marine_pests_in_paradise_capacity_building_awarene Is published in: 10.3391/mbi.2016.7.4.05 Downloaded from University of Tasmania Open Access Repository Downloaded from University of Tasmania Open Access Repository Please do not remove this coversheet as it contains citation and copyright information. Licence. Rights statement: Copyright 2016 The Author(s) Abstract Introduced marine species can have a large impact upon small countries that are reliant on marine tourism. Non-governmental organisations,
such as the World Conservation Union (IUCN), are working with technical experts to implement capacity building and awareness programs
that transfers introduced marine species knowledge to countries in need of aid. The Republic of Palau is reliant on tourism and as such is pro-
actively engaging in this process to ensure that it has the necessary skills to determine and manage its introduced marine species pathways
and vectors. The IUCN with the aid of technical experts implemented a four day training workshop that provided both theoretical and
practical field experience with introduced marine species port surveys. An outcome of this exercise was the detection of 11 introduced
marine species, the training of 10 Palau agencies and two international organisations, and recommendations for future implementation that
will aid Palau to address the problem of introduced marine species within their borders. Key words: non-indigenous species, non-native species, marine pests, biofouling, biosecurity, environmental management, species criteria,
Pacific Island Countries concern is the reliance of a number of small island
states, specifically Pacific Island Countries (PICs),
on their marine environments, to the extent that the
“marine economy” derived from tourism, artisanal and
commercial fisheries and aquaculture contributes
more than 10% of Gross National Income (GNI) in
many instances (Table 1). Marine pests in paradise: capacity building, awareness raising and preliminary
introduced species port survey results in the Republic of Palau Marnie L. Campbell1,2,*, Chad L. Hewitt1,3 and Joel Miles4
1National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Newnham, Tasmania, 7250, Australia
2Environmental Research Institute, University of Waikato, Hamilton 3240, New Zealand
3School of Science, Faculty of Science and Engineering, University of Waikato, Hamilton 3240, New Zealand
4National Invasive Species Committee, Bureau of Agriculture, Ministry of Natural Resources, Environment & Tourism,
969940 Republic of Palau Marnie L. Campbell1,2,*, Chad L. Hewitt1,3 and Joel Miles4
1National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Newnham, Tasmania, 7250, Australia
2Environmental Research Institute, University of Waikato, Hamilton 3240, New Zealand
3School of Science, Faculty of Science and Engineering, University of Waikato, Hamilton 3240, New Zealand
4National Invasive Species Committee, Bureau of Agriculture, Ministry of Natural Resources, Environment & Tourism,
969940 Republic of Palau *Corresponding author
E-mail addresses: marnie.campbell@waikato.ac.nz (MLC), chad.hewitt@waikato.ac.nz (CLH), joelmiles52@gmail.com (JM) Corresponding author
E-mail addresses: marnie.campbell@waikato.ac.nz (MLC), chad.hewitt@waikato.ac.nz (CLH), joelmiles52@gmail.com (JM) addresses: marnie.campbell@waikato.ac.nz (MLC), chad.hewitt@waikato.ac.nz (CLH), joelmiles52@gma Received: 12 April 2016 / Accepted: 19 July 2016 / Published online: 17 September 2016 Handling editor: Vadim Panov University of Tasmania Open Access Repository Library and Cultural Collections
University of Tasmania
Private Bag 3
Hobart, TAS 7005 Australia
E oa.repository@utas.edu.au CRICOS Provider Code 00586B | ABN 30 764 374 782 utas.edu.au Management of Biological Invasions (2016) Volume 7, Issue 4: 351–363
DOI: http://dx.doi.org/10.3391/mbi.2016.7.4.05
© 2016 The Author(s). Journal compilation © 2016 REABIC Management of Biological Invasions (2016) Volume 7, Issue 4: 351–363
DOI: http://dx.doi.org/10.3391/mbi.2016.7.4.05
© 2016 The Author(s). Journal compilation © 2016 REABIC Research Article Research Article Open Access Introduction * – estimate
Country
GDP
Currency
Marine component (%)
Cook Island
$171,599,000
NZ$
11.31
Federated States of Micronesia
$229,869,864
US$
4.70
Fiji Islands
$3,587,300,000
F$
2.34
Kiribati
$74,100,000
AUD$
11.98
Marshal Islands
$97,311,800
US$
7.40
Nauru*
$80,000,000
AUD$
2.12
Niue
$14,210,300
NZ$
1.58
Palau
$129,601,000
US$
2.69
Papua New Guinea
$8,780,800,000
Kina
0.56
Samoa
$705,914,000
Tala
7.99
Solomon Islands*
$1,352,700,000
SI$
0.01
Tonga
$251,135,000
T$
7.13
Tuvalu
$22,044,500
AUD$
6.77
Vanuatu
$29,206
Vt million
0.95 (Brazil, China, India, Iran, South Africa, and the
Ukraine). This training provided basic skills in ballast
water management, ballast water risk assessment,
and taxonomic identification (IMO 2000b). In
general, these initiatives targeted stakeholders such
as universities, museums, navy, and port authorities. GISP had maintained a broader, all-ecosystem
approach to invasive species providing for example,
public awareness (e.g., invasive species posters),
training workshops, and advice to decision makers
(e.g., Simons and de Poorter 2008). Similarly, the
IUCN provides introduced species awareness raising
in countries such as Chile (e.g., Hewitt et al. 2006),
the Seychelles, and Samoa, and have also undertaken
some targeted introduced species surveys (e.g.,
Tamelander et al. 2009). contribution (e.g., Gillett and Lightfoot 2001; Gibson
and Nero 2008). For many PICs, fisheries can account
for more than 10% contribution to GNI, with marine
based tourism having an equal or greater share. Despite a high reliance on the marine environment
and marine resources for economic well-being,
many of these PICs have limited understanding
about introduced marine species and the risks they
pose to their economies. As Nuñez and Pauchard
(2010) have discussed, these developing states have
limited capacity or capability to meet the demands
of marine biosecurity within their domain. To address
this, a number of non-governmental and intergovern-
mental organisations have established programs to
ensure that all countries have access to information
about marine introduced species (awareness raising)
and can receive expert training (capacity building) to
facilitate regional and international outcomes. )
One example of the IUCN efforts is a facilitation
and capacity building exercise conducted in the
Republic of Palau (herein referred to as Palau) in
2007 on introduced species port survey methods,
both theoretical and applied. This paper provides an
overview of the exercise, including outcomes derived
from the workshop. Introduction Within the last two decades research on introduced
species has expanded from a focus on ballast water
mediated transport (e.g., Williams et al. 1988; Carlton
and Geller 1993), to a more holistic view of potential
vectors (e.g., Carlton 2001; Hewitt et al. 2004;
Padilla and Williams 2004; Fofonoff et al. 2003;
Campbell and Hewitt et al. 2013; Williams et al. 2015). Marine invaders have now been detected in
virtually all regions of the world oceans, with
indications that multiple vectors contribute to these
broad-scale distributions (e.g., Hayes et al. 2005;
Hewitt and Campbell 2007). Not all countries have
the capacity to determine the current state of invasions
within their waters or the ability to implement pro-
active biosecurity measures (e.g., Bax et al. 2003;
Hewitt and Campbell 2007; Nuñez and Pauchard
2010; Azmi et al. 2015a, 2015b). This lack of capacity
has serious biosecurity implications. Of particular y
(
)
The marine economy is a useful term to capture
the direct and indirect benefits derived from marine
based activities to GNI, which need to be examined
in the context of marine biosecurity. Marine economy
activities include more traditional economic drivers,
such as commercial export based fisheries, aqua-
culture, aquarium species collection, and oil and
gas extraction; as well as subsistence fisheries and
marine based tourism (see Table 1). While traditional
economic drivers have previously been calculated
for contribution to GNI, it is commonly realised that
the ability to capture less traditional aspects in PICs
is limited, resulting in an under-estimation of GNI 351 M.L. Campbell et al. Table 1. Pacific Island countries Gross Domestic Product (GDP), currency, and the percentage “marine economy” component of the GDP,
with the Republic of Palau highlighted by italics (source: Gillett and Lightfoot 2001). * – estimate Table 1. Pacific Island countries Gross Domestic Product (GDP), currency, and the percentage “marine economy” component of the GDP,
with the Republic of Palau highlighted by italics (source: Gillett and Lightfoot 2001). * – estimate Table 1. Pacific Island countries Gross Domestic Product (GDP), currency, and the percentage “marine economy” component of the GDP,
with the Republic of Palau highlighted by italics (source: Gillett and Lightfoot 2001). Introduction The work in Palau involved a
number of different in-country agencies, working with
Australian marine biosecurity experts who provided
training that was facilitated by the IUCN and the
Office of Environmental Response and Coordination,
Palau. The activity in Palau focussed on providing
an awareness of introduced marine species and the
problems they cause, training interested agencies in
port survey techniques and taxonomic sorting, and
introducing the concepts of risk assessment. Two examples of this proactive international
stance include: 1) the work undertaken by the Global
Ballast Water Management Programme (referred to
as GloBallast) — a collaboration between the Global
Environment
Facility
(GEF),
United
Nations
Development Program (UNDP) and the International
Maritime Organisations (IMO) (IMO 2000a); and 2)
the IUCN’s marine programme (IUCN 2013), with a
third organisation unfortunately closing in 2011 (the
Global Invasive Species Programme [GISP]; BGCI
2011). These organisations have provided training
for introduced species surveys, identification and
taxonomy, risk assessment, ballast water manage-
ment, and public awareness in a number of countries. p
Phase I of the Globallast programme undertook
introduced species port survey training in six countries g
p
One of the main objectives of this work was to
ensure that Palau would receive training in skills that 352 Introduced marine species in Palau would allow them to undertake their own introduced
species baseline surveys and establish monitoring
programs, thus ensuring that knowledge transfer
occurred. It is anticipated that follow-up surveys
within Palau will further build capacity and increase
knowledge about introduced marine species in this
region. Outcomes from the training exercise provided
preliminary data on the introduced species present in
the region and associated vectors that may pose a
risk to Palau. This type of information can then form
the basis of informed introduced species manage-
ment programs. aim of the developed survey was to detect introduced
marine species through examination of marine bio-
diversity. Therefore, survey sites were selected based
on likely primary inoculation points (areas with
overseas linkages) and secondary sites with high
frequency of visitation from primary inoculation points
(i.e., secondary transfer locations). In addition, sites
of high environmental, economic or socio-cultural
importance were identified for monitoring. Upon return from the field a literature search was
undertaken to identify known introduced and crypto-
genic species from the region to inform the develop-
ment of a biosecurity framework specific to Palau. Training workshop Palau is an archipelago of over 586 islands (eight
main islands) located east of the Philippine island of
Mindanao; the first Europeans to see the islands
were the Spanish in the 1500’s (Faulkner et al. 2004;
Yukihira et al. 2007). It is a biodiversity hotspot
(Faulkner et al. 2004; Yukihira et al. 2007) and a
well-known international tourist destination based
on its Rock Islands, jellyfish lakes and unique
SCUBA diving opportunities (Yamashita 2000). The
government is proactive, with regards to introduced
pests and are concerned with the potential impact
introduced marine species pose to their tourism
(including charter fishing) industries. Between 25th and 28th July, 2007, 10 Palauan agencies
(Bureau of Marine Resources, Bureau of Agriculture,
CARP Dive Tour Company, Koror State Government,
Office of Environmental Response and Coordination
(OERC), Palau Conservation Society, Palau Inter-
national Coral Reef Centre, Coral Reef Research
Foundation, Peleliu State Government, Sam’s Tours)
and two international organisations (IUCN, The Nature
Conservancy) were trained in introduced marine
species baseline survey design and techniques,
including undertaking a preliminary survey of
Malakal Harbour, Koror State, Palau, sample sorting
and para-taxonomic species identification. It is
important to note that the para-taxonomic training
was focussed on identifying obvious non-native
species (building capability) and providing knowledge
about where to find taxonomic information, such as
relevant databases or how to contact taxonomic experts. The supplementary material in this paper provides a
list of taxonomic databases and references that we
typically provide during port survey capacity building
training. Introduced species survey methods The preliminary survey served two purposes: 1) to
train the survey team in introduced species survey
methods; and 2) to undertake a preliminary examina-
tion of the species present in Malakal Harbour. The
survey aimed to detect introduced marine species
and provide an indication of their spatial distribution. Collection of native biodiversity data was a secondary
aim of the survey plan. Within this paper we discuss
the detection of introduced marine species, not
native species. A component of the project included in-water
training of methods and post-survey taxonomic
sorting with species identification. The survey training
specifically covered the Hewitt and Martin (1996,
2001) protocols, with further information provided
during a workshop on alternative protocols such as
the Rapid Assessment Survey (RAS) surveys (e.g.,
Cohen et al. 2001, 2005; Pedersen et al. 2003), the
Bishop Museum protocols (e.g., Coles and Eldredge
2002), the Chilean aquaculture survey protocols (e.g.,
Hewitt et al. 2006), and passive sampling (Ruiz and
Hewitt 2002; Wyatt et al. 2005; deRivera et al. 2005). p
An initial suite of survey sites were selected by
introduced species port survey specialists for consi-
deration by the workshop participants. This selection
of sites was subsequently discussed with workshop
participants to add local knowledge and thereby
improve site selection by adding or removing sites
where needed. This ensured that selection of sites
was refined and prioritised based on the heuristic
knowledge of the workshop participants, coupled
with the prioritisation knowledge provided in the
Hewitt and Martin (2001) introduced species sampling
principles (Table 2). At the end of the heuristic
workshop process, a total of 38 sites were identified, Workshop participants designed a baseline survey,
grounded by their shared local knowledge coupled
with their training in the Hewitt and Martin protocols
(1996, 2001). As with all baseline port surveys; the 353 M.L. Campbell et al. Table 2. Sites selected for introduced marine species surveys, including method of sampling, heuristic priorities and introduced species
priorities. Italic font indicates sites that would be sampled under a typical introduced marine species survey using the Hewitt and Martin
(1996, 2001) protocols. Table 2. Sites selected for introduced marine species surveys, including method of sampling, heuristic priorities and introduced species
priorities. Italic font indicates sites that would be sampled under a typical introduced marine species survey using the Hewitt and Martin
(1996, 2001) protocols. priorities. Introduced species survey methods Italic font indicates sites that would be sampled under a typical introduced marine species survey using the Hewitt and Martin
(1996, 2001) protocols. Site name
Method
Heuristic priority
Introduced spp priority
Training sites covered
Neco floating dock
Qualitative
2
1
Neco marine dock
Quantitative
2
1
PMIC
Quantitative
1
1
Sam’s Tours
Quantitative
1
1
Channel marker
Semi-quantitative
2
1
Bureau of Marine Resources/Belau -Mariculture
Demonstration Center
Quantitative
1
1
Marine Law Enforcement
Quantitative
1
1
Commercial Berth 3 (south)
Quantitative
1
1
Commercial Berth end
Qualitative
1
1
Phase 1 – Malakal port sites
Malakal causeway
Quantitative
3
2
Palau Royal Resort
Quantitative
2
1
Carp Restaurant
Quantitative
2
1
Fisheries wharf
Quantitative
2
1
Palau Island Traders International
Quantitative
1
1
BRM - mariculture
Quantitative
1
2
Marine law
Quantitative
1
1
Barge wreck
Qualitative
2
3
CPR – dry dock
Quantitative
2
1
Chandelier cave
Qualitative
3
4
Recreational anchorage
Qualitative
1
1
Recreational anchorage – Chinese wreck
Qualitative
1
3
Recreational anchorage - wreck
Qualitative
1
3
Old Japanese dry dock
Qualitative
3
1
Phase 2 – external to port – Koror State
Channel buoys 1
Semi- quantitative
1
1
Channel buoys 2
Semi- quantitative
1
1
Channel buoys 3
Semi- quantitative
1
1
Derelict tugs
Qualitative
1
3
Large foreign vessel anchorage Pincher Bay
Qualitative
3
3
PPR
Qualitative
3
2
Sea plane anchorage
Qualitative
3
1
T-dock (historical jetty)
Qualitative
2
1
KB (JP) bridge - north
Quantitative
1
2
KB (JP) bridge - south
Qualitative
1
2
Fish & fins/PICRC
Quantitative
2
1
Dump site
Qualitative
3
2
Ngetkedam
Qualitative
3
3
Anchorage – near helmut dive site
Qualitative
3
1
MV “Pristine”
Qualitative
2
3
Phase 3 – beyond Koror State (To be designed) Phase 3 – beyond Koror State (To be designed) sampled during a full Hewitt and Martin style
survey. By sampling fewer sites but maintaining a
high detection limit (statistical ability to find an
introduced species based on the sampling effort
expended), the efficiency of the survey is increased
both with regards to resource use and statistical
robustness. However, when training participants, it
is often desirable to include more sites to ensure the
inclusion of stakeholder opinion. Training workshops A four day workshop was conducted that involved
10 Palauan and two international organisations. The
initial 2 days involved classroom activities, where
participants were trained using a more traditional
style “chalk and talk” information provision session
followed by information transfer with group activities. During the “chalk and talk” session experts discussed
what introduced marine species are, the different
field survey techniques that exist for detecting
introduced species (sensu Campbell et al. 2007), and
how risk analysis can be used for marine biosecurity
(e.g., Campbell 2008; Campbell and Hewitt 2011, 2013). (
p
,
p
)
The modified criteria include an additional five
criteria to those that Chapman and Carlton (1991,
1994) originally suggested. These new additions were
derived by evaluating native and introduced species
patterns from national and international port survey
data sets (Australia, South Africa, Brazil; Pollard
and Hutchings 1990a, 1990b; Hewitt et al. 1999, 2004;
Hewitt and Campbell 2001; Hewitt 2002; Campbell
2003; Campbell et al. 2004; Hewitt and Campbell,
unpubl. data) and readily available international
species data present in the published literature (e.g.,
Cohen and Carlton 1995; Ribera and Boudouresque
1995; Brattegard and Holthe 1997; Zaitsev and Mamaev
1997; Cranfield et al. 1998; Zaitsev and Alexandrov
1998; Coles et al. 1999; Boudouresque and Verlaque
2002; CIESM 2002; Galil et al. 2002; Leppäkoski et
al. 2002; Orensanz et al., 2002; Green and Short
2003; Occhipinti-Ambrogi and Savini 2003; Castilla
et al. 2005). To determine if a species was native,
introduced or cryptogenic, a “weight of evidence”
approach was applied. Three of the new criteria are
based on patterns linked to an introduced species
affinity with mediated transport mechanisms (such
as vessels and aquaculture) that lead to a broader
distribution than may be observed by naturally
dispersing natives and are summarised as: Within the group activities a “strawman” model
of a Hewitt and Martin port survey for Malakal and
surrounding regions was provided to participants. The workshop participants then used their newly
acquired introduced species and survey knowledge,
plus their local knowledge to modify sites and decide
the best sampling techniques for each subsequently
selected site (Campbell and Hewitt 2008). This com-
bined method resulted in participants stating that they
felt confident that they had the skills to understand,
plan and implement a baseline survey for introduced
species with minimal guidance from experts. Introduced species survey methods within a three phase program (phase 1 – Malakal Harbor;
phase 2 – surrounding regions within Koror State;
and phase 3 – areas outside of Koror State) being
suggested to complete the site survey. This paper
concentrates on phase 1, with phases 2 and 3 underway
via the Koror Rangers at times of their convenience. Typically, an introduced species survey for a
region of this size would sample fewer sites (e.g., 22
sites) but retaining the ability to detect low density
invasions (e.g., Hewitt and Martin 2001). Indicated
in Table 2 (via italic font) are the sites that would be A number of sites that were sampled through
visual inspection (qualitative sampling) were added 354 Introduced marine species in Palau Figure 1. A selection of the survey sites in Palau: Malakal Harbour (A, B), “pristine” areas (C, D), and tourist sites (E, F). Figure 1. A selection of the survey sites in Palau: Malakal Harbour (A, B), “pristine” areas (C, D), and tourist sites (E, F). and –7m) in triplicate (n = 9 samples) where depth
allowed. When depths were limited (too shallow),
two depths (–0.5m and –3m) were sampled using four
replicates (n = 8 samples). During this preliminary
survey no benthic cores, phytoplankton or pelagic
samples were collected and hence sampling focussed
on biofouling communities. to the survey in an ad-hoc fashion, with introduced
or suspect introduced species being noted at all ad
hoc sites. Samples from the hulls of three vessels
were also collected in a qualitative fashion. Fouling
communities were sampled at 16 sites; nine sites
within Malakal Harbour and seven sites outside of
the harbour, including three “pristine” sites in the
Rock Islands (Figure 1). Standard 0.10 m2 quadrats
were used to sample hard substrate using the methods
described in Hewitt and Martin (1996, 2001). Quadrats were sampled at three depths (–0.5m, –3m, Collected specimens were placed on ice until
taxonomic sorting and identification occurred on the
afternoon of the day that collection occurred. Specimens were preserved using 70% ethanol and 355 M.L. Campbell et al. M.L. Campbell et al. M.L. Campbell et al. — Criterion 8: The regional (100’s – 1000’s km)
distribution of the introduced species is wide
when compared to similar native species’
regional distribution; labels were used to ensure that site and specimen
integrity was maintained following the methods of
Hewitt and Martin (1996). Identifying a species status A modified version of the Chapman and Carlton
(1991, 1994) 10-point criteria was used to determine
a species status (Table 3; Campbell et al., in review). These criteria evaluate ecological, geographical and
evolutionary attributes of a species, using deductive
reasoning to aid in the determination of a species
status. Knowledge that spans across multiple discip-
lines, such as taxonomy, phylogeny, genetics,
ecology, biology, and biogeography, is used to
assess the species status. The modified criteria have
been trialled (e.g., Brazil, China, India, Iran, Samoa,
the Seychelles) in regions where a historical track
record of native species exist and worked relatively
well (M Campbell, unpublished data). — Criterion 15: This genus is not present in the
country/island/continent (higher taxonomic affi-
nities are lacking). Introduced species survey methods Specimens were identified
to least taxonomic unit, aiming to obtain a species
level identification, so that species status (is it a
native, introduced, cryptogenic species) could be
derived. A number of para-taxonomic experts were
involved in the species identifications. — Criterion
10:
Introduced
species
have
a
widespread global distribution; — Criterion 14: Only one sex of a dimorphic species
can be detected. Several introduced species have
been identified where a single sex is detected and
reproduction is limited to asexual means alone. Founder effects may result in a reduced proba-
bility of individuals of both sexes being
introduced to a locale; and Training workshops The remaining two-days of the workshop involved
field activities where a group of people that would
be involved in undertaking port surveys had in-water
training in the field sampling and laboratory sorting
techniques. This training resulted in nine sites being
surveyed with additional ad hoc collections. The
preliminary survey detected introduced species and as
such is considered to be a successful implementation
of a knowledge and skills capacity building exercise. — Criterion 7: The local (<10’s km) distribution of
the introduced species is wide when compared to
similar native species’ local distributions; — Criterion 7: The local (<10’s km) distribution of
the introduced species is wide when compared to
similar native species’ local distributions; 356 Introduced marine species in Palau Table 3. Criteria used to determine if a marine species is introduced, cryptogenic or native (from Campbell et al., in review). The criteria are
modified from Chapman and Carlton (1991), with the exceptions of new additions, which are highlighted in bold font and shaded background. Geography
Criterion
Description
Provincial
1
Sudden local appearance
Provincial
2
Subsequent local spread
Provincial
3
Distribution associated with human mechanisms of dispersal
Provincial
4
Trophic dependence (and symbioses) on known introduced marine species
Provincial
5
Most prevalent in, or restricted to, new or artificial environments
Provincial
6
Local distribution restricted compared to native species
Provincial
7
Local distribution wider when compared to native species
Provincial
8
Regional species distribution wider when compared to native species
Global
9
Disjunct global distribution
Global
10
Widespread global distribution
Global
11
Active dispersal mechanisms are inadequate to attain current global distribution without human aid
Global
12
Passive dispersal mechanisms are inadequate to attain current global distribution without human aid
Global
13
The species is most similar morphologically, or genetically, to species in other regions of the world
Global
14
Only one sex of a dimorphic species can be detected
Global
15
This genus is not present in the country/island/continent (higher taxonomic affinities are lacking) national Convention for the Control and Management
of Ships Ballast Water and Sediments Ballast Water
Convention in 2004. Baseline survey results The preliminary field survey detected 11 introduced
and two cryptogenic and seven potentially introduced
species (Table 4). The introduced species were
dominated by bryozoans (46%), followed by ascidians
(27%), hydroids (18%) and barnacles (9%). Taxa from
the ascidians, polychaetes and porifera are potentially
introduced and need further analysis to confirm their
identity. Training workshops These species were detected in association
with wharf facings, floating docks, channel markers,
and as biofouling on international vessel hulls. Fourteen percent of the species detected on international
vessel hulls were not detected in the port environs,
inferring that international vessels are a potential vector
of concern as they may transfer new species to the
region. A number of species of concern, that can become
pests, or are known as pests elsewhere, were detected
(e.g., the hydroids Eudendrium carneum Clarke,
1882 and Thyroscyphus fruticosus (Esper, 1793), the
bryozoan Watersipora subtorquata (d’Orbigny, 1852),
and a tentative identification of the Caribbean barnacle
Chthamalus proteus Dando and Southward, 1980). These species have the potential to impact on native
fauna and flora in Palau, due to their propensity to
heavily foul substrata. Both E. carneum and T. fructicosus were previously known and are believed
to have been introduced with a floating bridge that
came from China in 1996 (Lambert 2002; Colin
2009). The method of introduction for the other
species is unknown but likely associated with vessel
biofouling given that Palau receives little international
ballast water but it does receive a large number of
recreational vessels (such as touring yachts). Historically, and in modern times, biofouling is
one of the primary mechanisms that introduced marine
species are transferred between locations (Godwin 2003;
Hewitt et al. 2004; Ashton et al. 2006; Davidson et al. 2008; Hopkins and Forrest 2008; Mineur et al. 2008;
Lee and Chown 2009; Galil et al. 2014). This vector
is relatively poorly managed (Hewitt and Campbell
2007; Lee and Chown 2009), although a number of
countries have introduced guidelines regarding vessel
management that specifically targets introduced
marine species vectoring via biofouling (e.g., USA:
California Hull Fouling Legislation Assembly Bill
740 (AB 740)). It’s not surprising that a lag period
for development and implementation of biofouling
management guidelines exists, given the 14-year lag
period we’ve seen for the adoption of the Inter- (
g y
)
Of concern is that some of the species detected
have the potential to become pest species and
damage tourist destinations. A similar situation has
already occurred in Palau, with the introduction of
the cnidarian, Aiptasia sp., into one of the major
international tourist destinations, Ongeim’l Tketau
(Jelly Fish Lake; Colin 2009), located on the rock
island of Mecherchar. Training workshops Palau relies on the tourism
industry, with 11% of the gross domestic product
being tourist related (US Department of State 2012)
and thus impacts upon drawcard tourist destinations
could have serious implications for the country’s
economy. 357 M.L. Campbell et al. Table 4. Detected introduced, cryptogenic and potentially introduced species during the preliminary Palau introduced species survey. Species are listed with an indication of the sampling locations/sites (wharves, moorings, vessel hulls, or “pristine” locations without
commercial activity). A “●” indicates presence upon a substrate. Please note that scientific names and taxonomic authorities (for species
names) were verified using the WoRMS database (http://www.marinespecies.org/). Table 4. Detected introduced, cryptogenic and potentially introduced species during the preliminary Palau introduced species survey. Species are listed with an indication of the sampling locations/sites (wharves, moorings, vessel hulls, or “pristine” locations without
commercial activity). A “●” indicates presence upon a substrate. Please note that scientific names and taxonomic authorities (for species
names) were verified using the WoRMS database (http://www.marinespecies.org/). Phyla
Species
Status
Wharves
Vessels
Mooring
Pristine
Porifera
Haliclona caerulea? (Hechtel, 1965)
Potential
●
●
●
Mycale sp. (orange sponge)
Potential
●
●
Hydroida
Eudendrium carneum Clarke, 1882
Introduced
●
Obelia sp. Cryptogenic
●
●
●
Thyroscyphus fruticosus (Esper, 1793)
Introduced
●
●
Polychaeta
Sabellastarte sp. Potential
●
Serpulididae
Potential
●
●
Cirripedia
Amphibalanus amphitrite (Darwin, 1854)
Cryptogenic
(cosmopolitan)
●
●
Chthamalus proteus Dando and Southward, 1980
Introduced
●
●
Bryozoa
Amathia distans Busk, 1886
Introduced
●
●
Virididentula dentata (Lamouroux, 1816)
Introduced
●
Bugula neritina (Linnaeus, 1758)
Introduced
●
●
Tricellaria occidentalis (Trask, 1857) /
T. inopinata D’Hondt and Occhipinti Ambrogi, 1985
Introduced
●
●
Watersipora subtorquata (D’Orbigny, 1852)
Introduced
●
Ascidia
Ascidia sydneiensis Stimpson, 1855
Potential
●
Botryllus sp. (cf. niger)
Potential
●
●
Didemnum perlucidum Monniot F., 1983
Introduced
●
●
Phallusia nigra Savigny, 1816
Introduced
●
Diplosoma listerianum (Milne Edwards, 1841)
Introduced
●
Pisces
Gobiidae sp. Potential
●
● Styela plicata (Lesueur, 1823), a species that is
native to Palau, but has been introduced to Australia
(e.g., Glasby 1999; Wyatt et al. 2005) and elsewhere
(e.g., da Rocha and Kremer 2005; de Barros et al. 2009), was detected in moderate densities at the
commercial wharves on facings. This species is a pest
in some regions (e.g., Glasby 1999), reaching high
densities and fouling infrastructure (e.g., Glasby 1999;
Connell 2000). Training workshops Given the presence of this species in
the port environment, it is possible that Palau may
act as a donor region for this species, with a pathway
related to international recreational vessels that visit
Palau during larger treks. the timing of likely introduction, species were
assigned association with primary vectors of
introductions (e.g., ballast water, vessel biofouling,
intentional aquaculture species and hitch-hiker
aquaculture species). The majority of species (98%)
have traits that indicate vessel biofouling is the highest
likelihood vector, however the vectors of ballast
water (40%), aquaculture hitch-hikers (39%) and
intentional introductions (31%) have a substantial
proportion of species that may have been transported
by these means. Within Palau, six introduced and 13 cryptogenic
species have been identified through biodiversity work
undertaken by the Coral Reef Research Foundation
(Lambert 2002; Colin 2009; supplementary material
Table S1). Within (Western) Samoa and American
Samoa, nine introduced species have been detected
(P. Skelton, pers. comm.; Table S1). Similarly, within
Guam 40 introduced and 45 cryptogenic species have
been detected by surveys using the Bishop Museum
protocols (Paulay et al. 2002; Table S1). Surveys
using the Hewitt and Martin protocols have detected
25 introduced species and six cryptogenic species
within tropical Australia (Hewitt 2002; Table S1). Detecting and identifying introduced marine species Based on dialogue during the workshop and
outcomes of the field survey, the following recom-
mendations were made to the OERC and various
State Governments to improve introduced marine
species management in Palau: The combined literature review and preliminary
baseline survey identified a total of 11 introduced,
17 cryptogenic and 12 potentially introduced species
for Palau. These species were typically associated
with artificial substrates, such as wharves, floating
buoys and vessel hulls. 1. An evaluation of the roles and responsibilities of
national and state governments for marine
biosecurity delivery needs to occur; Pre-existing literature Alternatively, this lack of overlap may also be due to
the dearth of pathways shared between the regions;
however this seems unlikely given that these regions
are historically linked by wartime activity during
WWII, and currently linked by recreational traffic
(Oliver 1989; Marti 2004) and in some instances
commercial traffic (Campbell and Hewitt 1999; Ruiz
et al. 2015). We note however, that commercial traffic
has been reduced with modernisation of ports and
vessels (e.g., Ward 1989). position of National Invasive Species Coordinator
was created and filled; this position also acts as the
secretary for the NISC. In March 2016, the Palau
President signed into law the Biosecurity Act
(http://www.paclii.org/pw/legis/num_act/ba2014rn9582015241/). This new law contains an update to Quarantine laws
(http://www.islandtimes.us/index.php?option=com_content&vie
w=article&id=644:biosecurity-bill-becomes-law). Many aspects
of the early legislation is focussed or implemented in
an agricultural and land management context, with
an outward focus on preventing pest species
reaching Palau. After this initial training exercise (as
described in this paper), non-native marine species
and survey expertise was established and initiatives
were put in place to consider and improve the manage-
ment of marine ecosystems. The Palau National Invasive
Species Committee is very pro-active and involved
in further initiatives across the Pacific island region
(http://www.palaunisc.org/news--noteworthy/archives/06-2016). Pre-existing literature Previous introduced species and biodiversity research
from Palau (Lambert 2002; Golbuu et al. 2005;
Colin 2009), and the wider Indo-Pacific biogeographic
region including: tropical Australia (Hewitt 2002),
Guam (Paulay et al. 2002), Samoa and American
Samoa (P. Skelton, pers. comm.), had collectively
identified 127 introduced or cryptogenic marine or
estuarine taxa. Ascidians represent a substantial
portion (33%) of these introductions, followed by
bryozoans (11%), hydroids (11%) and then bivalves
(9%; Figure 2). Based on life-history characters and Of the potential and possible introduced species
in Palau, there are no shared introduced and crypto-
genic species with tropical Australia; however Palau 358 Introduced marine species in Palau Figure 2. Number of
introduced and cryptogenic
marine species by taxa
introduced into Palau,
Guam, Samoa and tropical
Australia. 0
5
10
15
20
25
30
35
40
45
50
Urochordata
Bryozoa
Cnidaria ‐ hydrozoa
Mollusca ‐ bivalvia
Polychaeta
Pisces
Porifera
Algae
Arthropods ‐ malacostrata
Cnidaria ‐ anthozoa
Molluscs ‐ gastropoda
Arthropodes ‐ cirripedia
Arthropods ‐ isopoda
Echinodermata
Numbers
Taxa Figure 2. Number of
introduced and cryptogenic
marine species by taxa
introduced into Palau,
Guam, Samoa and tropical
Australia. and Samoa share one introduced species (T. fruticosa),
and Palau and Guam, share three introduced species
(Didemnum perlucidum Monniot F., 1983, Diplosoma
listerianum (Milne Edwards, 1841), Lissoclinum fragile
(Van Name, 1902)). The lack of species-sharing between
regions may be an artefact of taxonomic effort, or
a result of the qualitative versus quantitative nature
of the survey sampling methods used in these areas. Alternatively, this lack of overlap may also be due to
the dearth of pathways shared between the regions;
however this seems unlikely given that these regions
are historically linked by wartime activity during
WWII, and currently linked by recreational traffic
(Oliver 1989; Marti 2004) and in some instances
commercial traffic (Campbell and Hewitt 1999; Ruiz
et al. 2015). We note however, that commercial traffic
has been reduced with modernisation of ports and
vessels (e.g., Ward 1989). and Samoa share one introduced species (T. fruticosa),
and Palau and Guam, share three introduced species
(Didemnum perlucidum Monniot F., 1983, Diplosoma
listerianum (Milne Edwards, 1841), Lissoclinum fragile
(Van Name, 1902)). The lack of species-sharing between
regions may be an artefact of taxonomic effort, or
a result of the qualitative versus quantitative nature
of the survey sampling methods used in these areas. Figure 2. Number of
introduced and cryptogenic
marine species by taxa
introduced into Palau,
Guam, Samoa and tropical
Australia. Recommendations 2. A full baseline port survey, including regions
beyond Koror State, should be implemented to
gauge the presence of introduced and cryptogenic
species that may cause high risk impacts to
economic, cultural, social and environmental
values. The preliminary findings suggest that
commercial and non-commercial vessel areas
(e.g., tourist destinations) are a high priority. We
note that since the initial sampling reported here, When this research was undertaken Palau had two
pieces of legislation relating to non-native species:
Palau National Code Title 25, Chapter 20 refers to
Quarantine laws; and in 2004 Palau published the
Palau National Invasive Species Strategy (https://www. cbd.int/doc/submissions/ias/ias-pw-strategy-2007-en.pdf),
which included the establishment of The National
Invasive Species Committee (NISC). In 2006, the which included the establishment of The National
Invasive Species Committee (NISC). In 2006, the 359 M.L. Campbell et al. M.L. Campbell et al. additional sampling has started to occur and is
ongoing (I. Olkeriil, pers. comm.). However, the
recommendation that a full baseline port survey
be conducted has not been acted upon; Acknowledgements 7. Eradication and management plans for a number
of potential high risk species should be
developed for rapid implementation in the
likelihood that a high risk introduced species is
detected; and This work was partially funded by the IUCN, TOTAL, the Marine
Biosecurity Education Consortium (funded by the Australian
Department of Environment), and the Australian Maritime
College. The Palau Office of Environmental Response and
Coordination and the Koror State Government provided funding
for extending the field survey and undertaking species
identification. We wish to acknowledge and thank the people that
took part in the training and provided information, particularly
Ilebrang Okeriil (regulatory and implementation information), and
Lori and Patrick Colin of the Coral Reef Research Foundation
(CRRF). Lori and Patrick strongly advocated for capacity
building in Palau that led to this work and provided substantive
pre-existing taxonomic information and identifications as part of
the US National Cancer Institute Marine Natural Products
Collection program. We also thank and acknowledge Carmen
Primo (University of Tasmania) for identification of ascidian
samples. Last, we thank and acknowledge the reviewers for their
constructive comments. 8. Mooring buoys and ropes at tourist destinations
need to be changed frequently to prevent the
transfer and establishment of introduced species
between tourist locations. For a number of reasons, including limited resources,
most of these recommendations have had no, or only
partial, implementation. Palau continues to explore
avenues for obtaining resources for this purpose. It should be noted that a Regional Biosecurity
Plan (RBP) for Micronesia and Hawaii has been
developed and adopted in association with build-up
of US Government resources in Guam. The assessment
for the RBP evaluated both direct and regional risks
and impacts across terrestrial, freshwater and marine
systems and included several elements of the
recommendations listed above in the marine assess-
ment (Ruiz et al. 2015) The Strategic Implementation
Plan (SIP) for the RBP outlines numerous activities
for implementation of the plan; activities specific to
Palau include a number of marine-related activities,
including development and adoption of hull fouling
regulations, and building capacity to enforce such
regulations. Conclusions additional sampling has started to occur and is
ongoing (I. Olkeriil, pers. comm.). However, the
recommendation that a full baseline port survey
be conducted has not been acted upon; Palau is a pristine destination that attracts a large
number of international tourists; however introduced
marine species are becoming prevalent in this region,
which could have economic implications for tourism. Palau is a pristine destination that attracts a large
number of international tourists; however introduced
marine species are becoming prevalent in this region,
which could have economic implications for tourism. The Palau government is pro-actively attempting to
prepare for the impacts that may be associated with
introduced species. As part of this preparation, an
introduced marine species capacity building exercise
that combined both theory and field experience was
implemented within Koror State. This was managed
via a workshop that trained 10 Palau and two
international agencies in introduced marine species
and port survey techniques. The survey and subsequent
literature review detected 11 introduced, two crypto-
genic and seven potentially introduced species. A
series of recommendations were made to aid Palau
in its ability to prepare and deal with introduced
marine species. This work is preliminary in nature
and continued efforts on introduced species manage-
ment are needed to ensure Palau is fully prepared for
this global problem of introduced marine species. 3. Risk assessments should occur to determine high
risk routes (pathways), and transport vectors into
Palau and into different Palau islands. By
understanding the pathways and vectors of intro-
duced species effective management strategies
can be formulated; 4. A risk assessment of high risk species that pose a
future threat to Palau should be conducted to aid
in the creation of effective preparedness plans
that include targeted surveillance, with rapid
response plans (also see points 6 and 7); 5. A programme for inspection of high risk vessels
(based on risk assessments) entering Koror State
should be considered. Consideration of domestic
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component of global change in stressed marine ecosystems. Marine Pollution Bulletin 46: 542–551, http://dx.doi.org/10.1016/
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surveys for introduced marine species – design considerations,
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JR, Zabin CJ (2015) A vector analysis of marine ornamental
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13–29, http://dx.doi.org/10.3391/mbi.2015.6.1.02 Ruiz GM, Zabin CJ, Ashton GV, Campbell M, Davidson IC,
Eldredge LG, Fofonoff PW, Hewitt CL, Minton M (2015)
Marine Invasive Species Risk Assessment for the Common-
wealth of the Northern Mariana Islands, Guam, Hawai’i, Palau,
the Federated States of Micronesia, and the Republic of the
Marshall Islands, Regional Biosecurity Plan for Micronesia and
Hawaii Vol III. Prepared by: University of Guam and the
Secretariat of the Pacific Community 2014, http://www.navfac. navy.mil/navfac_worldwide/pacific/about_us/regional-biosecurity-plan-
for-micronesia-and-hawaii-.html p
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Wyatt ASJ, Hewitt CL, Walker DI, Ward TJ (2005) Marine
introductions in the Shark Bay World Heritage Property,
Western Australia: a preliminary assessment. Diversity and Distri-
butions 11: 33–44, http://dx.doi.org/10.1111/j.1366-9516.2005.00109.x Yamashita S (2000) The Japanese Encounter with the South:
Japanese Tourists in Palau. The Contemporary Pacific 12: 437– Simons S, de Poorter M (2008) Best Practices in Pre-Import Risk
Screening for Species of Live Animals in International Trade. References Proceedings of an Expert Workshop on Preventing Biological
Invasions. University of Notre Dame, Indiana, USA, 9–11 April
2008, 32 pp 463, http://dx.doi.org/10.1353/cp.2000.0071 Yukihira H, Shimoike K, Golbuu Y, Kimura T, Victor S, Ohba H
(2007) Coral reef communities and other marine biotopes in
Palau. In: Kayanne H, Omori M, Fabricius K, Verheij E, Colin
P, Golbuu Y, Yukihira Y (eds), Coral Reefs of Palau. Palau
International Coral Reef Center, Palau, pp 10–29 Tamelander J, Campbell M, Lundin CG (2009) Detecting Bioinvasions
on Small Islands in the Indian Ocean – Project Completion
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Switzerland, 24 pp Zaitsev YP, Mamaev V (1997) Marine Biological Diversity in the
Black Sea. A Study of Change and Decline. United Nations
Publications, New York, 208 pp US Department of State (2012) US Relations with Palau, http://www. state.gov/r/pa/ei/bgn/1840.htm (accessed 22 October 2013) Zaitsev YP, Alexandrov BG (1998) Black Sea Biological Diversity,
Ukraine. United Nations Publications, New York, 351 pp Ward GR (1989) Earth’s empty quarter? The Pacific Islands in a
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vessel ballast water as a vector for the transport of non-
indigenous marine species. Estuarine, Coastal and Shelf Science
26: 409–420, http://dx.doi.org/10.1016/0272-7714(88)90021-2 Supplementary material The following supplementary material is available for this article: Table S1. Pre-existing knowledge of introduced and cryptogenic species in Palau, Guam, Western and American Samoa, and tropical Australia. Table S1. Pre-existing knowledge of introduced and cryptogenic species in Palau, Guam, Western and American S Table S1. Pre-existing knowledge of introduced and cryptogenic species in Palau, Guam, Western and American Samo Appendix 1. Taxonomic databases and networks (listed alphabetically). Appendix 1. Taxonomic databases and networks (listed alphabetically). Appendix 2. Taxonomic bibliography. This material is available as part of online article from: Table S1. Pre-existing knowledge of introduced and cryptogenic species in Palau, Guam, Western and American Samoa, and tropical Australia.
A
di 1 T
i d t b
d
t
k (li t d l h b ti
ll ) This material is available as part of online article from: http://www.reabic.net/journals/mbi/2016/Supplements/MBI_2016_Campbell_etal_Supplement.xls
http://www.reabic.net/journals/mbi/2016/Supplements/MBI_2016_Campbell_etal_Appendix1.pdf
http://www.reabic.net/journals/mbi/2016/Supplements/MBI_2016_Campbell_etal_Appendix2.pdf 363
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Supplementary Figure S5 Supplementary Figure S5 Chromosome 3 Copy Number and B-alleles 0.0
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SUPPLEMENTARY FIGURE S5: Copy number of the PIK3CA locus based on WES data. The
loss of one copy of PIK3CA in certain subclones detected by miFISH single cell analysis was not
detected by WES data analysis. A ~20% shift in cellularity for a single copy change is beyond the
resolution of WES. Supplementary Figure S5 Tumor Biopsy
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loss of one copy of PIK3CA in certain subclones detected by miFISH single cell analysis was not
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resolution of WES.
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Marine sediment as a likely source of methyl and ethyl iodides in subpolar and polar seas
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1 Faculty/Graduate School/School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan. 2 Arctic Research
Center, Hokkaido University, Kita-21 Nishi-11 Kita-ku, Sapporo, Hokkaido 001-0021, Japan. 3 Himeji City Science Museum, 1470-15 Aoyama, Himeji, Hyogo
671-2222, Japan. 4 Graduate School of Human Science and Environment, University of Hyogo, 1-1-12, Shinzaike-honcho, Himeji, Hyogo 670-0092, Japan.
5 National Institute of Polar Research, 10-3, Midori-cho, Tachikawa, Tokyo 190-8518, Japan. 6 Tokai University, Department of Marine Biology and Sciences,
5-1-1, Minamisawa, Minami-ku, Sapporo, Hokkaido 005-8601, Japan. ✉email: ooki@fish.hokudai.ac.jp MMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsenv ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 net (mesh = 100 μm) towing from the bottom layer (87 m depth)
to the surface on 15 March 2019. Other dominant species were
Chaetoceros spp. and other Thalassiosira sp. Although small-sized
phytoplanktons could not be detected by the microscopic analysis,
we considered that diatoms were the dominant species in the
spring bloom 2019 on the ground of the pigment analysis result
that diatoms occupied >90% of depth integrated chl-a within
euphotic layer in spring blooms 2010 and 2011 in the Funka
Bay24. In April of both years the concentrations dropped below
4 μg L–1, indicating termination of the spring bloom. In the bot-
tom
water
(80–96 m)
we
found
high
concentrations
of
chlorophyll-a (>7 μg L–1) from the beginning of the diatom bloom
in early March of both years until middle of March 2018 and April
2019. The bottom water was below the euphotic zone, so the
chlorophyll-a maximum in the dark bottom water was caused by
settling of diatom aggregates produced in the surface water during
the bloom. The concentration of C2H5I in the surface sediment
(Fig. 2b) showed annual minima on 4 March 2018 (25 pmolL–1)
and 14 December 2018 (24 pmolL–1), and dramatically increased
on 18 March 2018 (257 pmol L–1) and 15 March 2019 (178 pmol
L–1). These increases were found 11–14 d after the detections of
increases in bottom-water chlorophyll-a. The high concentrations
of C2H5I in the sediment continued through August and dropped
in October. In the water column, the annual minimum for C2H5I
(<1 pmolL–1) was during the winter and into the spring bloom. The bottom-water concentrations dramatically increased post-
bloom on 8 April 2018 (5.4 pmolL–1) and 14 April 2019
(5.4 pmolL–1), 16–30 d after the detections of dramatic increases
in the sediment. The high concentrations in the bottom water
lasted until August in 2018 and until June in 2019. T T
he ocean surface is the dominant source of atmospheric
iodine1,2. Volatile organic iodine compounds (VOIs) such
as methyl iodide (CH3I) contribute to 20–40% of supply of
the
reactive
iodine
in
the
upper
troposphere
and
lower
stratosphere3. Although another longer-lived VOI, C2H5I, is less
abundant in air and surface seawater worldwide4–7, first estimates
suggest that sea-to-air flux of C2H5I in the world coastal ocean
accounts for 10% of total monohaloalkane iodine emission8. Results and discussion
T
l
i i
f Temporal variations of VOIs profile in water column and
sediment in Funka Bay. Time-series cross sections of chlor-
ophyll-a, C2H5I, and CH3I in the water column and sediment in
Funka Bay, Hokkaido, Japan, (Fig. 2) indicate a potential source of
VOIs in the sea-floor sediment. All observation data are provided
in Supplementary Data 1–4. The annual maximum chlorophyll-a
concentration (>12 μg L–1) in the surface water was found in
March of both 2018 and 2019 (Fig. 2a). Such high levels of
chlorophyll-a indicate a spring diatom bloom in the bay23. The
maximum level of 29.7 μg L–1 on 4 March 2019 was much higher
than 12.5 μg L–1 on 18 March 2018. It suggests that the magnitude
of diatom bloom in 2019 was greater than 2018. From microscopic
image analysis, Thalassiosira nordenskioeldii occupied 14.2% of
number of phytoplankton cells (n = 1209) collected by plankton At the lower part of middle layer water (65–75 m) and bottom
layer water (85 m), the concentrations of C2H5I and CH3I
decreased by half from 12 June to 29 August in 2019 (squares
outlined with black-and-white dotted lines in Fig. 2b, c), where −170˚
E
65˚
2017
2017&18
2018
50 m
50 m
50 m
50 m
Bering Sea
Chukchi Sea
N
140˚
145˚
40˚
45˚
42°
140°30’
141°00’
St. 30
80 m
60 m
Funka Bay
Hokkaido
T
O
T: Subtropical Tsugaru current
O: Subpolar Oyashio current
Hakodate
N Pacific
Funka Bay
Chukchi and Bering Seas
N
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Hokkaido
Subarctic N Pacific Ocean
Hokkaido
Chukchi and
Bering Seas
N Bering Sea
St4
St8
St14
St22
a
b
c
d
St30
St19
St21
St23
Fig. 1 Sampling sites. a Map of North Pacific including study areas. b Map of Hokkaido Island in Japan. c Sampling site of Funka Bay in Hokkaido, Japan. Black circle indicates the station 30. d Sampling sites of Chukchi and Bering Seas. White and gray circles indicate sampling sites in 2017 and 2018,
respectively. Black circle indicates a common sampling site in 2017 and 2018. Northern Bering Sea stations and Bering Sea stations are 14, 19, 22 and 4, 8,
21, 23 respectively. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 T In the ocean the dominant producers of VOIs have been
thought to be marine plants such as macro algae9,10, micro
algae11–13, and photosynthetic bacteria14,15. Very few studies have
taken notice of VOI production by heterotrophic bacteria16–18. Abiotic photochemical production of CH3I has been suggested19. Most previous studies have focused on the production of VOIs in
the upper euphotic zone, where marine plants grow. High levels of
CH3I in surface water occur widely in the oligotrophic subtropical
open oceans, and relatively low levels occur in subpolar and polar
open oceans6. In contrast, concentrations of C2H5I in surface
water are high in subpolar and polar continental shelf areas
compared with the substantially lower levels in subtropical open
oceans6,20. Recent studies have shown high concentrations of
C2H5I and some VOIs in bottom water of the polar Chukchi Sea
and northern Bering Sea21, and subpolar Funka Bay, Japan22. These high concentrations have given rise to the question of
whether there is a VOI source near the sea floor. In this study, we
measure the concentrations of VOIs in the sea-floor sediment by
shipboard observations (Fig. 1). We also conducted laboratory
incubations to test the hypothesis that diatom aggregates settled
on the sea floor emit VOIs in darkness. As for CH3I, the timings of increases in the sediment and bottom
water (Fig. 2c) were consistent with those of C2H5I. The annual
average concentrations of surface-sediment C2H5I (118 pmol L–1)
and CH3I (51 pmol L–1) were 20 and 8 times those in the bottom
water, respectively. It is therefore obvious that the increases in
bottom water were led by transfer from much higher concentrations
in the surface sediment. The production of C2H5I and CH3I from
diatom aggregates in relation to bacterial activity in darkness has
already been reported18. We, therefore, assumed that the C2H5I and
CH3I in the sediment originates from diatom aggregates settled on
the sea floor. This assumption was confirmed by dark incubation
experiments using diatom aggregates collected in Funka Bay. Marine sediment as a likely source of methyl and
ethyl iodides in subpolar and polar seas
Atsushi Ooki
1,2✉, Keita Minamikawa1, Fanxing Meng1, Naoya Miyashita1,3,4, Toru Hirawake
1,5,
Hiromichi Ueno
1,2, Yuichi Nosaka6 & Tetsuya Takatsu
1
https://doi.org/10.1038/s43247-022-00513-7
OPEN Atsushi Ooki
1,2✉, Keita Minamikawa1, Fanxing Meng1, Naoya Miyashita1,3,4, Toru
Hiromichi Ueno
1,2, Yuichi Nosaka6 & Tetsuya Takatsu
1 The surface ocean is an important source of longer-lived volatile organic iodine compounds,
such as methyl and ethyl iodide. However, their production in deep waters is poorly con-
strained. Here, we combine shipborne seawater and sediment core analyses to investigate
the temporal and vertical distribution of methyl and ethyl iodide concentrations in the Bering
and Chukchi Seas in July 2017 and 2018, and Funka Bay, Japan, between March 2018 and
December 2019. We find the highest methyl and ethyl iodide concentrations in sediment
porewaters and at the sediment surface. In Funka Bay, concentrations in the sediment and
bottom waters strongly increased following the spring bloom and subsequent settling of
phytodetritus to the seafloor. Dark incubation experiments suggest that diatom aggregates
are a source of methyl and ethyl iodide to the ambient water. Our findings suggest that
phytodetritus at the seafloor is a potential source of volatile organic iodine compounds to the
water column in polar and subpolar coastal oceans. 1 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 White dotted lines separate bottom water below 80 m
outlined with black-and-white dotted lines indicate the temporal-vertical range where C2H5I and CH3I concentrations decreased with tim 5 (W
WO
t
)
i
d
t th
l
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b 9%
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Euphotic zone
Euphotic zone
s of vertical profiles in water column and sediment. a Chl-a in water column. b C2H5I in water column and sediment. c CH3I in water
nt. The dates of annual rises in concentration are marked with triangles below each panel. Solid black lines indicate the euphotic zone
osynthetically active radiation (PAR) is 1% of the surface PAR. White dotted lines separate bottom water below 80 m depth. Squares
and-white dotted lines indicate the temporal-vertical range where C2H5I and CH3I concentrations decreased with time. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 the water mass25 (W or WO waters) remained at the layers
between the two dates (Supplementary Fig
S1)
However
The biological loss rate for CH3I has been repor
day−1 at 7 °C in the subarctic Atlantic Ocean wh
0
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Annual rise
Annual rise
Annual rise
Annual rise
Annual rise
Annual rise
Annual rise
Bottom layer
Bottom layer
Bottom layer
Euphotic zone
Euphotic zone
Euphotic zone
Fig. 2 Time sections of vertical profiles in water column and sediment. a Chl-a in water column. b C2H5I in water column and sediment. column and sediment. The dates of annual rises in concentration are marked with triangles below each panel. Solid black lines indicate the
depth, at which photosynthetically active radiation (PAR) is 1% of the surface PAR. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 0
2
4
[cm]
Sediment
0
120
240
0
2
4
6
8
10
[pmol/L]
0
2
4
6
8
10
[μg/L]
CHI
20
40
60
80
[m]
Water
20
40
60
80
[m]
Water
M
M
J
2018
2019
N
J
S
M
M
J
N
S
0
Depth
M
M
J
2018
2019
N
J
S
M
M
J
N
S
0
Depth
(b)
(a)
Chl-a
CHI
3/4
3/4
3/18
3/15
4/8
4/14
Annual rise
Annual rise
Annual rise
Annual rise
Annual rise
Annual rise
Bottom layer
Bottom layer
Euphotic zone
Euphotic zone (b) 0
2
4
[cm]
Sediment
0
120
240
0
2
4
6
8
10
[pmol/L]
M
M
J
2018
2019
N
J
S
M
M
J
N
S
0
20
40
60
80
Water
[m]
Depth
CHI
(c)
3/18
3/15
4/8
4/14
Annual rise
Annual rise
Annual rise
Annual rise
Bottom layer
Euphotic zone (c) Fig. 2 Time sections of vertical profiles in water column and sediment. a Chl-a in water column. b C2H5I in water column and sediment. c CH3I in water
column and sediment. The dates of annual rises in concentration are marked with triangles below each panel. Solid black lines indicate the euphotic zone
depth, at which photosynthetically active radiation (PAR) is 1% of the surface PAR. White dotted lines separate bottom water below 80 m depth. Squares
outlined with black-and-white dotted lines indicate the temporal-vertical range where C2H5I and CH3I concentrations decreased with time. the water mass25 (W or WO waters) remained at the layers
between the two dates (Supplementary Fig. S1). However,
elevated concentrations of phosphate (PO43−) at the layers,
which were derived from decomposition of organic matter settled
on the sea floor, decreased by 0–17% only between the two dates
(Supplementary Fig. S2). As for the obvious decreases in C2H5I
and CH3I concentrations at the layers, we presumed that
biological losses of C2H5I and CH3I occurred in the water. In
2018, elevations of C2H5I concentration and following decreases
at 65–75-m depths were found between May and June, however
obvious elevation of CH3I concentration at the depths were not
detected. It is possible that biological losses of C2H5I and CH3I in
the middle layer had advanced in 2018 compared to 2019. the water mass25 (W or WO waters) remained at the layers
between the two dates (Supplementary Fig. S1). Results and discussion
T
l
i i
f Video recordings of the sea floor were taken at station 30 in Funka Bay and at stations 4, 8, 14, and 22 in the Bering Sea (Supplementary
Movie 1–4, 6). 140˚
145˚
40˚
45˚
Hokkaido
T
O
T: Subtropical Tsugaru current
O: Subpolar Oyashio current
N Pacific
Hokkaido
b 42°
140°30’
141°00’
St. 30
80 m
60 m
Funka Bay
Hakodate
Funka Bay
c −170˚
E
65˚
2017
2017&18
2018
50 m
50 m
50 m
50 m
Bering Sea
Chukchi Sea
N
Chukchi and Bering Seas
N Bering Sea
St4
St8
St14
St22
d
St30
St19
St21
St23 N
E
−500
−500
−500
−500
−500
−500
0
0
0
0
0
0
0
0
0
0
0
40˚
50˚
60˚
65˚
180˚
−160˚
160˚
140˚
Subarctic N Pacific Ocean
Hokkaido
Chukchi and
Bering Seas
a Subarctic N Pacific Ocean Hokkaido Chukchi and
Bering Seas b 45˚ E 145˚ Fig. 1 Sampling sites. a Map of North Pacific including study areas. b Map of Hokkaido Island in Japan. c Sampling site of Funka Bay in Hokkaido, Japan. Black circle indicates the station 30. d Sampling sites of Chukchi and Bering Seas. White and gray circles indicate sampling sites in 2017 and 2018,
respectively. Black circle indicates a common sampling site in 2017 and 2018. Northern Bering Sea stations and Bering Sea stations are 14, 19, 22 and 4, 8,
21, 23 respectively. Video recordings of the sea floor were taken at station 30 in Funka Bay and at stations 4, 8, 14, and 22 in the Bering Sea (Supplementary
Movie 1–4 6) OMMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsen 2 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 3 VOIs in water column and sediment in Bering and Chukchi Seas. a Proportional composition (%) of VOIs and their total concentrations
(pmol L–1) in the water column and sediment in Chukchi Sea. b Northern
Bering Sea. c Bering Sea. d Funka bay. Proportion of CH3I, C2H5I, CH2ClI,
and CH2I2 are shown in boxes with gray, white, shade, and black,
respectively. The water column was divided into two layers: near-bottom
water and the upper water column. The values for the northern Bering Sea
and Bering Sea are averages for each area, and the values for Funka Bay are
total averages for the time-series observations. Water depths of B (−5 m)
and B (−0.3 m) are depths 5 m and 0.3 m above the bottom, respectively. phytodetritus settled on the sea floor is a hotspot of VOIs
production in the ocean. Here, we briefly mention the obvious CH3I maxima, which
were found from May to August within the oligotrophic euphotic
zone (Fig. 2c). While the chlorophyll-a concentrations in that
period were much lower than those in the bloom, CH3I
concentrations (4.5–8.5 pmol L–1) in May–August were approxi-
mately five times those in the bloom. In contrast, C2H5I
concentrations (0.6–1.7 pmol L–1) in the euphotic zone during
the summer were similar to those in the bloom. We will address
these features in future work related to nutrients and oxygen
levels in the water column. Sediment sources of VOIs in subpolar and polar coastal oceans. We determined the total concentrations (pmol L–1) of VOIs and
their percent compositions in the water column and sediment of
the study area (Fig. 3). The predominant VOIs in the surface
sediments were C2H5I (65–83%) and CH3I (12–29%) at all sta-
tions, and the total VOI concentrations (125–252 pmol L–1) in
the surface sediments (0–1 cm) were several times higher than
those in the bottom water. The total VOI concentrations
(37–104 pmol L–1) were lower in the deeper sediment (1–5 cm)
than in the surface sediments, and C2H5I still predominated
among the four VOIs. As in Funka Bay, we attributed the elevated We also conducted dark incubations using a culture of the
diatom Thalassiosira nordenskioeldii, which predominates in the
spring bloom in Funka Bay28 (Supplementary Table S2). We
started with a sterile culture grown in f/2 medium in the light
with added iodide (I−). The diatom cultures with I−produced
C2H5I and CH3I when moved to dark conditions. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 3 VOIs in water column and sediment in Bering and Chukchi Seas. a Proportional composition (%) of VOIs and their total concentrations
(pmol L–1) in the water column and sediment in Chukchi Sea. b Northern
Bering Sea. c Bering Sea. d Funka bay. Proportion of CH3I, C2H5I, CH2ClI,
and CH2I2 are shown in boxes with gray, white, shade, and black,
respectively. The water column was divided into two layers: near-bottom
water and the upper water column. The values for the northern Bering Sea
and Bering Sea are averages for each area, and the values for Funka Bay are
total averages for the time-series observations. Water depths of B (−5 m)
and B (−0.3 m) are depths 5 m and 0.3 m above the bottom, respectively. Composition ratio of volatile organic iodine
C₂H₅I
CH₃I
CH₂ClI
CH₂I₂
Water
(pmol/L)
Total conc
Sediment
0 - 41 m
9
9
275
65
0 - 1 cm
1 - 5 cm
42 m
(%)
0
50
100
Water
Sediment
40
73
125
96
0 - 1 cm
1 - 5 cm
Water
(c) Bering Sea
Sediment
64
49
252
104
0 - 1 cm
1 - 5 cm
Water
(d) Funka bay
Sediment
14
18
179
37
0 - 1 cm
1 - 5 cm
0 - B (-5 m)
B (-0.3 m)
0 - B (-5 m)
B (-0.3 m)
0 - B (-5 m)
B (-0.3 m)
(b) Northern Bering Sea
(a) Chukchi Sea C2H5I and CH3I in the sediment and bottom water in the Bering
and Chukchi Seas to diatom aggregates settled on the sea floor. It
has been confirmed that diatoms occupied the great part of
phytoplankton communities in the Bering and Chukchi Seas27. At
the Chukchi Sea station located at downstream of the Bering
Strait, where vertical mixing was well developed throughout the
water column, the high ratio (32%) of C2H5I was found in the
total water column. We considered that the sediment source
VOIs were distributed throughout the water column by mixing
and have an impact on VOIs emission to the air at the place. p
p
From microscopic images, several types of centric diatoms and
pennate diatoms were found to occupy the phytoplankton
community of the plankton net sample collected in the bloom
of Funka Bay. We considered that diatom aggregates consisting of
various species produce C2H5I and CH3I in darkness. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 From the
underwater video recordings taken at the observation sites (see
Supplementary Movie 1–4), we could see many invertebrates
covering the sea floor of the Bering Sea at stations 4 and 14, but
there were far fewer animals on the sea floor at the other stations. C2H5I predominated in the sediment whether animals were
abundant or not on the sea floor. We concluded that surface
sediment was an important source of C2H5I and CH3I to the
bottom water of global subarctic coastal oceans where diatoms
flourish and their impact on VOIs emission to the air was limited
in well mixed areas. VOI production from diatom aggregates in darkness. We
confirmed VOI production from diatom aggregates by conduct-
ing bottle incubations in darkness using diatom samples collected
from Funka Bay during the bloom in 2019. This production is
visible in the time-series of the amounts of C2H5I and CH3I
collected from the incubation bottles and their total emissions
(Fig. 4). The peak amounts of both C2H5I and CH3I were col-
lected on day 3 under oxic conditions and on day 7 under anoxic
conditions. The total emissions of C2H5I (anoxic, 27 pmol; oxic,
5.7 pmol) were much greater than those of CH3I (anoxic,
1.6 pmol; oxic, 2.4 pmol). The incubation results are provided in
Supplementary Table S1a, b. Our result that C2H5I had the
highest production among the VOIs is consistent with a previous
incubation
study18. The
VOI
emissions
(pmol)
per
unit
chlorophyll-a (mg) were 6.8 pmol (mg chl-a)−1 for C2H5I and
2.8 pmol (mg chl-a)−1 for CH3I under oxic conditions. Iodide
addition (+8 μmol L−1) to the incubation bottles resulted in a
7-fold increase in C2H5I production and a 12-fold increase in
CH3I
production
under
oxic
conditions
(Supplementary
Table S1c). The iodide addition under anoxic condition resulted
in
further
increases
in
the
productions
(Supplementary
Table S1d). The temperature (8 °C) of dark incubation was set to
be an annual maximum of Funka Bay water at the bottom. Thus,
the production rates obtained from the experiments would be a
maximum if there is a temperature dependence on the produc-
tions associated with biological activity. Note that the dark
incubations using diatom aggregates collected in Funka Bay were
not sterile, thus the results of VOIs production mean net pro-
duction including both production and biological loss. Fig. 3 VOIs in water column and sediment in Bering and Chukchi Seas. Fig. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 However,
elevated concentrations of phosphate (PO43−) at the layers,
which were derived from decomposition of organic matter settled
on the sea floor, decreased by 0–17% only between the two dates
(Supplementary Fig. S2). As for the obvious decreases in C2H5I
and CH3I concentrations at the layers, we presumed that
biological losses of C2H5I and CH3I occurred in the water. In
2018, elevations of C2H5I concentration and following decreases
at 65–75-m depths were found between May and June, however
obvious elevation of CH3I concentration at the depths were not
detected. It is possible that biological losses of C2H5I and CH3I in
the middle layer had advanced in 2018 compared to 2019. The biological loss rate for CH3I has been reported to be 9%
day−1 at 7 °C in the subarctic Atlantic Ocean, where chemical
loss is negligible because of low temperature26. The net loss rates
(loss—supply) of our study were 2% day−1 (= Δ1.61 pmol L−1 /
68 days) for CH3I and 0.5% day−1 (= Δ1.19 pmol L−1 / 68 days)
for C2H5I at the layer between 12 June and 29 August (68 days
interval). The lower rates of our study would be due to
consecutive supplies of CH3I and C2H5I from the sediment
source. We considered that the biological losses in water column
near the bottom inhibited the transport of sediment source CH3I
and C2H5I to the surface mixed layer and air. And thus, the
sediment source CH3I and C2H5I would have a small impact on
the VOIs emission to the air, although we will conclude that 3 MMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsenv ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 Composition ratio of volatile organic iodine
C₂H₅I
CH₃I
CH₂ClI
CH₂I₂
Water
(pmol/L)
Total conc. Sediment
0 - 41 m
9
9
275
65
0 - 1 cm
1 - 5 cm
42 m
(%)
0
50
100
Water
Sediment
40
73
125
96
0 - 1 cm
1 - 5 cm
Water
(c) Bering Sea
Sediment
64
49
252
104
0 - 1 cm
1 - 5 cm
Water
(d) Funka bay
Sediment
14
18
179
37
0 - 1 cm
1 - 5 cm
0 - B (-5 m)
B (-0.3 m)
0 - B (-5 m)
B (-0.3 m)
0 - B (-5 m)
B (-0.3 m)
(b) Northern Bering Sea
(a) Chukchi Sea
Fig. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 We suggest that the large variability of C2H5I and CH3I in the
sediment surface is caused by their productions and biological losses
in the sediment; productions are stimulated by supplies of diatom
aggregates
settled
from
the
surface
and
iodide
from
the
deeper sediment as discussed in the next paragraph; biological
losses are stimulated by their accumulations in the sediment. seawater could be used for VOI production in darkness. Although
bacterial activity would promote VOI production from diatom
aggregates in darkness18, our results indicate that bacterial
activity is not necessary. Calculation of VOI production in the sediment of Funka Bay. Primary production in Funka Bay during the spring bloom was
25–73 g-C m–2[23], and the carbon content (μg-C) per unit
chlorophyll-a (μg chl-a) of diatoms during the bloom was 11.6 μg-C
(μg Chl-a)−1[28]. If all diatoms produced in the bloom had settled to
the sea floor, the productions of C2H5I (11–33 nmol m–2) and CH3I
(5–14 nmol m–2) on the sea floor could occur applying the emission
rates of 6.8 pmol (mg chl-a)–1 for C2H5I and 2.8 pmol (mg chl-a)–1
for CH3I under oxic conditions with no I−addition. Note that the
calculated emissions mean net production including production and
biological loss. If the emitted C2H5I and CH3I were supplied to the
bottom water (80–96 m), the increases of bottom water concentra-
tions were 0.92–2.7 pmol L–1 for C2H5I and 0.38–1.1 pmol L–1 for
CH3I. These calculated increases are lower than the actual increases
of C2H5I (4.3–4.4 pmol L–1) and CH3I (3.0–5.0 pmol L–1) in bottom
water between the middle of March and April of both years. Actually,
high levels of C2H5I and CH3I remained in the sediment and bottom
water until August. As we have discussed a possibility of biological
loss in water column near the bottom, the natural increases involved
impacts of production from diatom aggregates and biological loss. y
We conclude that the production of VOIs from diatom aggregates
in the sediment would have been sustained for several months after
the spring bloom by using extracellular I−in the surrounding
seawater. Normally, the I−concentration in deep water is much
lower than that of IO3−; however, I−could be supplied from
deeper sediment in which IO3−is reduced to I−under anoxic
conditions29. We have confirmed that I−concentrations in bottom
water obviously increased from March to June 2017 in Funka Bay,
which were coincident with decrease in IO3−concentrations
(Supplementary Fig. S3; unpublished data). COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 The pathways of
iodine cycle are indicated as arrows. Fig. 5 Diagram of the iodine cycle between water column and sediment. Fig. 5 Diagram of the iodine cycle between water column and sediment. Iodine cycle between water column and sediment is shown in relation to
C2H5I emissions from dark bottom sediment. Iodide in surface seawater is
accumulated in phytoplankton cell during their growth (1); the
phytoplankton aggregations sink to sea floor (2); IO3−in the
deeper sediment is reduced to I−(3); C2H5I and CH3I are produced from
the sedimented aggregations using intercellular I−(4) and extracellular I−
supplied from deeper sediment (5); large fraction of sediment source I−is
supplied to bottom water (6); sediment source C2H5I and CH3I are supplied
to the bottom water and lost by microbial activity (7). The pathways of
iodine cycle are indicated as arrows. Fig. 4 Time series of VOIs amounts by dark incubation experiment. a Collected amount and total emission of C2H5I from the dark incubation
bottle. b CH3I. Anoxic condition with N2 supply and oxic condition with air
supply are shown in gray square plots and black circle plots, respectively. VOIs were collected from the head-space of diatom incubation bottles in
darkness and their total emissions over 42 days. These emissions were
collected by flushing with either N2 (anoxic conditions) or air (oxic
conditions) as carrier gases. The plots and error bars are averages and
ranges of incubation results of the two bottles (n = 2), respectively. Many
of error bars are behind the symbols. The incubation results are shown in
Supplementary Tables S1–S3. incubation experiment, we considered that the productions of C2H5I
and CH3I in the sediment surface were affected by both the amount
of diatom aggregates settled on the sea floor and the supply of iodide
(I−) to the aggregates. Moreover, the concentrations of C2H5I and
CH3I in the sediment deeper than 1 cm depth was only 20% of the
sediment surface (0–1 cm) where the sediment source C2H5I and
CH3I were produced; and thus, we considered that the lower
concentrations in the deeper sediment were owing to biological
(probably bacterial) loss. If the biological losses of C2H5I and CH3I
are stimulated by their accumulations in the sediment, the
concentrations on the sediment surface should vary dramatically. COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 In contrast,
iodate (IO3−) addition did not result in remarkable C2H5I or
CH3I production. Clearly, I−was necessary for production of the
two VOIs. We concluded that both intracellular I−absorbed
earlier during their growth and extracellular I−dissolved in OMMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsenv 4 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 Control
Air
N₂ supply
C₂H₅I collected (pmol)
Total
(0－42 d; pmol)
(0－42 d; pmol)
emission
10
0
5
27
0
28
42
14
(days)
5.7
N₂
Air
0
28
42
14
(days)
2.4
N₂
Air
CH₃I collected (pmol)
Total
emission
0
0.5
1
1.6
(a)
(b)
Control
N₂ supply
Air
Air
N₂
Fig. 4 Time series of VOIs amounts by dark incubation experiment. Control
Air
N₂ supply
C₂H₅I collected (pmol)
Total
(0－42 d; pmol)
emission
10
0
5
27
0
28
42
14
(days)
5.7
N₂
Air
(a) I-
IO₃-
Sediment
Reduction
Anoxic
Water
Surface
mixed layer
Phytoplankton
CH₃I C₂H₅I
Biological loss
IO₃-
IO₃-
I-
I-
(1)
(2)
(3)
(6)
(5)
(7)
(4)
Fig. 5 Diagram of the iodine cycle between water column and sediment. Iodine cycle between water column and sediment is shown in relation to
C2H5I emissions from dark bottom sediment. Iodide in surface seawater is
accumulated in phytoplankton cell during their growth (1); the
phytoplankton aggregations sink to sea floor (2); IO3−in the
deeper sediment is reduced to I−(3); C2H5I and CH3I are produced from
the sedimented aggregations using intercellular I−(4) and extracellular I−
supplied from deeper sediment (5); large fraction of sediment source I−is
supplied to bottom water (6); sediment source C2H5I and CH3I are supplied
to the bottom water and lost by microbial activity (7). The pathways of
iodine cycle are indicated as arrows. I-
IO₃-
Sediment
Reduction
Anoxic
Water
Surface
mixed layer
Phytoplankton
CH₃I C₂H₅I
Biological loss
IO₃-
IO₃-
I-
I-
(1)
(2)
(3)
(6)
(5)
(7)
(4) C₂H₅I collected (pmol) (0－42 d; pmol)
0
28
42
14
(days)
2.4
N₂
Air
CH₃I collected (pmol)
Total
emission
0
0.5
1
1.6
(b)
Control
N₂ supply
Air
Air
N₂ (b) Fig. 5 Diagram of the iodine cycle between water column and sediment. Iodine cycle between water column and sediment is shown in relation to
C2H5I emissions from dark bottom sediment. Iodide in surface seawater is
accumulated in phytoplankton cell during their growth (1); the
phytoplankton aggregations sink to sea floor (2); IO3−in the
deeper sediment is reduced to I−(3); C2H5I and CH3I are produced from
the sedimented aggregations using intercellular I−(4) and extracellular I−
supplied from deeper sediment (5); large fraction of sediment source I−is
supplied to bottom water (6); sediment source C2H5I and CH3I are supplied
to the bottom water and lost by microbial activity (7). MMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsenv Methods
Shi b
d The seawater sampling depths were 0, 5, 10, 20, 30, 40, 50, 60, 65,
70, 75, 80, 85, 95 (+1 m above the sea floor), and 95.7 m (+0.3 m above the sea floor) in
Funka Bay, and 0, 10, 20, 30, 40, and 50 m depths, and +1 m and +0.3 m above the sea
floor, in the Chukchi and Bering Seas. Aliquots of seawater samples (125 mL) were
collected into dark glass bottles and approximately 250 mL of seawater was allowed to
overflow the bottles. Saturated HgCl2 solution (100 μL) was added to the bottles. The
bottles were sealed with a butyl rubber septum and an aluminum cap and kept in a
refrigerator until pre-treatment, typically within a month. We also conducted a dark incubation experiment to test if diatom aggregate
without prior freezing produces VOIs. We collected diatom aggregates in the
Funka-bay by plankton net towing from the depth of 85 m to the surface in spring
bloom 2021. The diatom sample was not frozen before the incubation. An aliquot
(9 mL) of diatom aggregate sample was put in a 200 mL glass bottle. We prepared
10 incubation bottles. The first VOIs collection from the bottle was conducted after
6 days of the net sampling. The head space of the bottle was collected on days 6, 20,
and 26 (Supplementary Table S3). Since we did not collect VOIs on day 0, the
initial concentration was not obtained. We confirmed that the diatom aggregates
without prior freezing produced CH3I (0.7–2.2 pmol) and C2H5I (11.5–16.5 pmol)
for 20 days or 26 days of incubation time. Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. Analytical procedures. Dissolved VOIs were collected from the pore water and
seawater samples by a purge-and-trap method. The total volume of seawater (125
or 32 mL) in the sample bottle was introduced into a purge vessel by a high-purity
nitrogen carrier at 20 mL min−1 for 7 min, and the dissolved gases were con-
secutively purged by bubbling with nitrogen at 65 mL min−1 for 35 min. The
purged gas sample was collected in a cold-trap containing adsorption resin (Tenax
TA, 10 mg) cooled to −90 °C. The cold-trap samples were stored in a freezer
(−30 °C) until analysis by gas chromatography–mass spectrometry (GC–MS). Methods
Shi b
d The sea-floor depths in the Bering
and Chukchi Seas were 40–50 m, and the depth of Funka Bay was 96 m. All sea
floors were in darkness below the euphotic zone. The sediment cores were cut into sections at intervals of 1 cm from the surface (sea
floor, 0 cm) to a depth of 5 cm. Each sediment section was collected in a polyethylene
bag. The pore water from each sediment section was collected by using a syringe and
pulling the water through a porous ceramic filter cup using a soil water sampler (DIK-
8393, Daiki Rica Kogyo Co. Ltd., Saitama, Japan) within two hours of the sediment core
collection. The pore-water sample in the syringe was filtered into a glass bottle (32 mL)
using a syringe filter with a pore size of 0.22 µm. To arrest microbial activity, 50 μL of
saturated mercuric chloride (HgCl2) solution was added to the bottle. The bottle was
crimp-sealed with 0.5 mL headspace using a butyl rubber septum and an aluminum
cap. The sample bottles containing pore water were kept in a refrigerator at 4 °C until
pre-treatment for VOI analysis, typically within a month. pp
y
We conducted another dark incubation experiment to examine effect of iodine
addition. A sterile culture of diatom, Thalassiosira nordenskioeldii, was grown in Si-rich
f/2 medium (NO3−, 700 μmol L−1; PO43−, 26 μmol L–1; Si(OH)4, 75 μmol L–1) with
addition of iodide (I−, 8 μmol L–1) or iodate (IO3−, 8 μmol L–1). Then, the cultures
were used for the bottle incubation in darkness under oxic condition. We set up two
bottles for each no iodine addition, I−addition, and IO3−addition. VOIs were collected
from the incubation bottles on days 0 and 11 (Supplementary Table S2). Seawater sampling. Seawater samples were collected in Niskin bottles attached to a
rosette multi-sampler along with a conductivity-temperature-depth (CTD) probe (SBE
19 plus, Sea-Bird Electronics, Inc., Bellevue, WA, USA). Surface water was collected
with a plastic bucket. Bottom waters were collected 1 m above the sea floor using a Van
Dorn sampling bottle or a Niskin bottle, and 0.3 m above the sea floor from the
sediment-core tubes. Methods
Shi b
d Shipboard observations. Shipboard observations were conducted over the Bering
Sea and Chukchi Sea continental shelves from the Training Vessel (T/V) Oshoro-
maru in July of 2017 and 2018. Time-series observations in Funka Bay, Hokkaido,
Japan, were conducted from the T/V Ushio-maru 17 times from March 2018 to
December 2019. Both vessels were operated by the Faculty of Fisheries Sciences,
Hokkaido University. Figure 1 shows the locations of the sampling stations. The
sea floor at the four stations in the Bering Sea were videotaped during the obser-
vations (see Supplementary Movie 1–4). The sea floor at the station 30 of the Funka
Bay and observation scenes were recorded in movie files and photos (see Supple-
mentary Movie 5–8 and Supplementary Fig. S5–S16). We set up two bottles each for anoxic conditions and oxic conditions. The 12
incubation bottles (A-1—D-3; see Supplementary Table S1a–d) were placed in a dark
incubator at 8 °C. Iodide solution was added (+8 μmol L–1) to the bottles C and D. Head-space gas of each bottle was sampled on days 0, 3, 7, 14, 21, 28, 35, and 42 after
the start of incubations. Carrier gas (N2 or pure air) was introduced to the bottle
through a needle at a flow rate of 20 mL min–1 for 30 min at each sampling time. The
head-space gas was collected through another needle. The water in the bottle was kept
at 8 °C and magnetically stirred during the gas collection. The water vapor in the gas
sample was removed by a glass-tube water trap at −25 °C. The VOIs in the gas sample
were collected in a cold trap with adsorption resin (Tenax TA) at −90 °C. The oxygen
level in the bottles was measured before and after the head-space gas collection. Analysis
for VOIs was the same as for the seawater samples. The collection efficiency of VOIs
from the bottles, based on one head-space gas collection, was 28%. The calculation of
the total amount of VOIs emission was described in explanatory notes of
Supplementary Table S1a–d. Sediment core sampling. Sediment core samples were collected in polycarbonate
core tubes (inner diameter 82 mm; length 600 mm) attached to a gravity-type core
sampler (Rigo Co. Ltd., Saitama, Japan) for Funka Bay and a multiple core sampler
(Rigo Co. Ltd.) for the Bering and Chukchi Seas. ARTICLE ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 from diatom aggregates in dark bottom sediment in relation to the
iodine cycle between the water column and sediment (Fig. 5). from diatom aggregates in dark bottom sediment in relation to the
iodine cycle between the water column and sediment (Fig. 5). then, the water-based standard was measured by purge and trap / GC-MS system. The analytical precisions of CH3I and C2H5I in water sample were ±2%. Details of
the GC–MS analysis are described elsewhere20. The amounts of CH3I and C2H5I in
the sample bottle (32 mL) reduced by 3−7% a month during the sample storage,
thus our results include approximately 3–7% of under estimation. Our results strongly support the idea that phytodetritus (e.g. diatom aggregates) on the sea floor is a hotspot of VOI production in
the ocean18. We concluded that the impact of the sediment source
VOIs is limited in the middle layer and bottom layer waters in
stratified coastal oceans because of biological losses below the surface
layer. However, it is possible that considerable amounts of sediment
source VOIs are supplied to the ocean surface and emitted to the air
in vertically well-mixed coastal areas, such as more coastal areas and
fast-flowing straits. Further research on the sediment source VOIs in
subtropical and tropical coastal oceans and more coastal areas of
global ocean is needed to evaluate their atmospheric impacts. Chl-a concentrations in discrete seawater samples (100 mL) were measured by a
fluorometer model 10-AU-005 (Turner Designs, San Jose, CA, USA). Nutrient
concentrations (NO3−, NO2–, NH4+, SiO2, and PO43−) in discrete seawater
samples were measured by colorimetric methods using a QuAAtro system (BL-Tec,
Osaka, Japan). Chl-a concentrations in discrete seawater samples (100 mL) were measured by a
fluorometer model 10-AU-005 (Turner Designs, San Jose, CA, USA). Nutrient
concentrations (NO3−, NO2–, NH4+, SiO2, and PO43−) in discrete seawater
samples were measured by colorimetric methods using a QuAAtro system (BL-Tec,
Osaka, Japan). VOI production from diatom aggregates in darkness in the laboratory. Diatom
aggregates were collected from the same location in Funka Bay (St. 30) on 15
March 2019 and 18 March 2021 during massive spring diatom blooms (>15 μg L–1
of chl-a). A plankton net (0.3 m diameter, 100 μm mesh) was towed vertically from
85 m depth to the surface. ARTICLE The diatom aggregates collected in 2019 were suspended
in 900 mL of seawater and frozen (−25 °C) in a polyethylene bag until preparation
for the incubation experiment. Supplementary Fig. S4 is a schematic showing the
preparation protocol for the incubation experiment. An aliquot (9 mL) of defrosted
diatom aggregate sample was put in a 200 mL glass bottle. Fluorometric analysis
showed that 9 mL of the diatom aggregate contained 0.84 mg chlorophyll-a. Fil-
tered seawater (100 mL) was added to the glass bottle. The diatom aggregates
collected by plankton net in 2021 were transferred to the incubation bottle within
2 h after sampling without freezing. A spot sensor for an optical O2 meter (Fire-
Sting, PyroScience GmbH, Hubertusstr, Germany) was attached to the inner wall of
the incubation bottles to measure the oxygen level in the bottle. The bottle was
sealed with a butyl rubber septum and an aluminum cap. Methods
Shi b
d The
purge efficiencies for CH3I, C2H5I, CH2ClI and CH2I2 were 94%, 95%, 83%, and
66%, respectively. Concentrated VOIs in the cold-trap were thermally desorbed
and transferred to an automated pre-concentration GC–MS analytical system
(capillary column: Porabond Q, 0.32 mm, 50 m; GC–MS: Agilent 5973, 6890). A
gravimetrically prepared standard gas (Taiyo Nissan, Inc., Tokyo) in a gas cylinder
containing CH3I, C2H5I and other halocarbons at concentrations of 10 ppb (dry air
mole fraction) was analyzed with the preconcentration-GC-MS system every
measurement date. The concentrations in the cylinder were calibrated by mea-
suring a methanol-based liquid standard containing these compounds every six
months. The methanol-based liquid standard was injected into pure water, and Data availability The datasets generated during the current study are available at https://doi.org/10.5281/
zenodo.6905361. Received: 5 November 2021; Accepted: 28 July 2022; Received: 5 November 2021; Accepted: 28 July 2022; Received: 5 November 2021; Accepted: 28 July 2022; COMMUNICATIONS EARTH & ENVIRONMENT | https://doi.org/10.1038/s43247-022-00513-7 The I−and IO3−
concentrations were measured according to the stripping voltam-
metry method30. We considered that continuous supply of I-, which
was derived from IO3−reduction in anoxic deeper sediment, to
diatom aggregates settled on the sea floor sustained the VOIs
productions until August. Because diatoms assimilate iodide faster
than iodate during their growth31, we thought that iodine cycle in
coastal area is driven by iodide accumulation by phytoplankton. A
small fraction of accumulated I−in diatom cell is used for the VOIs
productions. We prepared a diagram representing C2H5I emission If productions of C2H5I and CH3I from diatom aggregates depend
only on an amount of diatom settled on the sea floor, the
concentrations of C2H5I and CH3I in the sediment surface in 2019,
where the considerably high concentration of chl-a (maximum of
29.7 μg L–1) was found in the bloom 2019 compared with the bloom
2018 (maximum of 12.5 μg L–1) (Fig. 2a), should have become
higher than 2018. However, C2H5I and CH3I concentrations in the
sediment surface in 2019 were lower than 2018 (Fig. 2b, c). Since I−
addition stimulated the productions of C2H5I and CH3I from ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commsenv 5 Additional information Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s43247-022-00513-7. Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s43247-022-00513-7. 20. Ooki, A. & Yokouchi, Y. Determination of Henry’s law constant of
halocarbons in seawater and analysis of sea-to-air flux of iodoethane (C2H5I)
in the Indian and Southern oceans based on partial pressure measurements. Geochem. J. 45, e1–e7 (2011). Correspondence and requests for materials should be addressed to Atsushi Ooki. Peer review information Communications Earth & Environment thanks Stephen Archer
and the other, anonymous, reviewer(s) for their contribution to the peer review of this
work. Primary Handling Editors: Clare Davis. Peer reviewer reports are available. 21. Ooki, A. et al. Concentration maxima of volatile organic iodine compounds in
the bottom layer water and the cold, dense water over the Chukchi Sea in the
western Arctic Ocean: possibility of production related to the degradation of
organic matter. Biogeosciences 13, 133–145 (2016). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints 22. Shimizu, Y. et al. Seasonal variation of volatile organic iodine compounds in
the water column of Funka Bay, Hokkaido, Japan. J. Atmos. Chem. 74,
205–225 (2017). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 23. Kudo, I. & Matsunaga, K. Environmental factors affecting the occurrence and
production of the spring phytoplankton bloom in Funka bay, Japan. J. Oceanogr. 55, 505–513 (1999). 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
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Commons license, and indicate if changes were made. The images or other third party
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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/. 24. Isada, T. et al. References References
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University). This research was supported financially by FYs 2018–2019 Research Projects
from the Hokusui Society Foundation, Sapporo, Japan, the Japan Society for the Promotion
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for Sustainability (ArCS) (JPMXD1300000000) / ArCS II projects funded by the Ministry of
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phytoplankton cultures. Limnol. Oceanogr. 42, 142–147 (1997). A.O. designed the research, conducted observations and incubation experiment using sterile
diatom and diatom aggregation collected in 2021 and wrote the manuscript with con-
tributions from all co-authors. K.M. conducted observations in Funka Bay and Bering/
Chukchi Seas and VOIs analysis and supported the data analysis and discussion about VOIs
in Funka Bay 2018-2019. F.M. conducted the observations in Funka Bay and incubation
experiments using diatom aggregation collected in 2019 and supported the data analysis and
discussion about incubation experiment. N.M. conducted the observations in Funka Bay and
VOIs analysis and supported the data analysis and discussion about VOIs in Funka Bay
2019. T.H. designed the Oshoro-maru cruise and supported the data analysis about basic
parameter in Bering and Chukchi Seas. H.U. designed the Oshoro-maru cruise and sup-
ported the data analysis about basic parameter in Bering and Chukchi Seas. Y.N. supported
the sterile diatom incubations and the data analysis about the experiment. T.T. designed the
Ushio-maru cruise and supported the data analysis about the basic parameter in Funka Bay. y
y
p
phytoplankton cultures. Limnol. Oceanogr. 42, 142–147 (1997). 12. Tokarczyk, R. & Moore, R. M. Production of volatile organohalogens by
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gas brines and seawaters. Microb. Ecol. 49, 547–557 (2005). Competing interests p
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The authors declare no competing interests. Additional information Influence of hydrography on the spatiotemporal variability of
phytoplankton assemblages and primary productivity in Funka Bay and the
Tsugaru Strait. Estuarine Coastal Shelf Sci. 188, 199–211
(2017). 25. Ooki, A. et al. Isoprene production in seawater of Funka Bay, Hokkaido,
Japan. J Oceanogr. 75, 485–501 (2019). 26. Moore, R. M. Methyl halide production and loss rates in sea water from field
incubation experiments. Marine Chem. 101, 213–219 (2006). 27. Giesbrecht, K. E. et al. A decade of summertime measurements of
phytoplankton biomass, productivity and assemblage composition in the
Pacific Arctic Region from 2006 to 2016. Deep-Sea Res. Part II-Topical Studies
Oceanogr. 162, 93–113 (2019). 28. Ban, S. et al. In situ egg production and hatching success of the marine copepod
Pseudocalanus newmani in Funka Bay and adjacent waters off southwestern 28. Ban, S. et al. In situ egg production and hatching success of the marine copepod
Pseudocalanus newmani in Funka Bay and adjacent waters off southwestern 7 7 COMMUNICATIONS EARTH & ENVIRONMENT | (2022) 3:180 | https://doi.org/10.1038/s43247-022-00513-7 | www.nature.com/commse
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Preoperative serum selenium predicts acute kidney injury after adult cardiac surgery
|
Research Square (Research Square)
| 2,024
|
cc-by
| 4,586
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Guowei Fu
Changzhou Second People’s Hospital
Shuying Bai Guowei Fu
Changzhou Second People’s Hospital
Shuying Bai Abstract Background: The relationship between serum selenium (Se) and acute kidney injury after adult cardiac
surgery remains controversial. This study aimed to investigate the association of preoperative Se level
with incident CSA-AKI. Method and Results: A retrospective cohort study was conducted on patients who underwent cardiac
surgery. The primary outcome was incident CSA-AKI. Multivariable logistic regression models and natural
cubic splines were used to estimate the association of Se levels and primary outcome. A total of 453
patient with a mean age of 62.97 years were included. Among all patients, 159 (35.1%) incident cases of
CSA-AKI were identified. The level of preoperative Se concentration in patients with CSA-AKI was
significant lower than that in patients without CSA-AKI. In comparison with patients in the lowest quartile,
those with the highest quartile were associated with lower risk of CSA-AKI [adjusted OR (95% CI):
0.61(0.40-0.95)]. Dose-response relationship curve revealed a nearly L-shape correlation between serum
Se selenium levels and incident CSA-AKI. 0.61(0.40-0.95)]. Dose-response relationship curve revealed a nearly L-shape correlation between serum
Se selenium levels and incident CSA-AKI. Conclusion: Our study suggested that a higher level of serum Se was significantly associated with lower
risk of CSA-AKI. Further prospective studies are needed to clarify the causal relationship between serum
Se level and incident CSA-AKI. Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Cardiovascular Disorders on March
14th, 2024. See the published version at https://doi.org/10.1186/s12872-024-03825-y. Page 1/14 INTRODUCTION Page 2/14 Page 2/14 As an essential trace element, Selenium (Se) in the human body involves in lots of biological processes,
especially antioxidant reactions.[8] The biological functions of Se are achieved by 25 selenoproteins with
activating selenocysteine group.[9] The Se deficiency could impair human health and increase the risk of
Keshan disease,[10] cognitive impairment,[11] cardiovascular diseases.[12] The kidney absorbs plasma Se
via renal tubular epithelial cells and synthesizes GPx3 to maintain human selenium homeostasis.[13]
Animal study has shown that Se deficiency increases oxidative stress associated mitochondrial
dysfunction which causes renal injury in mice.[14] Current literatures indicated potential clinical benefit of
higher serum Se level in patients with chronic kidney disease (CKD).[15–17] However, there is few evidence
on the relationship between serum Se concentration and risk of CSA-AKI. Thus, this study aimed to
investigate the effect of serum Se on the incidence of CSA-AKI among Chinese population undergone
cardiac surgery. Study population Between January 2011 and December 2018, we initially screened 726 patients aged more than 18 year-
old who underwent cardiac surgery (including coronary artery bypass grafting [CABG] and open-heart
valve repair or replacement surgery) at Changzhou Second People’s Hospital in China. The carbon-
furnace atomic-absorption spectrometry and Zeeman compensation was used to determine the serum Se
concentrations by a Spectra AA 220 Z (Varian). For those having multiple cardiac surgeries, only the first
one was selected. The exclusion criteria are as following: (1) patients with preoperative CKD 5 stage, i.e.,
an estimated glomerular filtration rate (eGFR) < 15 mL/min/1.73 m2, or undergoing maintenance dialysis,
(2) with a prior AKI requiring dialysis within 1 year of index surgery, (3) died within 48 hours after surgery,
(4) with missing information on serum Se and creatinine levels. This single-center, retrospective,
observational cohort study was approved by the local Institutional Review Board (No. 2022105-10) and
was performed in adherence with the Declaration of Helsinki. The requirement for obtaining informed
consent was waived because of the retrospective design. INTRODUCTION Acute kidney injury (AKI) is the most common major postoperative event of cardiac surgery, acting as a
syndrome of sudden renal excretory dysfunction with high morbidity and mortality.[1] Cardiac surgery-
associated acute kidney injury (CSA-AKI) ranked second among the causes of AKI in the intensive care
setting,[2] and mild serum creatinine increase after cardiac surgery indicated a higher morbidity, a longer
length of hospital stay and increased healthcare costs.[3] Moreover, CSA-AKI is related to a long-term risk
of death in patients with cardiac surgery independent of other risk factors, which even persisting in
patients having complete renal recovery.[4] Given current varying AKI definitions and study population, the
incidence of CSA-AKI ranged from 5% to as high as 42%.[5] A meta-analysis including 320,086 patients
undergone cardiac surgery reported that the global incidence was 22.3% for all stages of CSA-AKI.[6] It’s
therefore imperative to deploy prevention strategies for CAS-AKI by early identifying high-risk patients,
ultimately improve the substantial health and socioeconomic burden. In addition to female sex and advanced age, several comorbidities such as previous chronic kidney
disease, diabetes mellitus, hypertension, cardiopulmonary diseases, and heart failure significantly
increase the risk of CSA-AKI development.[5] The multifactorial mechanisms underlying the
pathophysiology of CAS-AKI has been recognized but incompletely understood. In the setting of cardia
surgery, a variety of factors encompassing nephrotoxic drugs, hypotension, temporal renal ischemia,
hemolysis, inflammation, and oxidative stress,[7] may play roles in the development of AKI in different
ways and to differing extents. In addition to female sex and advanced age, several comorbidities such as previous chronic kidney
disease, diabetes mellitus, hypertension, cardiopulmonary diseases, and heart failure significantly
increase the risk of CSA-AKI development.[5] The multifactorial mechanisms underlying the
pathophysiology of CAS-AKI has been recognized but incompletely understood. In the setting of cardiac
surgery, a variety of factors encompassing nephrotoxic drugs, hypotension, temporal renal ischemia,
hemolysis, inflammation, and oxidative stress,[7] may play roles in the development of AKI in different
ways and to differing extents. Primary outcome The primary outcome of the current study was incident CSA-AKI. At our center, serum creatinine was
regularly measured the day before surgery. The change from the maximal postoperative serum creatinine
level within 7 days after surgery to the last baseline value was used to diagnosis CSA-AKI and determine
its stage. The Kidney Disease Improving Global Outcomes (KDIGO) criteria[1] was adopted to define CAS-
AKI: Stage 1 was defined as creatinine increase by 1.5–1.9 times baseline within 7 d or increase by ≥ 0.3
mg/dL within 48 h, Stage 2 as a 2-2.9 times baseline increase, and Stage 3 as ≥ 3 times baseline or
increase to ≥ 4 mg/dL or dialysis. Se determination and study covariates Page 3/14 Page 3/14 Demographic information, comorbidities, laboratory tests, and perioperative data were collected from
medical health recording. Blood samples for Se determination were taken from patients after a 12 h
fasting period preoperatively. The whole blood was centrifugated to obtain serum, which was
immediately stored at -80℃ until analysis. A Spectra AA 220 Z (Varian) based on the carbon-furnace
atomic-absorption spectrometry and Zeeman compensation was used to determine the serum Se
concentrations. Statistical analysis The numerical data are presented as mean ± standard deviations (SD) if normally distributed or otherwise
median with interquartile range (IQR), and groups comparisons were performed by the Mann-Whitney or
Kruskal-Wallis test as indicated. The categorical data are summarized as count with frequencies and
compared by the chi-square test. Apart from continuous form, preoperative serum Se levels were further
categorized into quartiles to assess a stepwise association. Multivariable logistic regression models were
used to estimate the association of Se levels and primary outcome by generated adjusted odds ratios
(ORs) with 95% confidence intervals (CIs). The variables in multivariable models included baseline
features showing statistical significance in the univariable analysis for CSA-AKI and those considered to
have clinical significance. Natural cubic splines were adopted to explore the concentration–response
curve between serum Se level and incident CSA-AKI events. All statistical analyses were performed using
R software, version 4.0.0 for Windows (R Foundation for Statistical Computing, Vienna, Austria, 2019),
and 2-tailed P values less than 0.05 was considered statistically significant. Baseline characteristics and primary outcome Among initially screened 726 patients undergone cardiac surgery, a total of 453 patents fulfilling
inclusion criteria were finally enrolled after assessment (Fig. 1). Table 1 depicted the baseline
characteristics of study patients. The 40.18% of patients were female, with a mean age of 62.97 ± 15.58
years. Regarding surgery type, 175 (38.63%) patients were treated by CABG, and the rest treated by valve
surgery. The mean serum creatinine, hemoglobin, albumin, and Se before surgery was 86.79 ± 9.53
µmol/L, 127.7 ± 8.05 g/dL, 4.02 ± 0.95 g/dL, and 158.92 ± 6.72µg/L, respectively. Page 4/14 Table 1
Baseline characteristics between patients with and without CSA-AKI. Total (n = 453)
Non-AKI (n = 294)
AKI (n = 159)
P value
Age, years
62.97 (15.58)
62 (15.84)
64.75 (14.96)
< 0.001
Female sex, %
182 (40.18)
104 (35.37)
78 (49.06)
0.005
BMI, kg/m2
26.85 (4.61)
26.31 (4.55)
27.85 (4.58)
< 0.001
Smoker, %
92 (20.31)
63 (21.43)
29 (18.24)
0.421
Hypertension, %
172 (37.97)
113 (38.44)
59 (37.11)
0.781
Diabetes, %
122 (26.93)
77 (26.19)
45 (28.3)
0.629
Chronic heart failure, %
149 (32.89)
53 (18.03)
96 (60.38)
< 0.001
Atrial fibrillation, %
59 (13.02)
14 (13.46)
45 (57.69)
< 0.001
COPD, %
9 (1.99)
5 (1.70)
4 (2.52)
0.553
Surgery type, %
0.750
CABG
175 (38.63)
112 (38.1)
63 (39.62)
Valve surgery
278 (61.37)
182 (61.9)
96 (60.38)
Anesthesia time, min
228.45 (40.52)
227.82 (39.23)
229.61 (42.9)
< 0.001
CPB time, min
120.96 (27.33)
115.54 (24.47)
131 (29.49)
< 0.001
Serum creatinine (µmol/L)
86.79 (9.53)
85.79 (8.02)
88.63 (11.63)
< 0.001
Hemoglobin, g/dL
127.7 (8.05)
129.16 (8.45)
125 (6.43)
< 0.001
Albumin, g/dL
4.02 (0.95)
4.17 (0.82)
3.73 (1.08)
< 0.001
Preoperative Se, µg/L
158.92 (6.72)
159.89 (5.7)
157.13 (8)
< 0.001
Data are presented as mean (SD) or n (%). CSA-AKI, cardiac surgery-associated acute kidney injury;
BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass
grafting; CPB, cardiopulmonary bypass; Se, selenium. Table 1 Data are presented as mean (SD) or n (%). CSA-AKI, cardiac surgery-associated acute kidney injury;
BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass
grafting; CPB, cardiopulmonary bypass; Se, selenium. Among all patients, 159 (35.1%) incident cases of CSA-AKI were identified. Of which, 126 (79.2%) were
recorded as KDIGO 1 stage, versus 33 (20.8%) as KDIGO 2–3 stage. Baseline characteristics and primary outcome Compared to those without CSA-AKI,
individuals with CSA-AKI were older and tend to have more comorbidities such as heart failure and atrial
fibrillation. Moreover, patients with CSA-AKI exhibited a higher preoperative serum creatinine level, a
longer anesthesia time and CPB time, lower level of hemoglobin and albumin. The level of preoperative
Se concentration in patients with CSA-AKI was significant lower than that in patients with patients
without CSA-AKI. Group differences according to serum Se level quartile Group differences according to serum Se level quartile Page 5/14 When patients were stratified by quartiles of serum Se level (Table 2), the lowest quartile group more
often presented as older age, females, higher BMI, longer anesthesia time, longer CPB time, higher serum
creatinine level, lower hemoglobin and albumin level. Page 6/14 Table 2
Baseline characteristics according to preoperative serum Se level quartile. Q1 (154.46–
163.30
µg/L)
Q2 (155.98-
158.33
µg/L)
Q3 (160.25-
162.33
µg/L)
Q4 (164.60-
168.74
µg/L)
P
value
No. of patients
114
113
113
113
Age, years
61.63 (16.52)
62.81 (16.81)
62.55 (14.74)
64.88 (14.07)
<
0.001
Female sex, %
60 (52.63)
49 (43.36)
37 (32.74)
36 (31.86)
0.003
BMI, kg/m2
27.65 (4.74)
26.8 (4.82)
26.69 (4.56)
26.24 (4.25)
<
0.001
Smoker, %
20 (17.54)
18 (15.93)
28 (24.78)
26 (23.01)
0.286
Hypertension, %
42 (36.84)
33 (29.2)
43 (38.05)
54 (47.79)
0.039
Diabetes
28 (24.56)
33 (29.2)
28 (24.78)
33 (29.2)
0.757
Chronic heart
failure, %
46 (40.35)
38 (33.63)
31 (27.43)
34 (30.09)
0.185
Atrial fibrillation, %
18 (30)
18 (36.73)
9 (24.32)
14 (38.89)
0.240
COPD, %
2 (1.75)
2 (1.77)
2 (1.77)
3 (2.65)
0.951
Surgery type, %
0.039
CABG
43 (37.72)
43 (38.05)
55 (48.67)
34 (30.09)
Valve surgery
71 (62.28)
70 (61.95)
58 (51.33)
79 (69.91)
Anesthesia time,
min
226.51 (38.79)
230.46
(43.48)
233.95
(42.24)
222.88
(36.94)
<
0.001
CPB time, min
125.17 (27.67)
118.93
(26.17)
123.29
(26.89)
116.43
(28.02)
<
0.001
Serum creatinine
(µmol/L)
88.9 (10.53)
85.65 (9.17)
85.49 (8.86)
87.1 (9.18)
<
0.001
Hemoglobin, g/dL
126.37 (7.67)
128.58 (7.54)
127.82 (8.42)
128.05 (8.46)
<
0.001
Albumin, g/dL
3.83 (0.94)
4.16 (0.98)
4.07 (0.95)
4 (0.89)
<
0.001
Data are presented as mean (SD) or n (%). CSA-AKI, cardiac surgery-associated acute kidney injury;
BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass
grafting; CPB, cardiopulmonary bypass; Se, selenium. Associations between serum Se levels and CSA-AKI Data are presented as mean (SD) or n (%). CSA-AKI, cardiac surgery-associated acute kidney injury;
BMI, body mass index; COPD, chronic obstructive pulmonary disease; CABG, coronary artery bypass
grafting; CPB, cardiopulmonary bypass; Se, selenium. Associations between serum Se levels and CSA-AKI Associations between serum Se levels and CSA-AKI Page 7/14 Page 7/14 Table 3 presented the associations between preoperative Se levels and incident CSA-AKI from
multivariable regression analyses. Group differences according to serum Se level quartile In multivariable analyses, we found that a high preoperative Se level
(adjusted OR 0.61, 95% CI: 0.40–0.95) was significantly associated with decreased risk of CSA-AKI, after
adjusting age, sex, BMI, smoking, drinking, hypertension and diabetes. Dose-response relationship curve
revealed a nearly L-shape correlation between serum Se selenium levels and incident CSA-AKI (Fig. 2). Table 3
Logistic regression analyses on clinical outcomes for preoperative Se levels. Serum Se levels
Crude model
Adjusted model*
OR (95% CI)
P value
OR (95% CI)
P value
Q1
Ref
Ref
Q2
0.37 (0.21–0.64)
< 0.001
0.38 (0.22–0.67)
0.001
Q3
0.30 (0.17–0.52)
< 0.001
0.32 (0.18–0.56)
< 0.001
Q4
0.39 (0.22–0.67)
< 0.001
0.43 (0.24–0.76)
0.004
P for trend#
< 0.001
0.002
*Adjusted for age, sex, BMI, smoking, drinking, hypertension, and diabetes. #P for trend: calculated by treating quartiles as a continuous variable in each model. DISCUSSION In this retrospective cohort study, we investigated the association between preoperative Se levels and
CSA-AKI development amongst patients undergoing cardiac surgery. We observe a relatively high
incidence of CSA-AKI in patents with low Se levels, and found that a higher serum Se level was
significantly associated with decreased risk of incident CSA-AKI even after adjusting for confounding
variables. Serum Se concentration largely varies among different population partially due to the differential
geographical distribution of Se in soil. The Se deficiency is common globally especially in China, and it
has been the well-defined etiology of an endemic cardiomyopathy called Keshan disease.[10] The main
sources of dietary Se are meat and eggs, followed by grains such as flour and rice.[18] Generally, Se is
enriched in protein-rich foods where also containing high bioavailability of Se.[19] In human body, the
intestinal tract is the important site for Se absorption. Elderly people who often with impaired digestive
capacity hence are at a high risk of an insufficient selenium intake.[16] Benefiting from the increasing
epidemiological knowledge, the role of Se (including organic or inorganic forms) has been related to a
number of clinical diseases.[20] Different from observation findings, interestingly, current intervention
studies with Se supplements in population all failed to prove the preventive effect of Se to reduce the risk Page 8/14 Page 8/14 of type 2 diabetes or cancers.[21, 22] These controversial results suggest the complexity
pathophysiological effects of Se in vivo and potential threshold effects. Several studies have reported the associations between serum Se and morbidity and mortality of kidney
disorders, mainly focusing on CKD. Patients with CKD are always characterized by active inflammation
and oxidative stress, as well as abnormal metabolisms of microelements. Previous observational studies
demonstrated that patients with CKD had a lower serum Se levels than those of healthy adults.[17, 23] Xie
et al. found that Se intake seemed to have an inverse relationship on CKD development.[16] Zhu et al. analyzing 3,063 CKD adults from NHANES database reported that a higher serum selenium concentration
could attenuate the risk of all-cause and CVD mortality in patients with CKD, albeit without estimated
adequate dose recommendation.[15] In contrast, the evidence regarding the relationship between serum
Se level or Se supplementation and CSA-AKI development after cardiac surgery is still scarce. DISCUSSION A joint
supplement intervention trial comprising Se supplements found that Se 600 mg twice a day did not
reduce the risk of AKI in patients with elective off-pump CABG.[24] In our study, we firstly reported that a
higher serum Se level could significantly reduce the risk of CSA-AKI after cardiac surgery. We conjecture
that heterogeneity in study designs, included patients, serum Se levels, and surgery (CABG and valve
surgery in our study) have contributed to differences between the two studies. These preliminary evidence
calling for future large-sample research to clarify the possible association between Se and prevention of
CSA-AKI. Several underlying mechanisms of selenium involvement in kidney health have been discussed. The
most noteworthy aspect is the antioxidant effect of Se by acting as a cofactor of antioxidant GPx
enzymes.[9] Se nanoparticles may alleviate AKI induced by ischemia reperfusion injury by upregulated the
(GPx)-1 levels and suppressed NLRP3 inflammasome.[25] When selenoprotein expression are saturated,
however, excessive serum Se will bind to nonspecific selenium-containing proteins and exert harmful
effects through selenomethionine (SeMet) in place of methionine.[26] The metabolites of SeMet including
selenols/selenates can product superoxide radicals and selenyl sulphides/disulphides, causing protein
aggregation, endoplasmic reticulum stress, and inactivation of transcription factors.[27] This could to
some extent explain the negative results in the Se supplementation trails.[21, 22, 24] In addition, Se is
involved in the synthesis and activity of deiodinases, and then through regulating thyroid hormone
influences renal hemodynamics.[28] Moreover, Se has been found related to cellular immunity and
humoral immunity, and Se depletion could impaired lymphocytic proliferation, macrophage activation,
cytokine generation, and neutrophil chemotaxis.[29, 30] LIMITATIONS Several limitations should be considered when interpreting our findings. First, like other observational
studies, we cannot derive a causal relationship between Se level and AKI after cardiac surgery. And the
generalization of our findings should be with caution because of the sample size and only Chinese
patients. Second, residual confounders or unmeasured factors may interfere the results even adjusting Page 9/14 Page 9/14 for multiple baseline variables. However, we have further validated the step-wise associations by Se
quartile and a dose-response curve, which lowers the chances of bias. Third, we only assessed the serum
Se concentration at baseline because of the design of a short-term exposure-outcome study. Future
research should take the dynamic changes of Se concentration into account. for multiple baseline variables. However, we have further validated the step-wise associations by Se
quartile and a dose-response curve, which lowers the chances of bias. Third, we only assessed the serum
Se concentration at baseline because of the design of a short-term exposure-outcome study. Future
research should take the dynamic changes of Se concentration into account. Competing interests All authors had no conflicts of interest to disclose. Data Availability The datasets used and analyzed during the present study are available from the corresponding author on
reasonable request. Ethics approval and consent to participate This study was approved by the Institutional Review Board of the Changzhou Second People’s Hospital. The requirement for obtaining informed consent was waived because of the retrospective design. ACKNOWLEDGMENTS None. CONCLUSIONS In this study, preoperative higher serum Se level was significantly associated with lower risk of incident
CSA-AKI in patients undergoing cardiac surgery. This findings suggested that Se deficiency maybe a
modifiable risk factor for CSA-AKI and be used to identify patients at the high risk of CSA-AKI
development. Further prospective studies are needed to clarify the causal relationship between serum Se
level and incident CSA-AKI. Contributors SYB designed the study and performed analyses, and accepts full responsibility for the work and/or the
conduct of the study, had access to the data, and controlled the decision to publish. GWF collected the
data and wrote the manuscript. All authors reviewed and approved the final manuscript. Page 10/14 References 1. Brown JR, Baker RA, Shore-Lesserson L, Fox AA, Mongero LB, Lobdell KW, LeMaire SA, De Somer F,
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M, et al. Cardiac surgery-associated acute kidney injury. Cardiorenal Med. 2013;3(3):178–99. 3. Ortega-Loubon C, Tamayo E, Jorge-Monjas P. Cardiac Surgery-Associated Acute Kidney Injury:
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kidney injury. Curr Opin Anaesthesiol. 2017;30(1):60–5. 8. Weekley CM, Harris HH. Which form is that? The importance of selenium speciation and metabolism
in the prevention and treatment of disease. Chem Soc Rev. 2013;42(23):8870–94. 8. Weekley CM, Harris HH. Which form is that? The importance of selenium speciation and metabolism
in the prevention and treatment of disease. Chem Soc Rev. 2013;42(23):8870–94. 9. Zachara BA. Selenium and selenium-dependent antioxidants in chronic kidney disease. Adv Clin
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in China. Am J Clin Nutr. 1993;57(2 Suppl):259s–63. 11. Li S, Sun W, Zhang D. Association of Zinc, Iron, Copper, and Selenium Intakes with Low Cognitive
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the Risk of Acute Kidney Injury after Off-Pump CABG: a Randomized Clinical Trial. Brazilian J
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1):1457s–9. Figures Page 12/14 Figure 1
Study flowchart of the patients selection. Study flowchart of the patients selection. Page 13/14 Figure 2
Natural cubic spline analysis between serum selenium concentrations and CSA-AKI. Figure 2 Natural cubic spline analysis between serum selenium concentrations and CSA-AKI. p
y Page 14/14 Page 14/14
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The characteristics of the synonymous codon usage in hepatitis B virus and the effects of host on the virus in codon usage pattern
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Virology journal
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* Correspondence: mamingren@yahoo.com.cn; haxq@yahoo.com
Experimental Center of Medicine, Lanzhou General Hospital, Lanzhou Military
Area Command; Key lab of Stem cells and Gene Drugs of Gansu Province,
Lanzhou 730000, China Abstract Background: Hepatitis B virus (HBV) infection is one of the main human health problem and causes a large-scale
of patients chronic infection worldwide.. As the replication of HBV depends on its host cell system, codon usage
pattern for the viral gene might be susceptible to two main selections, namely mutation pressure and translation
selection. In this case, a deeper investigation between HBV evolution and host adaptive response might assist
control this disease. Result: Relative synonymous codon usage (RSCU) values for the whole HBV coding sequence were studied by
Principal component analysis (PCA). The characteristics of the synonymous codon usage patterns, nucleotide
contents and the comparison between ENC values of the whole HBV coding sequence indicated that the
interaction between virus mutation pressure and host translation selection exists in the processes of HBV evolution. The synonymous codon usage pattern of HBV is a mixture of coincidence and antagonism to that of host cell. But
the difference of genetic characteristic of HBV failed to be observed to its different epidemic areas or subtypes,
suggesting that geographic factor is limited to influence the evolution of this virus, while genetic characteristic
based on HBV genotypes could be divided into three groups, namely (i) genotyps A and E, (ii) genotype B, (iii)
genotypes C, D and G. Conclusion: Codon usage patterns from PCA for identification of evolutionary trends in HBV provide an alternative
approach to understand the evolution of HBV. Further more, a combined selection of mutation pressure with
translation selection on codon usage might shed a light on understanding the evolutionary trends of HBV
genotypes. Keywords: Hepatitis B virus, codon usage pattern, evolution, mutation pressure, translation selectio evolution of the virus with its point mutations, because
the incidence of recombination is rare and any point
mutation could effect the genetic characteristics of two
overlapped genes [3]. The evolution of HBV should be
interactional and constrained by the overlap of genes
[4]. In some cases, the evolution of one overlapping-
gene protein may evolve more rapidly as a consequce of
negative selection to the other,[5]. And the overlapping
genes might be subject to different selections [6]. Furthermore, independent adaptive selection for both
overlapping genes has been reported [7]. One of the
main features of HBV are its genetic heterogeneity [8]. There are four main subtypes, namely ayw, adw, adr
and ayr [9]. According to phylogenetic analysis of the © 2011 Ma 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 characteristics of the synonymous codon
usage in hepatitis B virus and the effects of host
on the virus in codon usage pattern Ming-ren Ma*, Xiao-qin Ha*, Hui Ling, Mei-liang Wang, Fang-xin Zhang, Shang-di Zhang, Ge Li and Wei Yan Open Access Open Access Ma et al. Virology Journal 2011, 8:544
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t/8/1/54 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Introduction Hepatitis B virus (HBV) disease is one of the main glo-
bal health problems that two billion people are infected
and 350 million people undergo chronic infection as
well [1]. HBV belongs to the protyotype member of the
family Hepadnaviridae, and has a compact and circular
DNA genome of about 3.2 kb in length, with four over-
lapping open reading frames including large S region
(PreS/S), PreC/C, × and P [2,3]. Moreover, the overlap-
ping regions on the genome are helpful to study the Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 2 of 10 Page 2 of 10 complete HBV genomic sequence, 9 genotype of HBV
from genotype A to I have been determined and divided
into approximately twenty-five subgenotypes [10-14]. HBV genotypes show distinct geographical distributions
at the level of nucleotide different more than 8% each
other [11,15,16]. It is noticed that nucleotide composition
comprising of HBV coding sequence with various genetic
diversities is selective rather than random, because the
natural selection from host is responsible for selection of
various strains shaped by mutation. In previous reports,
translation selection and compositional constraints under
the mutational pressure are thought to be the major fac-
tors accounting for codon usage variation among gen-
omes in microorganisms [17-24]. In some RNA viruses,
compared with natural selection, mutation pressure plays
a more important role in synonymous codon usage pat-
tern [25,26]. Although it is known that compositional
constraints and translation selection are the more gener-
ally accepted mechanisms accounting for codon usage
bias [27-30], other selection forces have also been pro-
posed such as fine-tuning translation kinetics selection as
well as escape of cellular antiviral responses [23,31-34]. Thus, the codon usage pattern may be important in dis-
closing the molecular mechanism and evolutionary pro-
cess of HBV to avoid host cell response. To our
knowledge, it is the first systemic study to analysis the
synonymous codon usage pattern and evolutional
dynamics of HBV as well as the relationship between
codon usage pattern of HBV and its host. Table 1 The overall nucleotide contents and nucleotide
contents at the synonymous third position of sense
codons in the whole coding sequence of HBV contents at the synonymous third position of sense
codons in the whole coding sequence of HBV
No. Synonymous coodn usage in HBV The C% and U% were higher than A% and G%, and C3%
and U3% were higher than A3% and G3% in HBV (Table 1). The C% and U% were higher than A% and G%, and C3%
and U3% were higher than A3% and G3% in HBV (Table 1). The overall nucleotide composition never affects the
nucleotide contents in the third site of codon in HBV
coding sequence, suggesting that composition con-
straints may be one of the factors in affecting the codon
usage pattern of HBV. For the synonymous codon usage
pattern of HBV, the over-represented synonymous
codons are rare in HBV coding sequence, only including
UCU for Ser, in addition, the under-represented ones
contain AUA for Ile, CCC for Pro, ACC for Thr, GCC
for Ala, CGU and CGG for Arg (Table 2). The codon usage bias of HBV suggests that some
synonymous codons are not chosen equally and
randomly. Introduction T%
C%
A%
G%
T3%
C3%
A3%
G3%
1
27.88
28.16
22.21
21.75
29.94
27.27
21.23
21.56
2
28.28
28.04
21.59
22.09
29.75
27.90
20.57
21.78
3
27.35
28.20
22.04
22.42
29.40
27.04
21.19
22.37
4
27.56
28.33
21.96
22.15
29.44
27.58
20.91
22.07
5
27.85
27.87
22.26
22.02
30.23
26.33
21.75
21.69
6
27.56
28.50
21.93
22.02
29.20
27.73
21.12
21.95
7
27.60
28.50
21.93
21.97
29.27
27.73
21.12
21.89
8
27.60
28.50
21.87
22.04
29.27
27.73
21.05
21.95
9
28.09
27.38
21.88
22.64
29.94
26.10
21.55
22.40
10
28.35
27.23
23.29
21.13
31.95
25.71
22.51
19.83
11
28.12
27.48
21.88
22.52
29.73
26.53
21.48
22.26
12
27.54
27.83
21.87
22.77
28.68
27.54
21.22
22.57
13
28.21
26.97
23.04
21.78
29.04
27.58
21.65
21.73
14
27.95
28.17
21.52
22.35
29.96
27.41
20.76
21.88
15
27.95
28.11
21.79
22.15
30.14
27.04
21.19
21.63
16
27.43
28.01
21.98
22.58
29.58
26.79
21.07
22.56
17
28.65
27.79
21.75
21.82
30.55
27.04
21.14
21.28
18
28.72
27.91
21.39
21.98
29.75
26.45
21.75
22.05
19
28.57
28.40
21.21
21.82
28.25
28.35
21.15
22.25
20
28.55
28.41
21.22
21.82
28.23
28.34
21.19
22.24
21
28.57
28.39
21.22
21.82
28.23
28.34
21.19
22.24
22
28.34
28.27
21.62
21.77
29.48
27.47
21.19
21.86
23
28.22
28.07
21.66
22.04
29.66
27.05
21.24
22.04
24
28.43
28.00
21.65
21.91
30.25
27.24
21.22
21.29
25
27.57
27.99
21.81
22.62
29.71
26.79
20.94
22.56
26
28.74
27.98
21.56
21.72
29.78
26.53
21.96
21.74
27
28.60
28.24
21.39
21.77
29.34
27.14
21.70
21.83
28
28.68
28.17
21.64
21.52
29.56
27.01
21.96
21.48
29
28.78
28.14
21.43
21.65
29.60
26.97
21.74
21.70
30
28.66
28.25
21.36
21.72
29.47
26.97
21.56
22.00
31
28.78
27.68
22.10
21.45
30.76
25.77
22.13
21.34
32
29.07
27.45
21.85
21.63
30.85
25.90
21.91
21.34
33
29.08
27.56
21.65
21.71
30.76
26.25
21.65
21.34
34
28.81
27.42
22.08
21.69
30.63
25.68
22.34
21.34
35
28.39
27.90
21.71
22.01
29.67
26.72
21.52
22.08
36
28.72
27.77
21.92
21.59
30.59
26.12
22.08
21.21
37
28.98
27.52
21.89
21.61
30.63
26.16
21.87
21.34
38
28.95
27.61
21.89
21.55
30.67
26.25
22.04
21.04
39
29.05
27.42
21.85
21.68
30.89
25.81
22.08
21.21
40
28.95
27.59
21.91
21.55
30.85
25.99
21.74
21.43
41
28.42
27.75
21.79
22.04
29.89
26.46
21.78
21.87
42
28.88
27.45
21.89
21.78
30.76
25.55
22.08
21.61
43
28.47
27.65
22.25
21.63
30.91
26.21
21.74
21.14
44
28.99
27.49
21.92
21.59
30.37
26.03
22.21
21.39
45
28.99
27.45
21.95
21.61
30.55
25.90
22.12
21.42
46
28.71
27.72
21.97
21.61
30.28
26.38
22.17
21.17
47
28.71
27.72
21.97
21.61
30.28
26.38
22.17
21.17
48
27.51
27.78
22.15
22.55
29.81
26.46
21.52
22.22
49
27.43
27.93
21.98
22.66
29.21
26.91
21.19
22.68 Genetic relationship based on synonymous codon usage
in HBV The PCA detected the first principal component (f1’)
which can account for 23.65% of the total synonymous
codon usage variation, and the second principal Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 3 of 10 Table 1 The overall nucleotide contents and nucleotide
contents at the synonymous third position of sense
codons in the whole coding sequence of HBV (Continued) Table 1 The overall nucleotide contents and nucleotide
contents at the synonymous third position of sense
codons in the whole coding sequence of HBV (Continued)
50
27.43
27.93
21.98
22.66
29.21
26.91
21.19
22.68
51
27.41
27.95
21.92
22.73
29.33
26.66
21.26
22.75
52
27.45
27.99
21.86
22.71
29.52
26.72
21.01
22.75
53
27.49
27.95
21.84
22.73
29.52
26.60
21.26
22.62
54
27.64
27.97
21.83
22.56
29.44
27.23
20.85
22.49
55
27.45
27.91
21.96
22.68
29.33
26.72
21.32
22.62
56
27.41
27.91
22.04
22.64
29.40
26.72
21.44
22.44
57
27.60
28.50
21.87
22.04
29.27
27.73
21.05
21.95
58
27.79
28.33
22.20
21.68
29.80
27.41
21.55
21.24 Table 2 The relationship of the synonymous codon usage
pattern between HBV and human cell pattern between HBV and human cell
Codon/Amino acid
HBV
Humana
TTT(F)
1.06
0.87
TTC(F)
0.94
1.13
TTA(L)
0.67
0.39
TTG(L)
1.08
0.73
CTT(L)
1.11
0.73
CTC(L)
1.22
1.22
CTA(L)
0.85
0.40
CTG(L)
1.06
2.53
ATT(I)
1.27
1.04
ATC(I)
1.26
1.52
ATA(I)
0.48
0.44
GTT(V)
1.27
0.69
GTC(V)
0.91
1.00
GTA(V)
0.65
0.42
GTG(V)
1.17
1.90
TCT(S)
1.69
1.11
TCC(S)
1.48
1.39
TCA(S)
1.28
0.84
TCG(S)
0.58
0.33
AGT(S)
1.48
0.84
AGC(S)
1.01
1.50
CCT(P)
0.99
1.12
CCC(P)
0.51
1.35
CCA(P)
1.37
1.07
CCG(P)
1.38
0.46
ACT(T)
0.89
0.94
ACC(T)
0.37
1.52
ACA(T)
1.32
1.07
ACG(T)
1.24
0.46
GCT(A)
0.99
1.09
GCC(A)
0.45
1.64
GCA(A)
1.27
0.85
GCG(A)
0.73
0.42
TAT(Y)
1.05
0.84
TAC(Y)
0.95
1.16
CAT(H)
1.21
0.81
CAC(H)
0.79
1.19
CAA(Q)
1.08
0.51
CAG(Q)
0.92
1.49
AAT(N)
1.36
0.89
AAC(N)
0.64
1.11
AAA(K)
0.73
0.82
AAG(K)
1.27
1.18
GAT(D)
1.04
0.89
GAC(D)
0.96
1.11
GAA(E)
1.23
0.81 component (f2’) for 19.47% of the total variation. Based
on the geographical factor in influencing HBV evolution
potentially, there is an obviously geographical distribu-
tion. For example, the overall codon usage pattern of
HBV isolated from Philippines and South Korea is far
from those of China and Indonesia, and the HBV iso-
lated from Germany and Iran has a similar genetic
diversity with that isolated from South Africa (Figure 1). a the synonymous codon usage pattern of human cell was calculated based
on the data of the synonymous codon usage frequencies of human cell. Genetic relationship based on synonymous codon usage
in HBV Based on the subtypes of HBV, the plots for the sub-
type adw were generally divided into two groups, while
the other three subtypes seem to have a similar genetic
characteristic (Figure 2). It is worth noting that the plots for different HBV
genotypes were generally separated from each other. Moreover, the genotypes A and B have an obviously dif-
ferent genetic characteristic with the rest, while geno-
types C, D and G appear to have a relationship of
evolution (Figure 3). These results indicated that the geographic distribu-
tion might be a limited factor to effect the codon usage
of the whole HBV coding sequence, and the subtypes
did not reflect the characteristic of HBV evolution to
some degree. In this case, the codon usage variation
might be one of factors to drive HBV evolution. The effect of mutation pressure on codon usage of HBV p
g
To analyze if the evolution of HBV is shaped by muta-
tion pressure from virus itself or by translation selection
from host, G+C content at the first and second codon
positions (GC12%) was compared with that at synon-
ymous third codon positions (GC3%) (Figure 4). A highly significant correlation was observed (r =
0.432, P < 0.01), implying that mutation pressure from
base composition of HBV is a main factor in shaping
genetic diversity of this virus, since the effects are pre-
sent at all codon positions. In addition, the ENC values
were calculated for each strain and the plot was made
by ENC value against GC3% (Figure 5). The Figureure 5 represented that the plots of HBV
aggregated below the expected curve, suggesting other
selections take part in the process of HBV evolution. 0.81 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 4 of 10 Table 2 The relationship of the synonymous codon usage
pattern between HBV and human cell (Continued)
GAG(E)
0.77
1.19
TGT(C)
0.80
0.86
TGC(C)
1.06
1.14
CGT(R)
0.48
0.51
CGC(R)
0.78
1.20
CGA(R)
0.61
0.63
CGG(R)
0.37
1.20
AGA(R)
1.49
1.20
AGG(R)
1.39
1.26
GGT(G)
0.60
0.64
GGC(G)
0.81
1.40
GGA(G)
1.36
0.98
GGG(G)
1.22
0.98 Table 2 The relationship of the synonymous codon usage
pattern between HBV and human cell (Continued) Comparative analysis of the RSCU values between HBV
and human cell Comparative analysis of the RSCU values between HBV
and human cell There is a resemblance of synonymous codons usage
pattern between this virus and human cell, for example,
the similar synonymous codon usage pattern includes all
synonymous codons for Phe, Ile, Val, Ser, Ala, Tyr, His,
Lys, Asp, Cys and Gly (Table 1). This may be explained
that the codon usage of HBV adapting to its host under
translation selection could result in the multiplication of
progeny virus. This phenomenon possibly implies that
the resemblance of codon usage is favorable for HBV
replication in human cells. But if compared with the
under-represented codons in human cells, CCG for Pro,
ACG for Thr, CAA for Gln and CUA for Leu in HBV
are highly used (Table 1). The result suggested that
these codons could influence the translational rate of
the context flanking them, resulting in the viral product
correct fold. Figure 1 The genetic characteristic of HBV isolated different countries. Figure 1 The genetic characteristic of HBV isolated different countries. Page 5 of 10 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Figure 2 The genetic characteristic of HBV based on the main four subtypes. Figure 3 The genetic characteristic of HBV based on different genotypes. Figure 2 The genetic characteristic of HBV based on the main four subtypes. Figure 2 The genetic characteristic of HBV based on the main four subtypes. Figure 3 The genetic characteristic of HBV based on different genotypes. Figure 3 The genetic characteristic of HBV based on different genotypes. Figure 3 The genetic characteristic of HBV based on different genotypes. Figure 3 The genetic characteristic of HBV based on different genotypes. Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 6 of 10 Figure 4 Correlation between GC content at first and second codon positions (GC2%) with that at synonymous third codon
positions (GC3%). Figure 4 Correlation between GC content at first and second codon positions (GC2%) with that at synonymous third codon
positions (GC3%). in the formation of the different optimal codons with
any nucleotide-ended, the codon usage pattern of HBV
is likely influenced by composition constraints. The
codon usage pattern of PV is mostly coincident with
that of its host, while the codon usage pattern of HBV
is antagonistic to that of its host [37,38]. The codon
usage pattern of HBV is a mixture of the two types of
codon usage. Comparative analysis of the RSCU values between HBV
and human cell The coincident portion of codon usage
pattern for HBV enables the corresponding amino acids
to be translated rapidly, the other antagonistic portion
of codon usage pattern likely enable viral proteins to be
folded properly, although the translation efficiency of
the corresponding amino acids is decreased. Latent
genes in Epstein-Barr virus deoptimize codon usage in
order to evade competition for host protein translation
[28] and attenuation of PV activity was performed by
rare codon pairs inducing poor translation for sequences
of viral proteins [27]. These results suggested that disfa-
vored codons coding for amino acids may not be a dele-
terious factor for viruses to adapt to its host cells. Discussion The ENC values calculated for HBV indicated that
although a significantly lower bias of codon usage exists
in HBV, the codon usage is not mainly affected by
mutation pressure. As for some viruses, previous study
reported that the major factor in shaping codon usage
patterns appears to be mutation pressure rather than
natural selection [19,21,24,35]. However, the comparison
of the synonymous codon usage between HBV and
human cells suggested that the interaction of mutation
pressure with translation selection exists in the process
of HBV evolution, although ENC values for the whole
HBV coding sequence to represent mutation pressure is
one of the factors in influencing codon usage pattern. This characteristic of HBV confers adaptive advantages
which result in a highly efficient dissemination of the
virus through different ways of transmission. The pattern of codon usage is a genetic characteristic
of various organisms in Previous study [19,20,27,
31,32,35,36]. Because C%, U%, U3% and C3% play roles Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 7 of 10 Figure 5 Distribution of the codon usage index, ENC, and GC content at synonymous third codon positions (GC3%). The curve shows
the expected codon usage of GC compositional constraints alone account for codon usage bias. f the codon usage index, ENC, and GC content at synonymous third codon positions (GC3%). The curve shows
e of GC compositional constraints alone account for codon usage bias. Figure 5 Distribution of the codon usage index, ENC, and GC content at synonymous third codon positions (GC3
he expected codon usage of GC compositional constraints alone account for codon usage bias. Figure 5 Distribution of the codon usage index, ENC, and GC content at synonymous third codon positions (GC3%). The curve shows
the expected codon usage of GC compositional constraints alone account for codon usage bias. According to the data of codon usage pattern of HBV
isolated from different countries, the geographic factor
fails to influence the formation of codon usage pattern
of HBV. After all, with development of international
communication and highly efficient dissemination of
HBV through various approaches of transmission, the
affection of geographic factor seems to be weak on the
limitation of HBV distribution in different countries. It
is interesting that the main four subtypes of HBV have
no significant difference in genetic characteristic shaped
by different human races. Materials and methods
Sequence data Table 3 The information of HBV strains in this study The 58 complete RNA sequences of HBV were down-
loaded from the National Center for Biotechnology
Information (NCBI) http://www.ncbi.nlm.nih.gov/Gen-
bank/ and detailed information about the viruses were
listed in Table 3 Each general nucleotide composition (U%, A%, C% and
G%) and each nucleotide composition in the third site of
codon (U3%, A3%, C3% and G3%) in HBV coding sequence
were calculated by biosoftware DNAStar 7.0 for windows. Discussion f’1a
f’2a
ENC value
1
AF405706
-0.79
1.32
56.41
2
X04615
-0.82
0.50
55.88
3
AB033554
-1.11
-0.90
55.78
4
AY741798
-0.82
1.31
56.17
5
AY741797
-0.82
1.15
55.82
6
AY741796
-0.72
1.23
56.62
7
AY741795
-0.75
1.26
56.59
8
AY741794
-0.73
1.26
56.61
9
AF100309
-1.02
-1.17
55.92
10
M57663
0.87
-1.05
55.48
11
AF100308
-1.16
-1.69
55.70
12
U87747
-0.38
-0.96
57.29
13
U87746
0.49
-0.27
55.71
14
AY123041
-0.69
0.77
55.94
15
AF068756
-0.48
0.70
56.39
16
AF282918
-0.84
-1.22
55.98
17
U95551
-0.99
0.62
56.36
18
GQ872210
-0.02
1.01
56.07
19
GQ161818
0.54
0.11
56.88
20
GQ161805
0.56
0.08
56.87
21
GQ161799
0.56
0.11
56.88
22
AY796032
-0.49
1.42
56.08
23
AY796031
-0.43
1.17
56.08
24
AY796030
-0.47
0.68
56.67
25
AF282917
-1.07
-1.45
55.70
26
AY233296
-0.07
1.39
55.62
27
AY23329
-0.38
1.30
56.04
28
AY233294
-0.33
1.62
55.95
29
AY233293
-0.39
1.51
55.92
30
AY233291
-0.45
1.29
55.95
31
AY233290
1.42
0.25
56.75
32
AY233289
1.57
-0.49
56.66
33
AY233288
1.39
-0.33
56.84
34
AY233287
1.55
-0.14
56.82
35
AY233286
1.03
0.00
56.78
36
AY233285
1.26
-0.54
56.52
37
AY233284
1.38
-0.24
56.78
38
AY233283
1.49
-0.45
56.54
39
AY233282
1.35
-0.17
56.73
40
AY233281
1.31
-0.08
56.95
41
AY233280
1.19
0.18
56.82
42
AY233279
1.34
0.04
56.90
43
AY233278
0.86
-0.56
56.37
44
AY233277
1.55
-0.15
56.88
45
AY233276
1.38
-0.38
56.83
46
AY233275
1.87
0.03
56.79
47
AY233274
1.34
-0.30
56.60
48
AY233273
-0.49
-0.80
56.45
49
DQ448628
-1.07
-1.31
55.84
50
DQ448627
-1.07
-1.56
55.84
51
DQ448625
-1.07
-1.56
55.68 Table 3 The information of HBV strains in this study
No. Accession No. Analysis of codon usage bias The ‘effective number of codons’ (ENC), the useful esti-
mator of absolute codon usage bias, was a measure
quantifying the codon usage bias of the whole coding
sequence of HBV. The ENC value ranges from 20
(when only one synonymous codon is chosen by the
corresponding amino acid) to 61 (when all synonymous
codons are used equally) [48]. In this study, this mea-
sure was used to evaluate the degree of codon usage
bias of coding sequences for HBV. The calculation of the relative synonymous
codon usage (RSCU) The relative synonymous codon usage (RSCU) values for
the whole 58 coding sequence of HBV were calculated
as previously described [46]. RSCU values do not
depend on the factors of amino acid composition and
the size of the coding sequence, because the two factors
can be eliminated in the process of calculation. When
RSCU value is equal to 1.0, it means that this codon is
chosen equally and randomly. The RSCU value for a
synonymous codon more than 1.0 or less than 1.0 indi-
cates the more frequency or less frequency, respectively. The synonymous codons with RSCU more than 1.6
were thought to be over-represented, while the synon-
ymous codons with RSCU less than 0.6 were regarded
as under-represented [47]. Discussion This result might suggested
that translation selection from human is not a single
factor to shape the overall codon usage pattern of this
virus and mutation pressure from HBV itself is a main
force to drive HBV evolution. Genotyping of HBV is of
high interest because there is increasing evidence that
HBV genotypes may be associated with HBeAg sero-
conversion rates, mutation occurring in the procure and
core promoter region, severity of liver disease and treat-
ment response [15,16,39,40]. There is a significant dif-
ference of the overall codon usage pattern of HBV
between genotypes A, B, E and C, D, G. HBV genotypes and subgenotypes have been associated with differences
in clinical and virological characteristics, showing that
they may play a role in the virus-host relationship [41]. It has been shown that genotypes C and D are asso-
ciated with more serious liver injuries and with a higher
incidence of HCC than genotypes A and B [42-44]. In
addition, genotype C and D have a much lower rate in
response to interferon therapy than those infected with
A or B genotypes [40,45]. Moreover, subtle differences
in frequency and type of lamivudine resistant variants
occur in genotype A and D infectious [15]. An evolu-
tionary approach to HBV infection, based on the princi-
ples of natural selection, may offer explanation for how
modes of transmission may favor some genotypes and
subgenotypes over others and influence HBV virulence. The genetic diversity and codon usage patterns we
proposed here are helpful to understand the processes
of HBV evolution, especially the roles played by transla-
tion selection from host and mutation pressure from
virus. Additionally, such information might benefit to
understand the roles of geographic and subtype factors
in influencing the process of HBV evolution. Page 8 of 10 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 quences of HBV were down-
l Center for Biotechnology
/www.ncbi.nlm.nih.gov/Gen-
ation about the viruses were
omposition (U%, A%, C% and
omposition in the third site of
G3%) in HBV coding sequence
are DNAStar 7.0 for windows. ve synonymous
odon usage (RSCU) values for
ence of HBV were calculated
[46]. RSCU values do not
amino acid composition and
ence, because the two factors
process of calculation. When
0, it means that this codon is
omly. Discussion The RSCU value for a
han 1.0 or less than 1.0 indi-
or less frequency, respectively. with RSCU more than 1.6
epresented, while the synon-
less than 0.6 were regarded
as
odons’ (ENC), the useful esti-
usage bias, was a measure
age bias of the whole coding
ENC value ranges from 20
ous codon is chosen by the
to 61 (when all synonymous
[48]. In this study, this mea-
e the degree of codon usage
or HBV. is
sis (PCA), which was a com-
atistical method [24], was car-
major trend in codon usage
rains of HBV. PCA involves a
at transforms some correlated
o a smaller number of uncor-
principal components. Each
a 59 dimensional vector, and
nded to the RSCU value of
only included several synon-
ular amino acid, excluding the
hree stop codons. Table 3 The information of HBV strains in this study
No. Accession No. Discussion f’1a
f’2a
ENC value
1
AF405706
-0.79
1.32
56.41
2
X04615
-0.82
0.50
55.88
3
AB033554
-1.11
-0.90
55.78
4
AY741798
-0.82
1.31
56.17
5
AY741797
-0.82
1.15
55.82
6
AY741796
-0.72
1.23
56.62
7
AY741795
-0.75
1.26
56.59
8
AY741794
-0.73
1.26
56.61
9
AF100309
-1.02
-1.17
55.92
10
M57663
0.87
-1.05
55.48
11
AF100308
-1.16
-1.69
55.70
12
U87747
-0.38
-0.96
57.29
13
U87746
0.49
-0.27
55.71
14
AY123041
-0.69
0.77
55.94
15
AF068756
-0.48
0.70
56.39
16
AF282918
-0.84
-1.22
55.98
17
U95551
-0.99
0.62
56.36
18
GQ872210
-0.02
1.01
56.07
19
GQ161818
0.54
0.11
56.88
20
GQ161805
0.56
0.08
56.87
21
GQ161799
0.56
0.11
56.88
22
AY796032
-0.49
1.42
56.08
23
AY796031
-0.43
1.17
56.08
24
AY796030
-0.47
0.68
56.67
25
AF282917
-1.07
-1.45
55.70
26
AY233296
-0.07
1.39
55.62
27
AY23329
-0.38
1.30
56.04
28
AY233294
-0.33
1.62
55.95
29
AY233293
-0.39
1.51
55.92
30
AY233291
-0.45
1.29
55.95
31
AY233290
1.42
0.25
56.75
32
AY233289
1.57
-0.49
56.66
33
AY233288
1.39
-0.33
56.84
34
AY233287
1.55
-0.14
56.82
35
AY233286
1.03
0.00
56.78
36
AY233285
1.26
-0.54
56.52
37
AY233284
1.38
-0.24
56.78
38
AY233283
1.49
-0.45
56.54
39
AY233282
1.35
-0.17
56.73
40
AY233281
1.31
-0.08
56.95
41
AY233280
1.19
0.18
56.82
42
AY233279
1.34
0.04
56.90
43
AY233278
0.86
-0.56
56.37
44
AY233277
1.55
-0.15
56.88
45
AY233276
1.38
-0.38
56.83
46
AY233275
1.87
0.03
56.79
47
AY233274
1.34
-0.30
56.60
48
AY233273
-0.49
-0.80
56.45
49
DQ448628
-1.07
-1.31
55.84
50
DQ448627
-1.07
-1.56
55.84
51
DQ448625
-1.07
-1.56
55.68 References 26. Levin DB, Whittome B: Codon usage in nucleopolyhedroviruses. J Gen
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syndrome virus. Infect Genet Evol 2010, 10:797-803. Principal component analysis Principal component analysis (PCA), which was a com-
monly used multivariate statistical method [24], was car-
ried out to analyze the major trend in codon usage
pattern among different strains of HBV. PCA involves a
mathematical procedure that transforms some correlated
variable (RSCU values) into a smaller number of uncor-
related variables called principal components. Each
strain was represented as a 59 dimensional vector, and
each dimension corresponded to the RSCU value of
each sense codon, which only included several synon-
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codon of AUG, UGG and three stop codons. Ma et al. Virology Journal 2011, 8:544
http://www.virologyj.com/content/8/1/544 Page 9 of 10 Table 3 The information of HBV strains in this study
(Continued)
52
DQ448623
-1.07
-1.34
55.76
53
DQ448622
-0.81
-1.44
55.90
54
DQ448621
-0.94
-1.10
56.24
55
DQ448620
-1.02
-1.46
55.77
56
DQ448620
-0.90
-1.29
56.01
57
AY373432
-0.73
1.26
56.61
58
AY373430
-0.93
0.82
55.86
a f’1 and f’2, respectively, were calculated by PCA method. Table 3 The information of HBV strains in this study
(Continued) Authors’ contributions RMM and HL carried out the molecular genetic studies, participated in the
sequence alignment and drafted the manuscript., MLW and FXZ participated
in the sequence alignment. SDZ, GL and YW participated in the design of
the study and performed the statistical analysis. XQH conceived of the study,
and participated in its design and coordination and helped to draft the
manuscript. All authors read and approved the final manuscript. RMM and HL carried out the molecular genetic studies, participated in the
sequence alignment and drafted the manuscript., MLW and FXZ participated
in the sequence alignment. SDZ, GL and YW participated in the design of
the study and performed the statistical analysis. XQH conceived of the study,
d
d
d
d
d
d h l
d
d f
h 21. Zhou T, Gu W, Ma J, Sun X, Lu Z: Analysis of synonymous codon usage in
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usage model of the context flanking each cleavage site in the
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h
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usage in foot-and-mouth disease virus. Vet Res Commun 2010, 34:393-404. Received: 6 September 2011 Accepted: 15 December 2011
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87:23-29. doi:10.1186/1743-422X-8-544
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Validation of a deep-learning-based retinal biomarker (Reti-CVD) in the prediction of cardiovascular disease: data from UK Biobank
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BMC medicine
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Validation of a deep-learning-based retinal biomarker (Reti-CVD) in the prediction of
cardiovascular disease
data from UK Biobank data from UK Biobank Tseng, Rachel Marjorie Wei Wen; Rim, Tyler Hyungtaek; Shantsila, Eduard; Yi, Joseph K.;
Park, Sungha; Kim, Sung Soo; Lee, Chan Joo; Thakur, Sahil; Nusinovici, Simon; Peng,
Qingsheng; Kim, Hyeonmin; Lee, Geunyoung; Yu, Marco; Tham, Yih-Chung; Bakhai, Ameet;
Leeson, Paul; Lip, Gregory Y. H.; Wong, Tien Yin; Cheng, Ching-Yu
Published in:
BMC Medicine g,
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Park, Sungha; Kim, Sung Soo; Lee, Chan Joo; Thakur, Sahil; Nusinovici, Simon; Peng,
Qingsheng; Kim, Hyeonmin; Lee, Geunyoung; Yu, Marco; Tham, Yih-Chung; Bakhai, Ameet;
Leeson, Paul; Lip, Gregory Y. H.; Wong, Tien Yin; Cheng, Ching-Yu
Published in:
BMC Medicine ,
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Leeson, Paul; Lip, Gregory Y. H.; Wong, Tien Yin; Cheng, Ching-Yu
Published in: DOI (link to publication from Publisher):
10.1186/s12916-022-02684-8 Document Version
Publisher's PDF, also known as Version of record Citation for published version (APA):
Tseng, R. M. W. W., Rim, T. H., Shantsila, E., Yi, J. K., Park, S., Kim, S. S., Lee, C. J., Thakur, S., Nusinovici,
S., Peng, Q., Kim, H., Lee, G., Yu, M., Tham, Y.-C., Bakhai, A., Leeson, P., Lip, G. Y. H., Wong, T. Y., & Cheng,
C.-Y. (2023). Validation of a deep-learning-based retinal biomarker (Reti-CVD) in the prediction of
cardiovascular disease: data from UK Biobank. BMC Medicine, 21(1), Article 28. https://doi.org/10.1186/s12916-
022-02684-8 Citation for published version (APA):
Tseng, R. M. W. W., Rim, T. H., Shantsila, E., Yi, J. K., Park, S., Kim, S. S., Lee, C. J., Thakur, S., Nusinovici,
S., Peng, Q., Kim, H., Lee, G., Yu, M., Tham, Y.-C., Bakhai, A., Leeson, P., Lip, G. Y. H., Wong, T. Y., & Cheng,
C.-Y. (2023). Validation of a deep-learning-based retinal biomarker (Reti-CVD) in the prediction of
cardiovascular disease: data from UK Biobank. BMC Medicine, 21(1), Article 28. https://doi.org/10.1186/s12916-
022-02684-8 Aalborg Universitet eneral rights
opyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners
nd it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research.
- You may not further distribute the material or use it for any profit-making activity or commercial gain Validation of a deep‑learning‑based
retinal biomarker (Reti‑CVD) in the prediction
of cardiovascular disease: data from UK Biobank Validation of a deep‑learning‑based
retinal biomarker (Reti‑CVD) in the prediction
of cardiovascular disease: data from UK Biobank Rachel Marjorie Wei Wen Tseng1,2†, Tyler Hyungtaek Rim1,3,4*† , Eduard Shantsila5, Joseph K. Yi6, Sungha Park7,
Sung Soo Kim8, Chan Joo Lee7, Sahil Thakur1, Simon Nusinovici1,3, Qingsheng Peng1,9, Hyeonmin Kim4,
Geunyoung Lee4, Marco Yu1,3, Yih‑Chung Tham1,3,10,11, Ameet Bakhai12,13, Paul Leeson14, Gregory Y.H. Lip15,
Tien Yin Wong1,16 and Ching‑Yu Cheng1,3,10,11 Rachel Marjorie Wei Wen Tseng1,2†, Tyler Hyungtaek Rim1,3,4*† , Eduard Shantsila5, Joseph K. Yi6, Sungha Park7,
Sung Soo Kim8, Chan Joo Lee7, Sahil Thakur1, Simon Nusinovici1,3, Qingsheng Peng1,9, Hyeonmin Kim4,
Geunyoung Lee4, Marco Yu1,3, Yih‑Chung Tham1,3,10,11, Ameet Bakhai12,13, Paul Leeson14, Gregory Y.H. Lip15,
Tien Yin Wong1,16 and Ching‑Yu Cheng1,3,10,11 © 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
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C
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d General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners
nd it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. Y
f
h
di
ib
h
i l
i f
fi
ki
i i
i l
i - Users may download and print one copy of any publication from the public portal for the purpose of private
- You may not further distribute the material or use it for any profit-making activity or commercial gain You may not further distribute the material or use it for any profit making activity o
- You may freely distribute the URL identifying the publication in the public portal - BMC Medicine Tseng et al. BMC Medicine (2023) 21:28
https://doi.org/10.1186/s12916-022-02684-8 Open Access Abstract Background Currently in the United Kingdom, cardiovascular disease (CVD) risk assessment is based on the QRISK3
score, in which 10% 10-year CVD risk indicates clinical intervention. However, this benchmark has limited efficacy in
clinical practice and the need for a more simple, non-invasive risk stratification tool is necessary. Retinal photography
is becoming increasingly acceptable as a non-invasive imaging tool for CVD. Previously, we developed a novel CVD
risk stratification system based on retinal photographs predicting future CVD risk. This study aims to further validate
our biomarker, Reti-CVD, (1) to detect risk group of ≥ 10% in 10-year CVD risk and (2) enhance risk assessment in indi‑
viduals with QRISK3 of 7.5–10% (termed as borderline-QRISK3 group) using the UK Biobank. Methods Reti-CVD scores were calculated and stratified into three risk groups based on optimized cut-off values
from the UK Biobank. We used Cox proportional-hazards models to evaluate the ability of Reti-CVD to predict CVD
events in the general population. C-statistics was used to assess the prognostic value of adding Reti-CVD to QRISK3 in
borderline-QRISK3 group and three vulnerable subgroups. Results Among 48,260 participants with no history of CVD, 6.3% had CVD events during the 11-year follow-up. Reti-CVD was associated with an increased risk of CVD (adjusted hazard ratio [HR] 1.41; 95% confidence interval [CI],
1.30–1.52) with a 13.1% (95% CI, 11.7–14.6%) 10-year CVD risk in Reti-CVD-high-risk group. The 10-year CVD risk of the
borderline-QRISK3 group was greater than 10% in Reti-CVD-high-risk group (11.5% in non-statin cohort [n = 45,473],
11.5% in stage 1 hypertension cohort [n = 11,966], and 14.2% in middle-aged cohort [n = 38,941]). C statistics
increased by 0.014 (0.010–0.017) in non-statin cohort, 0.013 (0.007–0.019) in stage 1 hypertension cohort, and 0.023
(0.018–0.029) in middle-aged cohort for CVD event prediction after adding Reti-CVD to QRISK3. Conclusions Reti-CVD has the potential to identify individuals with ≥ 10% 10-year CVD risk who are likely to benefit
from earlier preventative CVD interventions. For borderline-QRISK3 individuals with 10-year CVD risk between 7.5 and †Rachel Marjorie Wei Wen Tseng and Tyler Hyungtaek Rim contributed equally
to this work. *Correspondence:
Tyler Hyungtaek Rim
tyler.rim.academia@gmail.com
Full list of author information is available at the end of the article RESEARCH ARTICLE RESEARCH ARTICLE
Open Access
Validation of a deep‑learning‑based
retinal biomarker (Reti‑CVD) in the prediction
of cardiovascular disease: data from UK Biobank
Rachel Marjorie Wei Wen Tseng1,2†, Tyler Hyungtaek Rim1,3,4*† , Eduard Shantsila5, Joseph K. Yi6, Sungha Park7,
Sung Soo Kim8, Chan Joo Lee7, Sahil Thakur1, Simon Nusinovici1,3, Qingsheng Peng1,9, Hyeonmin Kim4,
Geunyoung Lee4, Marco Yu1,3, Yih‑Chung Tham1,3,10,11, Ameet Bakhai12,13, Paul Leeson14, Gregory Y.H. Lip15,
Tien Yin Wong1,16 and Ching‑Yu Cheng1,3,10,11 Validation of a deep‑learning‑based
retinal biomarker (Reti‑CVD) in the prediction
of cardiovascular disease: data from UK Biobank Study population
d
l
l We used clinical data and retinal photographs from the
UK Biobank, a prospective population-based cohort in
the UK [12]. The UK Biobank protocol is available online
[13]. Despite these updated guidelines, their effect on
clinical practice has been limited, with little change in
statin prescription behavior [6]. Moreover, considering
that 10-year CVD risk might underestimate the lifetime
probability of developing CVD, NICE recommends that
antihypertensive treatment should also be considered
for people under 60 who have a predicted 10-year CVD
risk below 10%. Practice-changing guidelines would
require time-consuming longitudinal prospective stud-
ies. A simple and personalized CVD risk refinement
tool may be helpful in not only minimizing time and
various resource costs, but also finding consensus on
statin and antihypertensive treatment initiation.f We excluded (1) duplicated retinal photographs
(n = 18,423), (2) those who had type 1 diabetes (n = 288),
(3) those with pre-existing CVD at baseline (n = 7624),
(4) poor-quality photographs (n = 11,115), and (5) those
who were < 40 years old (n = 1) (Additional file 1: eFig-
ure 1). Pre-existing CVD was defined as previous history
of coronary heart disease, other heart diseases, stroke,
transient ischemic attack, peripheral arterial disease, or
cardiovascular surgery, and patients who have undergone
cardiovascular procedures based on the Classification of
Interventions and Procedures version 4 (OPCS-4) [10]. Retinal photography is a simple, effective, and non-
invasive imaging tool that provides fast and accurate
information on the human vasculature that may not
be clearly visible to the human eye especially in recent
years with the advent of deep learning (DL) [7–9]. In
our previous study, we developed a DL-based three-tier
CVD risk stratification system, RetiCAC, using coro-
nary artery calcium (CAC) as a ground truth, which
allowed us to stratify CVD risk in the UK Biobank and
a diversified Asian population [10]. However, this study
had no comparison of RetiCAC with QRISK3 and was
not UK-specific, thereby limiting its application in A total of 48,260 participants, representing the general
population without a history of CVD, were included for
analysis. In addition, we also defined three at-risk sub-
groups: (1) the non-statin cohort, individuals not taking
a statin; (2) the stage 1 hypertension cohort, individuals
with stage 1 hypertension and not taking any antihyper-
tensive medications; and (3) the middle-aged cohort,
individuals aged 40–64 years at baseline (Additional
file 1: eFigure 1). Ethics statementh This retrospective study was deemed exempt from insti-
tutional review board (IRB) review by the SingHealth
Centralised Institutional Review Board (CIRB). This
study adhered to the tenets of the Declaration of Hel-
sinki. Written informed consent was obtained from the
participants of the original studies [10, 11]. Background In the United Kingdom (UK), around 1 in 9 people
live with cardiovascular disease (CVD) [1, 2]. A for-
mal risk assessment tool, QRISK3, was developed using
data from a cohort of 1.28 million individuals, and it
is currently recommended by the National Institute
for Health and Care Excellence (NICE) for CVD risk
assessment and guidance for primary prevention strat-
egies [3]. In 2014, NICE updated the requirements for
statin initiation in individuals at risk of developing
CVD, increasing the number of eligible individuals for
statin treatment [4]. NICE also updated the require-
ments for antihypertensive initiation in individuals
with stage 1 hypertension (clinically-measured blood
pressure ranging from 140/90 to 159/99 mmHg) in
2019 [5]. The current recommendation for initiating
statins for primary CVD prevention and antihyperten-
sives for stage 1 hypertension is a calculated QRISK3
score of 10% [4, 5]. Therefore, the benchmark of 10%
for 10-year CVD risk is very important in determining
clinical intervention.f In this study, we aimed to optimize the operating
threshold of RetiCAC, herein referred to as Reti-CVD,
via simple retinal imaging. Using Reti-CVD, we aimed (1)
to detect risk group of ≥ 10% in 10-year CVD risk and (2)
to evaluate the potential of Reti-CVD as a risk enhancer
when applied alongside QRISK3 for borderline-QRISK3
groups (i.e., individuals with a 10-year CVD risk of 7.5–
10%) in the UK Biobank. © 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. Tseng et al. BMC Medicine (2023) 21:28 Page 2 of 10 Tseng et al. BMC Medicine 10%, Reti-CVD could be used as a risk enhancer tool to help improve discernment accuracy, especially in adult groups
that may be pre-disposed to CVD.i Keywords Artificial intelligence, Cardiovascular disease, Deep learning, Retinal imaging, Retinal photograph, Risk
stratification, Risk stratification system, UK Biobank clinical decision-making such as statin and anti-hyper-
tensive medication initiation in the UK. Study population
d
l
l Since the purpose of Reti-CVD is aimed
at primary prevention of CVD, we focus on individuals Tseng et al. BMC Medicine (2023) 21:28 Tseng et al. BMC Medicine (2023) 21:28 Page 3 of 10 intervals so that a more detailed comparison could be
done with Reti-CVD. who display a lack of awareness towards the risk factors
associated with cardiovascular disease. In a previous
publication, the authors reported based on age-stratified
analyses that unawareness rates were highest in indi-
viduals aged between 40 and 49 years old and lowest in
individuals aged above 70 years old [14]. Therefore, our
middle-aged cohort is classified based on vulnerability. Statistical analysis
A
l
d Analyses were done using p < 0.05 as the significance
level, Stata/MP version 14.0 for survival analysis, and R
version 3.4.4 for estimation of net reclassification index
(NRI) using the R package survIDINRI [18]. Descrip-
tive statistics are provided for all participants and also
according to the three risk groups by Reti-CVD. New retinal‑based cardiovascular disease risk stratification
system ≈ Details of RetiCAC model development and previous
validations have been described elsewhere [9]. Briefly, the
RetiCAC score was defined based on a probability score
derived from our deep-learning algorithm of binary clas-
sification (absence vs presence of coronary artery cal-
cium [CAC]). The probability scores ranged from zero to
one, with a high value indicating a high probability of the
presence of CAC. First, we enhanced the RetiCAC algo-
rithm using more datasets which includes both retinal
photographs and CT scans from Korea (Additional file 3:
eDocument 1). Second, we proposed new cardiovascu-
lar disease risk stratification groups (i.e., Reti-CVD) with
optimized cut-off values based on 40th percentile and
95th percentile of Reti-CVD score among 48,260 par-
ticipants after exclusion. The cut-off values were deter-
mined to have similar incidence rate: the low-risk group
of Reti-CVD was designed to have similar incidence
rate to those of the 0 to 5% QRISK3 risk group (i.e., 2.5
per 1000 person-years), and the moderate-risk of Reti-
CVD group was designed to have similar incidence rate
to those of the 5 to 10% QRISK3 risk group (i.e., 7.0 per
1000 person-years). We used these proposed cut-off val-
ues to further stratify the CVD risk in the UK Biobank
participants. i
Retinal photographs included in the study were taken
using the Topcon 3D OCT-1000 Mark II (Topcon Cor-
poration) between 7 December 2009 and 21 July 2010. Retinal cameras used in the training set include AFP-210
non-mydriatic auto retinal camera (NIDEK Corporation,
Aichi, Japan), TRC-NW8 non-mydriatic retinal camera
(Topcon Corporation, Tokyo, Japan), and Nonmyd A-D
(Kowa Co. Ltd., Shizuoka, Japan). We did not include
Topcon 3D OCT-1000 Mark II (Topcon Corporation) in
our training set.hi The other variables used in this study were defined as
follows. Pre-diabetes and diabetes were defined based on
(1) medical history and (2) glucose ≥ 5.5 mmol/L. Medi-
cal history of high cholesterol, type 1 diabetes, antihyper-
tensive were self-declared and collected from a baseline
assessment questionnaire on medical conditions. Smok-
ing status was self-declared as well and categorized into
“life-time smoker” and “never.” Definition of cardiovascular disease events In the UK Biobank, we used hospitalization and mortal-
ity data provided by the National Health Service (NHS)
registers. The main outcome of interest in the current
study reflected the outcome used in the QRISK3 risk
score: fatal CVD events (ICD-10 I00-99, F01, Q20-Q28,
C38.0, P29, G45) [15] or nonfatal coronary heart disease,
ischemic stroke, or transient ischemic attack (ICD-10
G45, I20–24, and I63–64) [16]. Study population characteristics Table 1 details the clinical characteristics of the partici-
pants by the three groups of Reti-CVD score (categorized
as low, moderate, and high-risk groups). Among 48,260 included participants from the UK
Biobank, the median QRISK3 10-year CVD risk was
4.5% (IQR 2.2–8.0%, SD, 4.2%); CVD events occurred in
2766 (5.7%) participants during the follow-up. The Spear-
man’s rank correlation coefficient between Reti-CVD and
QRISK3 score was 0.50 (p < 0.001). The incremental prognostic value of the Reti-CVD
over the QRISK3 in the prediction of CVD events was
assessed using C-statistics and continuous net reclas-
sification index (NRI) [18]. In addition, decision curve
analysis was used to compare the net-benefit of models
at different thresholds over QRISK3 model and Reti-
CVD plus age and gender model. Age and gender were
included for fair comparison because QRISK3 is based
on survival model including various risk factors includ-
ing age, gender, smoking, and comorbidities. Continu-
ous models were presented as decision-analysis curves CVD events rate were 2.8% (545/19,304) in the low-risk
group, 7.2% (1900/26,543) in the moderate-risk group,
and 13.3% (321/2413) in the high-risk group of Reti-
CVD. Among the low-risk group of Reti-CVD, 84.3%
had QRISK3 score of 0 to < 5%, and among the high-risk
group of Reti-CVD, 8.6% had QRISK3 score of 0 to 5%,
40.1% had QRISK3 score of ≥ 5 to < 10%, and 36.3% had
QRISK3 score of ≥ 10 to < 15%. Table 1 Characteristics of the study population
Data are presented as n, n (% of participants), mean (standard deviation [SD]), or median (interquartile range [IQR]). CVD cardiovascular disease. Reti-CVD deep-
learning-based retinal CVD biomarker
Reti-CVD score
Characteristics
Low risk
Moderate risk
High risk
No. Calculation of the QRISK3 score and borderline‑QRISK3 For each individual, the QRISK3 score was calculated
using R package, version 3.6 [17]. The distribution of the
QRISK3 score is provided in Additional file 2: eFigure 2. For comparison with three-strata Reti-CVD groups, we
divided the subjects into 5 groups based on the QRISK3
score (%) (≥ 0 to < 5; ≥ 5 to > 10; ≥ 10 to > 15; ≥ 15 to < 20;
and ≥ 20). In addition, as the recommended threshold for
initiating statins and antihypertensive medication from a
10-year risk of CVD is 10%, we defined “borderline-risk”
group as those who had QRISK3 score between 7.5 and
10%. Specifically, we divided the subjects into 5 groups
as compared to the previous three-risk-strata group of
QRISK3 as outlined in the NICE guidelines. Herein lies
the difference in intervals used to evaluate CVD events. We used 5% intervals compared to the previous 10% In the UK Biobank, hospitalization and mortality data
were available up to May 05, 2021, at the time of analysis
and each participant was followed up to 11.4 years from
the date of baseline visit. In survival analysis, each patient
was followed up to 11.4 years (median follow-up, 11.0
years) from the date of baseline visit to the last follow-up
date or the date of the CVD events. In all populations, the cumulative incidence of car-
diovascular events rate was evaluated across the three
groups (low, moderate, and high risk) defined by the
Reti-CVD using Kaplan-Meier method. Cox proportional
hazards model was used to estimate the hazard ratios
(HRs) and trends in HRs and respective p-values were
examined by fitting a linear model for the three catego-
ries. Unadjusted HR was provided according to three risk Tseng et al. BMC Medicine (2023) 21:28 Page 4 of 10 groups of Reti-CVD, and QRISK3-ajudted HR trend was
provided. groups of Reti-CVD, and QRISK3-ajudted HR trend was
provided. (demonstrating potential outcomes of using any thresh-
old in that model) and models with higher net-benefit
were considered higher performing. To help future patients make an informed decision on
statin and antihypertensive medication initiation, we
only included the borderline-QRISK3 group who had
QRISK3 score between 7.5 and 10% of 10-year CVD risk. In the borderline QRISK3 group, cumulative incidence of
cardiovascular events rate was evaluated across the three
groups (low, moderate, and high risk) according to the
Reti-CVD and compared with participants with QRISK3
5–7.5% and 10–12.5%. Calculation of the QRISK3 score and borderline‑QRISK3 The same analysis was repeated
for middle-aged group (40 to 64 years). Data are presented as n, n (% of participants), mean (standard deviation [SD]), or median (interquartile range [IQR]). CVD cardiovascular disease. Reti-CVD deep-
learning-based retinal CVD biomarker (% of participants), mean (standard deviation [SD]), or median (interquartile range [IQR]). CVD cardiovascular disease. Reti-CVD deep-
D biomarker Study population characteristics of participants
19,304
26,543
2413
Cardiovascular outcomes
Nonfatal and fatal CVD events (QRISK)
545 (2.8%)
1900 (7.2%)
321 (13.3%)
QRISK3
QRISK3 score, mean (SD)
3.0 (2.5)
7.0 (4.1)
10.6 (4.6)
QRISK3 score
≥ 0 to < 5
16,282 (84.3%)
9646 (36.3%)
208 (8.6%)
≥ 5 to < 10
2572 (13.3%)
11,393 (42.9%)
967 (40.1%)
≥ 10 to < 15
384 (2.0%)
4408 (16.6%)
876 (36.3%)
≥ 15 to < 20
55 (0.3%)
880 (3.3%)
272 (11.3%)
> 20
11 (0.1%)
216 (0.8%)
90 (3.7%)
Clinical biomarkers
Age, mean (SD)
50.8 (7.0)
59.8 (6.7)
64.4 (4.5)
Gender
Female, n (%)
12,702 (65.8%)
13,622 (51.3%)
741 (30.7%)
Male, n (%)
6602 (34.2%)
12,921 (48.7%)
1672 (69.3%)
Other factors
Antihypertensive medication, n (%)
1637 (8.5%)
5642 (21.3%)
846 (35.1%)
Stage 1 hypertension, n (%)
4199 (21.8%)
7178 (27.0%)
589 (24.4%)
Pre-diabetes and diabetes, n (%)
269 (1.4%)
677 (2.6%)
86 (3.6%)
Statin, n (%)
694 (3.6%)
1923 (7.2%)
170 (7.0%)
Current smoker, n (%)
6451 (33.4%)
11,221 (42.3%)
1219 (50.5%) Table 1 Characteristics of the study population Tseng et al. BMC Medicine (2023) 21:28 Page 5 of 10 Tseng et al. BMC Medicine 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0
2
4
6
8
10
Time (years)
≥0 to <5
≥5 to <10
≥10 to <15
≥15 to <20
≥20
QRISK3 score
e
t
a
r t
n
e
v
e
e
s
a
e
sid
r
alu
c
s
a
v
oid
r
a
C 0.00
0.05
0.10
0.15
0.20
0.25
0.30
2413
2374
2309
2210
2119
2001
26543 26243 25855 25374 24803 24088
19304 19216 19093 18958 18779 18579
. 0
2
4
6
8
10
Time (years)
Low
Moderate
High
0.00
0.05
0.10
0.15
0.20
0.25
0.30
317
308
289
265
240
216
1207
1175
1140
1086
1024
943
5668
5547
5397
5221
5019
4769
14932 14770 14547 14254 13931 13510
26136 26033 25884 25716 25487 25230
. 0
2
4
6
8
10
Time (years)
≥0 to <5
≥5 to <10
≥10 to <15
≥15 to <20
≥20
QRISK3 score
QRISK3
≥0 to <5
≥5 to <10
≥10 to <15
≥15 to <20
≥20
Number at risk
e
t
a
r t
n
e
v
e
e
s
a
e
sid
r
alu
c
s
a
v
oid
r
a
C
RetiCVD
RetiCVD
Low
Moderate
High
Number at risk
Fig. 1 Kaplan-Meier curves according to Reti-CVD and QRISK3. Study population characteristics Cardiovascular disease (CVD) event rate according to QRISK3-five groups (A), and
Reti-CVD-three groups (B) in all participants (n = 48,260) were presented. In the general population, the high-risk group of Reti-CVD (B) shows
a similar incident CVD rate to that of the QRISK 10–15% group (A), indicating that if a participant is identified as Reti-CVD-high risk by retinal
photography, participants may be advised to undergo a full CVD risk assessment via the NHS primary care service 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0
2
4
6
8
10
Time (years)
Low
Moderate
High
RetiCVD Fig. 1 Kaplan-Meier curves according to Reti-CVD and QRISK3. Cardiovascular disease (CVD) event rate according to QRISK3-five groups (A), and
Reti-CVD-three groups (B) in all participants (n = 48,260) were presented. In the general population, the high-risk group of Reti-CVD (B) shows
a similar incident CVD rate to that of the QRISK 10–15% group (A), indicating that if a participant is identified as Reti-CVD-high risk by retinal
photography, participants may be advised to undergo a full CVD risk assessment via the NHS primary care service a Based on multivariable model after adjusting QRISK3 five groups. Incidence per 1000 person-years. CI confidence interval. CVD cardiovascular disease. HR hazard
ratio. N number at risk. Reti-CVD deep-learning-based retinal CVD biomarker Performance of Reti‑CVD in prediction of CVD event the general population of the UK Biobank (Fig. 1B). Based on Reti-CVD, the incidence of CVD per 1000 per-
son-years was 2.6 (95% CI, 2.4–2.8) in the low-risk group,
6.8 (6.5–7.1) in the moderate-risk group, and 13.1 (11.7–
14.6) in the high-risk group (Table 2), indicating a 13.1%
10-year CVD risk in Reti-CVD-high-risk group. Analysis
for non-statin cohort and stage 1 hypertension cohort are
provided in Additional file 4: eFigure 3 and Additional
file 5: eTable 1. Kaplan-Meier curves were described in all participants
according to five groups of QRISK3 and three groups
of Reti-CVD (Fig. 1). During the follow-up (median
11.0 years; interquartile range [IQR], 10.9–11.1 years),
513,714.3 person-years were analyzed. As expected,
QRISK3 stratified CVD risk well in general population of
the UK Biobank (Fig. 1A). Reti-CVD also shows distinct
CVD risk stratification based on the three groups within Table 2 Risk of cardiovascular events by the deep-learning-based retinal CVD biomarker (Reti-CVD) in all participants
a Based on multivariable model after adjusting QRISK3 five groups. Incidence per 1000 person-years. CI confidence interval. CVD cardiovascular disease. HR hazard
ratio. N number at risk. Reti-CVD deep-learning-based retinal CVD biomarker
Risk predictor
N
Cases
Person-years
Incidence (95% CI)
Unadjusted
hazard ratio (95%
CI)
Reti-CVD
Low
19,304
545
209,186
2.6 (2.4–2.8)
1 (reference)
Moderate
26,543
1900
280,021
6.8 (6.5–7.1)
2.62 (2.38–2.88)
High
2413
321
24,508
13.1 (11.7–14.6)
5.11 (4.45–5.86)
Adjusted HR trenda
1.41 (1.30–1.52)
QRISK3 score
≥ 0 to < 5
26,136
702
283,604
2.5 (2.3–2.7)
1 (reference)
≥ 5 to < 10
14,932
1099
157,442
7.0 (6.6–7.4)
2.84 (2.58–3.12)
≥ 10 to < 15
5668
695
57,723
12.0 (11.2–13.0)
4.93 (4.44–5.48)
≥ 15 to < 20
1207
196
11,988
16.3 (14.2–18.8)
6.75 (5.76–7.91)
≥ 20
317
74
2957
25.0 (19.9–31.4)
10.50 (8.27–13.35)
Total
48,260
2766
513,714
5.4 (5.2–5.6) ovascular events by the deep-learning-based retinal CVD biomarker (Reti-CVD) in all participants Tseng et al. BMC Medicine (2023) 21:28 Tseng et al. BMC Medicine (2023) 21:28 Page 6 of 10 Additional file 6: eTable 2 describes the performance
of Reti-CVD in predicting the incidence of CVD
events when applied to at-risk subgroup populations. Incidence of CVD and QRISK3-adjusted HR trends
were provided in 3 subgroups: individuals with body-
mass index (BMI) above 25 kg/m2, hypertension, and
pre-diabetes/diabetes. Our results show that the three-
strata Reti-CVD system can further stratify CVD risk
in all at-risk subgroup populations. Discussion In this study, we optimized the operating threshold of
RetiCAC, which was originally based on the Korean
cohort. This optimized biomarker, Reti-CVD, strati-
fied the general UK population into three risk groups
and presented with significant hazard ratio (HR) trends. Specifically, the high-risk group in Reti-CVD adequately 0.00
0.05
0.10
0.15
0.20
3353
3285
3207
3107
2992
2857
518
508
498
482
462
443
4526
4466
4386
4275
4166
4019
652
643
638
624
601
587
7842
7777
7672
7544
7402
7201
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
0.00
0.05
0.10
0.15
0.20
974
956
932
907
867
831
186
183
179
171
165
158
1525
1497
1466
1427
1393
1335
230
226
226
221
211
208
2622
2602
2562
2518
2475
2417
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
0.00
0.05
0.10
0.15
0.20
1327
1301
1271
1234
1195
1141
287
282
277
263
252
237
2856
2812
2770
2691
2623
2536
497
493
489
479
465
458
7097
7033
6948
6822
6692
6511
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
No. at risk
QRISK3 & RetiCVD
Cardiovascular disease event rate
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
No. at risk
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
QRISK3 & RetiCVD
No. at risk
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
QRISK3 & RetiCVD
Fig. 2 Kaplan-Meier curves according to Reti-CVD in borderline-QRISK3 group. Cardiovascular disease (CVD) event rate according to Reti-CVD-three
groups in borderline-QRISK3 group who had QRISK3 score between 7.5 and 10% were presented in non-statin cohort (A), stage 1 hypertension
cohort (B), and middle-aged (40–64 years) cohorts (C). Performance of Reti‑CVD in prediction of CVD event Specifically, in the
subgroup of individuals who were taking antihyperten-
sive medication, the incidence of CVD was as high as
17.7 (95% CI, 15.0–20.8) in Reti-CVD-high-risk group. Risk discrimination and incremental value We then tested the incremental value of Reti-CVD when
applied alongside QRISK3 in predicting incident CVD
events among non-statin, stage 1 hypertension, and mid-
dle-aged cohorts (Table 4). By adding Reti-CVD score to
QRISK3, C statistics increased by 0.014 (95% CI, 0.010–
0.017) in non-statin cohort, 0.013 (0.007–0.019) in stage
1 hypertension cohort, and 0.023 (0.018–0.029) in mid-
dle-aged cohort for prediction of CVD events. Also, by
adding Reti-CVD score to QRISK3, the continuous NRI
was 0.133 (95% CI, 0.088–0.173) in non-statin cohort,
0.094 (0.008–0.174) in stage 1 hypertension cohort, and
0.248 (0.190–0.301) in middle-aged cohort.ii Additional file 7: eFigure 4 summarizes net-benefit
comparisons using a decision-analysis curve. In entire
UK Biobank participants, Additional file 7: eFigure 4A
presents the decision-analysis curves for QRISK3 score
and Reti-CVD plus age, and gender model, indicating
that the two models are almost similar in terms of net-
benefit. In the hypertensive patients and the pre-diabetes
and diabetes subgroup (Additional file 7: eFigure 4B and
4C), the Reti-CVD plus age, and gender model dem-
onstrated a higher net-benefit than QRISK3 score in
general. Performance of Reti‑CVD as a risk enhancer
in borderline‑QRISK3 group The Kaplan-Meier analysis of CVD events in the UK
Biobank according to the three strata of Reti-CVD
among borderline group individuals (QRISK3 score
between 7.5 and 10%) is provided in Fig. 2. Although
QRISK3 was less than 10% in the borderline group,
the cumulative CVD events rate in Reti-CVD-high-
risk group was consistently higher than 10% of 10-year
CVD risk. Specifically, in this borderline-QRISK3 group,
the 10-year CVD risk was higher than 10% in Reti-
CVD-high-risk group with 11.5% (8.9–15.0%) in
non-statin cohort (n = 45,473), 11.5% (7.4–17.8%) in
stage 1 hypertension cohort (n = 11,966), and 14.2%
(10.3–19.5%) in middle-age cohort (n = 38,941)
(Table 3). Discussion Considering that statin and antihypertensive pharmacotherapy initiation is recommended
at QRISK of ≥ 10%, Reti-CVD can be used as a risk enhancer in borderline-QRISK3 groups of 7.5–10% to reach consensus on statin initiation. In
addition, although most of risk assessment systems were derived from cohorts of primarily middle-aged people and typically well-functioning
individuals, Reti-CVD can still be a risk enhancer in in borderline-QRISK3 groups of 7.5–10% middle-aged people 0.00
0.05
0.10
0.15
0.20
3353
3285
3207
3107
2992
2857
518
508
498
482
462
443
4526
4466
4386
4275
4166
4019
652
643
638
624
601
587
7842
7777
7672
7544
7402
7201
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
No. at risk
QRISK3 & RetiCVD
Cardiovascular disease event rate
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
≥ 0.00
0.05
0.10
0.15
0.20
974
956
932
907
867
831
186
183
179
171
165
158
1525
1497
1466
1427
1393
1335
230
226
226
221
211
208
2622
2602
2562
2518
2475
2417
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
No. at risk
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
QRISK3 & RetiCVD 0.00
0.05
0.10
0.15
0.20
1327
1301
1271
1234
1195
1141
287
282
277
263
252
237
2856
2812
2770
2691
2623
2536
497
493
489
479
465
458
7097
7033
6948
6822
6692
6511
. 0
2
4
6
8
10
Time (years)
≥5 to <7.5
≥7.5 to <10 & RetiCVD-Low risk
≥7.5 to <10 & RetiCVD-Moderate risk
≥7.5 to <10 & RetiCVD-High risk
≥10 to <12.5
No. at risk
RetiCVD
& Low
& Moderate
& High
QRISK3
≥5 to <7.5
≥7.5 to <10
≥7.5 to <10
≥7.5 to <10
≥10 to <12.5
QRISK3 & RetiCVD Fig. 2 Kaplan-Meier curves according to Reti-CVD in borderline-QRISK3 group. Discussion Reti-CVD deep-learning-
based retinal CVD biomarker
QRISK3
Reti-CVDa
N
Cases
Person-years
Incidence rate at
year 10 (95% CI)
Non-statin cohort (n = 45,473)
≥ 5 to < 7.5
7842
478
83,295
5.6 (5.1–6.1)
≥ 7.5 to < 10
Low
652
47
6868
7.2 (5.4–9.6)
≥ 7.5 to < 10
Moderate
4526
401
47,286
7.9 (7.1–8.8)
≥ 7.5 to < 10
High
518
62
5318
11.5 (8.9–15.0)
≥ 10 to < 12.5
3353
394
34,314
11.0 (9.9–12.2)
Total
16,891
1382
177,081
7.8 (7.4–8.2)
Stage 1 Hypertension cohort (n = 11,966)
≥ 5 to < 7.5
2622
154
27,891
5.5 (4.6–6.5)
≥ 7.5 to < 10
Low
230
16
2427
6.8 (4.1–11.3)
≥ 7.5 to < 10
Moderate
1525
143
15,821
8.3 (6.9–9.9)
≥ 7.5 to < 10
High
186
22
1903
11.5 (7.4–17.8)
≥ 10 to <1 2.5
974
115
9990
10.9 (9.0–13.3)
Total
5537
450
58,032
7.8 (7.1–8.5)
Middle–aged cohort (40 to 64 years, n = 38,941)
≥ 5 to < 7.5
7097
442
75,354
5.7 (5.2–6.3)
≥ 7.5 to < 10
Low
497
30
5291
5.8 (4.0–8.4)
≥ 7.5 to < 10
Moderate
2856
272
29,813
8.5 (7.5–9.7)
≥ 7.5 to < 10
High
287
43
2914
14.2 (10.3–19.5)
≥ 10 to < 12.5
1327
153
13,658
11.2 (9.5–13.2)
Total
12,064
940
127,030
7.4 (6.9–7.9) Table 3 Risk of cardiovascular events by the deep-learning-based retinal CVD biomarker (Reti-CVD) in participants who had 7.5–10%
QRISK3 score Table 3 Risk of cardiovascular events by the deep-learning-based retinal CVD biomarker (Reti-CVD) in participants who had 7.5–10%
QRISK3 score Table 4 Prognostic performance with the addition of Reti-CVD to the QRISK3 in the UK biobank dataset
Data are C statistic (95% CI), unless stated otherwise. The Reti-CVD plus QRISK3 model is a logistic model fit on the UK Biobank. Discussion Cardiovascular disease (CVD) event rate according to Reti-CVD-three
groups in borderline-QRISK3 group who had QRISK3 score between 7.5 and 10% were presented in non-statin cohort (A), stage 1 hypertension
cohort (B), and middle-aged (40–64 years) cohorts (C). Considering that statin and antihypertensive pharmacotherapy initiation is recommended
at QRISK of ≥ 10%, Reti-CVD can be used as a risk enhancer in borderline-QRISK3 groups of 7.5–10% to reach consensus on statin initiation. In
addition, although most of risk assessment systems were derived from cohorts of primarily middle-aged people and typically well-functioning
individuals, Reti-CVD can still be a risk enhancer in in borderline-QRISK3 groups of 7.5–10% middle-aged people Fig. 2 Kaplan-Meier curves according to Reti-CVD in borderline-QRISK3 group. Cardiovascular disease (CVD) event rate according to Reti-CVD-three
groups in borderline-QRISK3 group who had QRISK3 score between 7.5 and 10% were presented in non-statin cohort (A), stage 1 hypertension
cohort (B), and middle-aged (40–64 years) cohorts (C). Considering that statin and antihypertensive pharmacotherapy initiation is recommended
at QRISK of ≥ 10%, Reti-CVD can be used as a risk enhancer in borderline-QRISK3 groups of 7.5–10% to reach consensus on statin initiation. In
addition, although most of risk assessment systems were derived from cohorts of primarily middle-aged people and typically well-functioning
individuals, Reti-CVD can still be a risk enhancer in in borderline-QRISK3 groups of 7.5–10% middle-aged people Page 7 of 10 Page 7 of 10 Tseng et al. BMC Medicine (2023) 21:28 Tseng et al. BMC Medicine Table 3 Risk of cardiovascular events by the deep-learning-based retinal CVD biomarker (Reti-CVD) in participants who had 7.5–10%
QRISK3 score
a Reti-CVD only applied on borderline-QRISK3 groups. Incidence per 1000 person-years at year 10. CI confidence interval. N number at risk. Discussion Like other areas of medicine, there is now a paradigm
shift in cardiovascular medicine, towards using digital
innovations especially those related to artificial intel-
ligence to enhance diagnosis and risk stratification [19]. Retinal imaging has been posited as a potential imaging
application for risk assessment in a wide variety of dis-
eases. Reti-CVD, as a further extension of RetiCAC,
when applied alongside QRISK3, provides a good reflec-
tion of CVD severity in the UK population. This under-
scores the importance of the retinal imaging-based
Reti-CVD tool for clinical CVD risk stratification. An additional finding that we want to highlight is the
fact that Reti-CVD identified high-risk individuals who
were otherwise missed by QRISK3. 8.6% of patients
identified as high-risk by our new stratification tool had
a QRISK3 score between 0 to 5 and 40.1% of them were
categorized in the QRISK3 ≥ 5 to < 10% group. This dem-
onstrates the potential of Reti-CVD as an adjunct tool to
identify future high-risk individuals who may be left out
if CVD risk stratification is dependent solely on QRISK3
scoring. Consequently, when we incorporate only two
basic demographic factors of age and gender to Reti-
CVD, this model was superior to QRISK3 model across
the range of risk thresholds. Moreover, in our explorative
analysis in eTable 3, we confirmed that upon conducting a
more detailed QRISK3 analysis in the borderline QRISK3
group where individuals were divided based on 0.5 inter-
vals between 7.5 and 10%, more than 1.6% of individuals
were classified in the Reti-CVD-high-risk groups in all
subgroups. This indicates that Reti-CVD can be used as
an independent risk enhancer in this range of QRISK3. i
Previous studies using retinal imaging as a tool for pre-
dicting CVD risk have focused largely on other risk fac-
tors such as age, hypertension, and dyslipidemia [20–22]. Other studies have investigated the use of retinal imaging
to determine the risk of specific CVD subtypes such as
coronary heart disease [23] and peripheral arterial dis-
ease [24]. Compared to these studies, our study focuses
on predicting future CVD events in general instead of
diagnosis of current subtypes of CVD. Studies that have
used DL-based retinal imaging to predict coronary artery
calcium (CAC) scores were either applied on small sam-
ple sizes and/or did not evaluate the ability of the deep
learning systems to predict future CVD events [25,
26]. Discussion NRI for Reti-CVD plus QRISK3 versus
QRISK3 models was provided
NRI net reclassification index, CI confidence interval, CVD cardiovascular disease, Reti-CVD deep-learning-based retinal CVD biomarker
Non-statin cohort (n = 42,473)
Stage 1 hypertension
cohort (n = 11,966)
Middle-aged Cohort
(n = 38,941)
Models
C statistic (95% CI)
p value
C statistic (95% CI)
p value
C statistic (95% CI)
p value
Reti-CVD
0.624 (0.615–0.633)
NA
0.591 (0.575–0.608)
NA
0.611 (0.600 -0.622 )
NA
Reti-CVD plus age, gender
0.699 (0.690–0.709)
NA
0.663 (0.644–0.681)
NA
0.681 (0.669–0.692)
NA
QRISK3
0.682 (0.672–0.692)
NA
0.639 (0.620–0.658)
NA
0.650 (0.638–0.662)
NA
Reti-CVD plus QRISK3
0.696 (0.686–0.706)
NA
0.652 (0.633–0.671)
NA
0.674 (0.661–0.686)
NA
Δ Reti-CVD plus QRISK3
versus QRISK3
0.014 (0.010–0.017)
< 0.001
0.013 (0.007–0.019)
< 0.001
0.023 (0.018–0.029)
< 0.001
NRI
Continuous NRI (95% CI)
0.133 (0.088–0.173)
< 0.001
0.094 (0.008–0.174)
0.033
0.248 (0.190–0.301)
< 0.001 Table 4 Prognostic performance with the addition of Reti-CVD to the QRISK3 in the UK biobank dataset identified individuals who had a 10-year CVD risk
greater than 10%, with a CVD incidence of 13.1 per 1000
person-years. Moreover, we quantified the cumulative
CVD events and prognostic value for risk stratification
when adding Reti-CVD alongside QRISK3 in the bor-
derline-QRISK3 group (10-year CVD risk of 7.5–10%). Due to the new cut-off threshold of 10% for statin and antihypertensive treatment initiation, we focused on
individuals who had a QRISK3 score between 7.5 and
10% and showed that Reti-CVD could further stratify
the CVD risk into low, moderate, and high-risk groups. Specifically, in the borderline-risk group, individuals who
were stratified as high-risk using Reti-CVD had a 10-year
CVD risk greater than 10% and was comparable to the Tseng et al. BMC Medicine (2023) 21:28 Page 8 of 10 CVD incidence of QRISK3 (10 to 12.5%) group, indicat-
ing that Reti-CVD could be used as a risk enhancer. CVD incidence of QRISK3 (10 to 12.5%) group, indicat-
ing that Reti-CVD could be used as a risk enhancer. monitoring patients’ blood pressure and cholesterol lev-
els to better predict CVD risk. If identified as high-risk by
Reti-CVD, patients can be encouraged to undergo a NHS
health check-up for further evaluation. This could boost
the compliance rate for NHS health screening, which has
been estimated to be less than 50% [29]. This low-cost
opportunistic CVD risk screening based on routine eye
checks may help to include ‘hard to reach’ populations in
CVD risk assessment. Discussion In particular, a study by Chang et al. evaluated the
value-added ability of its DL-based system in predicting
CVD mortality from retinal imaging when added to the
Framingham risk scoring (FRS) system [27]. However,
Hippisley-Cox et al has suggested that the FRS algorithm
has ethnicity bias and may be less suited to the UK cohort
compared to QRISK algorithms [3]. Therefore, our novel
focus in applying Reti-CVD to QRISK3 reflects both the
UK Biobank demographic population and NICE guide-
lines, as QRISK3 was recommended by NICE. By comparing Reti-CVD to QRISK3, this study dem-
onstrates the potential value and scope of Reti-CVD to
improve discernment accuracy in the UK population
since QRISK3 is part of the up-to-date guidelines that
reflects the demographic profile of the UK Biobank. Study limitations include a possibility of systematic
errors in the form of misclassification bias, since hospi-
talization and mortality data provided by the NHS reg-
isters were used for CVD definition. However, a study by
Kivimaki et al. has analyzed the validity of and reported
good agreement between CVD event predictions and the
use of UK National Health Electronic Records, therefore
minimizing the likelihood of CVD event misdiagnoses
[30]. We also excluded poor quality images during the
development and validation of Reti-CVD. The quality
of retinal photographs available in clinical settings may
vary, and therefore this may pose challenges for real-
world implementation. y
In the general population, we have shown that Reti-
CVD can identify individuals with a CVD risk greater
than 10% in our high-risk Reti-CVD group. This finding
is useful for two reasons because a CVD risk of 10% is the
latest threshold for statin and anti-hypertensive medica-
tion initiation. First, at the primary care level, Reti-CVD
provides GPs with a simple and effective way of provid-
ing an initial assessment on asymptomatic patients. Ophthalmologists, optometrists, and opticians can also
use existing retinal photographs for opportunistic eye
screening. Second, Reti-CVD can aid the CVD preven-
tion program that NHS England has developed. The NHS
CVD primary prevention focusses on three main com-
ponents: atrial fibrillation, blood pressure, and choles-
terol [28]. Given that our study included individuals who
are not on statin or anti-hypertensive medication in the
general population, physicians can use Reti-CVD while Funding
Thi
j g
This project is supported by the Agency for Science, Technology and Research
(A*STAR) under its RIE2020 Health and Biomedical Sciences (HBMS) Industry
Alignment Fund Pre-Positioning (IAF-PP) Grant No. H20c6a0031. Any opinions,
findings, and conclusions or recommendations expressed in this material are
those of the author(s) and do not reflect the views of the A*STAR. 4. Cardiovascular disease: risk assessment and reduction, including lipid
modification [https://www.nice.org.uk/guidance/cg181/resources]. 2014 5. Jones NR, McCormack T, Constanti M, McManus RJ. Diagnosis and man‑
agement of hypertension in adults: NICE guideline update 2019. Br J Gen
Pract. 2020;70(691):90–1. 6. Pate A, Emsley R, van Staa T. Impact of lowering the risk threshold for sta‑
tin treatment on statin prescribing: a descriptive study in English primary
care. Br J Gen Pract. 2020;70(700):e765–e71. Conclusions In conclusion, a DL and retinal photograph-derived new
CVD biomarker, Reti-CVD, could effectively stratify
CVD risk in the general population. We also confirmed
that Reti-CVD has the potential to identify individuals Tseng et al. BMC Medicine (2023) 21:28 Tseng et al. BMC Medicine (2023) 21:28 Page 9 of 10 with ≥ 10% 10-year CVD risk who are likely to ben-
efit from preventative CVD interventions. In addition,
for people with a QRISK3 score between 7.5 and 10%,
Reti-CVD could be used as a risk stratification enhancer
for identifying high risk patients for aggressive CVD
prevention. Declarations with ≥ 10% 10-year CVD risk who are likely to ben-
efit from preventative CVD interventions. In addition,
for people with a QRISK3 score between 7.5 and 10%,
Reti-CVD could be used as a risk stratification enhancer
for identifying high risk patients for aggressive CVD
prevention. Acknowledgements
Not applicable. 1. Deshmukh M, Liu YC, Rim TH, Venkatraman A, Davidson M, Yu M, et al. Automatic segmentation of corneal deposits from corneal stromal dys‑
trophy images via deep learning. Comput Biol Med. 2021;137:104675. Competing interests T.H.R. was a former scientific adviser and owns stock of Mediwhale Inc. H.K. and G.L. are employees of Mediwhale Inc. T.H.R. and G.L. hold the follow‑
ing patents that might be affected by this study: 10–2018–0166720(KR),
10–2018–0166721(KR), 10–2018–0166722(KR), 62/694,901(US), 62/715,729(US),
and 62/776,345 (US). All other authors declare no competing interests. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s12916-022-02684-8. org/10.1186/s12916 022 02684 8. Additional file 1: eFigure 1. Study flow chart. Additional file 2: eFigure 2. Distribution of the QRISK3 score in the UK
Biobank. Additional file 3: eDocument 1. RetiCAC model updates. Additional file 4: eFigure 3. Kaplan-Meier curves according to Reti-CVD
and QRISK3. Additional file 5: eTable 1. Risk of cardiovascular events by the deep-
learning-based retinal CVD biomarker (Reti-CVD). Additional file 6: eTable 2. Subgroup analysis in the UK Biobank. Additional file 7: eFigure 4. Decision curve analysis. Additional file 8: eTable 3. Detailed distribution of Reti-CVD risk groups
among those between 7.5% and 10% QRISK3 score. Additional file 1: eFigure 1. Study flow chart. Additional file 2: eFigure 2. Distribution of the QRISK3 score in the UK
Biobank. Additional file 3: eDocument 1. RetiCAC model updates. Additional file 4: eFigure 3. Kaplan-Meier curves according to Reti-CVD
and QRISK3. Additional file 5: eTable 1. Risk of cardiovascular events by the deep-
learning-based retinal CVD biomarker (Reti-CVD). Additional file 6: eTable 2. Subgroup analysis in the UK Biobank. Additional file 7: eFigure 4. Decision curve analysis. Additional file 8: eTable 3. Detailed distribution of Reti-CVD risk groups
among those between 7.5% and 10% QRISK3 score. Received: 12 July 2022 Accepted: 28 November 2022 Consent for publication
Not applicable. Abbreviations
CVD
Cardiovascular disease
UK
United Kingdom
NICE
National Institute for Health and Care Excellence
DL
Deep-learning
CAC
Coronary artery calcium
IRB
Institutional Review Board
CIRB
SingHealth Centralized Institutional Review Board
NHS
National Health Service
NRI
Net Reclassification Index
BMI
Body-mass index
HR
Hazard ratio
FRS
Framingham risk score
GP
General practitioner Authors’ contributions 2. Ng WY, Zhang S, Wang Z, Ong CJT, Gunasekeran DV, Lim GYS, et al. Updates in deep learning research in ophthalmology. Clin Sci (Lond). 2021;135(20):2357–76. T.H.R. conceptualized the study. G.L. developed the algorithm. R.M.W.W.T. and T.H.R. wrote the manuscript and researched its contents and edited all
versions. All authors substantially revised the manuscript, read, and approved
the final manuscript. 3. Hippisley-Cox J, Coupland C, Vinogradova Y, Robson J, May M, Brindle
P. Derivation and validation of QRISK, a new cardiovascular disease risk
score for the United Kingdom: prospective open cohort study. BMJ. 2007;335(7611):136. Ethics approval and consent to participate The UK Biobank dataset was obtained ethically from UK Biobank (applica‑
tion number 68428). The UK Biobank has also received Research Tissue Bank
approval from North West Multi-centre Research Ethics Committee and the
Human Tissue Authority license [31]. 2.
Ng WY, Zhang S, Wang Z, Ong CJT, Gunasekeran DV, Lim GYS, et al.
Updates in deep learning research in ophthalmology. Clin Sci (Lond).
2021;135(20):2357–76. Author details
1 1 Singapore Eye Research Institute, Singapore National Eye Centre, Singapore,
Singapore. 2 Duke-NUS Medical School, Singapore, Singapore. 3 Ophthalmol‑
ogy and Visual Sciences Academic Clinical Program (Eye ACP), Duke-NUS
Medical School, Singapore, Singapore. 4 Mediwhale Inc., Seoul, South Korea. 5 Department of Primary Care and Mental Health, University of Liverpool,
Liverpool, UK. 6 Albert Einstein College of Medicine, New York, NY, USA. 7 Division of Cardiology, Severance Cardiovascular Hospital, Yonsei University
College of Medicine, Seoul, South Korea. 8 Division of Retina, Severance Eye
Hospital, Yonsei University College of Medicine, Seoul, South Korea. 9 Clinical
and Translational Sciences Program, Duke-NUS Medical School, Singapore,
Singapore. 10 Center for Innovation and Precision Eye Health, Yong Loo Lin
School of Medicine, National University of Singapore, Singapore, Singapore. 11 Department of Ophthalmology, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore. 12 Royal Free Hospital London
NHS Foundation Trust, London, UK. 13 Cardiology Department, Barnet General
Hospital, Thames House, Enfield, UK. 14 Cardiovascular Clinical Research Facil‑
ity, RDM Division of Cardiovascular Medicine, University of Oxford, Oxford,
UK. 15 Liverpool Centre for Cardiovascular Science, University of Liverpool
and Liverpool John Moores University and Liverpool Heart & Chest Hospital,
Liverpool, United Kingdom; and Department of Clinical Medicine, Aalborg
University, Aalborg, Denmark. 16 Tsinghua Medicine, Tsinghua University,
Beijing, China. Publisher’s Note
S
N Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. 11. Sudlow C, Gallacher J, Allen N, Beral V, Burton P, Danesh J, et al. UK
biobank: an open access resource for identifying the causes of a
wide range of complex diseases of middle and old age. PLoS Med. 2015;12(3):e1001779. 12. Palmer LJ. UK Biobank: bank on it. Lancet. 2007;369(9578):1980–2. 13. UK Biobank: Protocol for a large-scale prospective epidemiological
resource. 2007. 14. Man REK, Gan AHW, Fenwick EK, Gan ATL, Gupta P, Sabanayagam C, et al. Prevalence, determinants and association of unawareness of diabetes,
hypertension and hypercholesterolemia with poor disease control in
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purposed implementation of ClinRisk’s QRISK3 algorithm into R. F1000Re‑
search. 2020;8:2139. 18. Pencina MJ, D’Agostino RB Sr, D’Agostino RB Jr, Vasan RS. Evaluating the
added predictive ability of a new marker: from area under the ROC curve
to reclassification and beyond. Stat Med. 2008;27(2):157–72 discussion
207-12. 19. Vardas PE, Asselbergs FW, van Smeden M, Friedman P. The year in
cardiovascular medicine 2021: digital health and innovation. Eur Heart J. 2022;43(4):271–9. 20. Zhang L, Yuan M, An Z, Zhao X, Wu H, Li H, et al. Prediction of hyperten‑
sion, hyperglycemia and dyslipidemia from retinal fundus photographs
via deep learning: A cross-sectional study of chronic diseases in central
China. PLoS One. 2020;15(5):e0233166. 21. Poplin R, Varadarajan AV, Blumer K, Liu Y, McConnell MV, Corrado GS, et al. Prediction of cardiovascular risk factors from retinal fundus photographs
via deep learning. Nat Biomed Eng. 2018;2(3):158–64. 22. Gerrits N, Elen B, Craenendonck TV, Triantafyllidou D, Petropoulos IN,
Malik RA, et al. Age and sex affect deep learning prediction of cardio‑
metabolic risk factors from retinal images. Sci Rep. 2020;10(1):9432. 23. Wang JJ, Liew G, Wong TY, Smith W, Klein R, Leeder SR, et al. Retinal vas‑
cular calibre and the risk of coronary heart disease-related death. Heart. 2006;92(11):1583–7. 24. Mueller S, Wintergerst MWM, Falahat P, Holz FG, Schaefer C, Schahab N,
et al. Availability of data and materials The UK Biobank test dataset was obtained from UK Biobank (application
number 68428). Data cannot be shared publicly due to the violation of patient
privacy and the absence of informed consent for data sharing. The UK Biobank test dataset was obtained from UK Biobank (application
number 68428). Data cannot be shared publicly due to the violation of patient
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prediction for a multiethnic population via deep learning across different
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Med Inform. 2021;9(8):e25165. Tseng et al. BMC Medicine (2023) 21:28 Tseng et al. BMC Medicine (2023) 21:28 Page 10 of 10 Page 10 of 10 30. Kivimäki M, Batty GD, Singh-Manoux A, Britton A, Brunner EJ, Shipley MJ. Validity of cardiovascular disease event ascertainment using linkage to
UK hospital records. Epidemiology. 2017;28(5):735–9. 30. Kivimäki M, Batty GD, Singh-Manoux A, Britton A, Brunner EJ, Shipley MJ. Validity of cardiovascular disease event ascertainment using linkage to
UK hospital records. Epidemiology. 2017;28(5):735–9. 8. Tseng RMWW, Rim TH, Cheung CY, Wong TY. Artificial intelligence using
the eye as a biomarker of systemic risk. Artificial Intelligence in Ophthal‑
mology: Springer; 2021. p. 243–55. p
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31. UK Biobank research ethics approval. 2021. [https://www.ukbiobank.ac.
uk/learn-more-about-ukbiobank/about-us/ethics]. 31. UK Biobank research ethics approval. 2021. [https://www.ukbiobank.ac.
uk/learn-more-about-ukbiobank/about-us/ethics]. 9. Rim TH, Lee G, Kim Y, Tham YC, Lee CJ, Baik SJ, et al. Prediction of systemic
biomarkers from retinal photographs: development and validation of
deep-learning algorithms. Lancet Digit Health. 2020;2(10):e526–e36. 10. Rim TH, Lee CJ, Tham Y-C, Cheung N, Yu M, Lee G, et al. Deep-learning-
based cardiovascular risk stratification using coronary artery calcium
scores predicted from retinal photographs. Lancet Digital Health. 2021;3(5):e306–e16. p
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31. UK Biobank research ethics approval. 2021. [https://www.ukbiobank.ac.
uk/learn-more-about-ukbiobank/about-us/ethics]. •
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Validity of cardiovascular disease event ascertainment using linkage to
UK hospital records. Epidemiology. 2017;28(5):735–9. Publisher’s Note
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N Multiple instance learning detects peripheral arterial disease from
high-resolution color fundus photography. Sci Rep. 2022;12(1):1389. 25. Barriada RG, Simó-Servat O, Planas A, Hernández C, Simó R, Masip D. Deep Learning of Retinal Imaging: A Useful Tool for Coronary Artery Cal‑
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? Choose BMC and benefit from: 26. Son J, Shin JY, Chun EJ, Jung K-H, Park KH, Park SJ. Predicting high coro‑
nary artery calcium score from retinal fundus images with deep learning
algorithms. Transl Vis Sci Technol. 2020;9(2):28. 27. Chang J, Ko A, Park SM, Choi S, Kim K, Kim SM, et al. Association of cardio‑
vascular mortality and deep learning-funduscopic atherosclerosis score
derived from retinal fundus images. Am J Ophthalmol. 2020;217:121–30. 28. Arnould L, Guenancia C, Bourredjem A, Binquet C, Gabrielle PH, Eid P,
et al. Prediction of cardiovascular parameters with supervised machine
learning from singapore “I” vessel assessment and OCT-angiography: a
pilot study. Transl Vision Sci Technol. 2021;10(13):20. 29. Martin A, Saunders CL, Harte E, Griffin SJ, MacLure C, Mant J, et al. Delivery
and impact of the NHS health check in the first 8 years: a systematic
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Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores
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www.zora.uzh.ch Year: 2021 mmon variants in Alzheimer’s disease and risk stratification by polygenic risk
scores Rojas, Itziar ; Moreno-Grau, Sonia ; Tesi, Niccolo ; Grenier-Boley, Benjamin ; et al ; Clark, Christopher ;
Grünblatt, Edna ; Popp, Julius DOI: https://doi.org/10.1038/s41467-021-22491-8 Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-204834
Journal Article
Published Version
The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at:
de Rojas, Itziar; Moreno-Grau, Sonia; Tesi, Niccolo; Grenier-Boley, Benjamin; et al; Clark, Christopher; Grünblatt,
Edna; Popp, Julius (2021). Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores. Nature Communications, 12:3417. DOI: https://doi.org/10.1038/s41467-021-22491-8 Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-204834
Journal Article
Published Version
The following work is licensed under a Creative Commons: Attribution 4.0 Interna ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at:
de Rojas, Itziar; Moreno-Grau, Sonia; Tesi, Niccolo; Grenier-Boley, Benjamin; et al; Clark, Christopher; Grünblatt,
Edna; Popp, Julius (2021). Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores. Nature Communications, 12:3417. DOI: https://doi.org/10.1038/s41467-021-22491-8 ARTICLE Common variants in Alzheimer’s disease and risk
stratification by polygenic risk scores
https://doi.org/10.1038/s41467-021-22491-8
OPEN Genetic discoveries of Alzheimer’s disease are the drivers of our understanding, and together
with polygenetic risk stratification can contribute towards planning of feasible and efficient
preventive and curative clinical trials. We first perform a large genetic association study by
merging
all
available
case-control
datasets
and
by-proxy
study
results
(discovery
n = 409,435 and validation size n = 58,190). Here, we add six variants associated with
Alzheimer’s disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants
in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal
a 4 to 5.5 years difference in median age at onset of Alzheimer’s disease patients in APOE ɛ4
carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the
amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk
of Alzheimer’s disease. A full list of authors and their affiliations appears at the end of the paper. A full list of authors and their affiliations appears at the end of the paper. A full list of authors and their affiliations appears at the end of the paper. NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 T
hus far, multiple loci associated with Alzheimer’s disease
(AD) have been described next to causal mutations in two
subunits of γ-secretases, membrane-embedded aspartyl
complexes (PSEN1, PSEN2 genes), and the gene encoding one
target protein of these proteases, the amyloid precursor protein
gene (APP). The most prominent locus, APOE, was detected
almost 30 years ago using linkage techniques1. In addition,
genome-wide association studies (GWAS) of AD case-control
datasets and by-proxy AD case-control studies have identified 30
genomic loci that modify the risk of AD2–7. These signals account
for ~31% of the genetic variance of AD, leaving most of the
genetic risk as yet uncharacterized8. Further disentangling the
genetic constellation of common genetic variations underlying
AD can drive our biological insights of AD and can point toward
novel drug targets. T data, by-proxy case-control data, and the data from the Genome
Research at Fundació ACE (GR@ACE) study19. We confirm the
observed associations in a large independent replication study. Then, we construct an update of the PRS and test whether the
effects of the PRS are influenced by diagnostic certainty, sex and
AAO groups. Results Meta-GWAS of AD. We combined data from three AD GWASs:
the summary statistics calculated from the GR@ACE19 case-
control study (6331 AD cases and 6055 controls), the IGAP20
case-control study (up to 30,344 AD cases and 52,427 controls)
and the UKB AD-by-proxy case-control study21 (27,696 cases of
maternal AD with 260,980 controls, and 14,338 cases of paternal
AD with 245,941 controls, Fig. 1, Supplementary Data 1). Although we observed inflation in the resulting summary statis-
tics (λ median = 1.08; see Supplementary Fig. 1d), it was not
driven by an un-modeled population structure (LD score
regression intercept = 1.036). The full details of the studies are
described in methods. After study-specific variant filtering and
quality-control procedures, we performed a fixed effects inverse-
variance-weighted meta-analysis22 on the summary statistics of
the three studies. Using this strategy, we identified a genome-wide
significant (GWS) association (p < 5 × 10−8) for 36 independent
genetic variants in 35 genomic regions (the APOE region contains
signals for ɛ4 and ɛ2). As a sensitivity analysis, we removed the
AD-by-proxy study and compared the resulted effect estimates
with and without this dataset. We found a high correlation
between the effect estimates from the case-control and by-proxy There are over 50 million people living with dementia and the
global cost of dementia is well above 1 trillion US$9. This means
there is a medical and economical urgency to efficiently test
interventions that are under development. Therefore, to increase
power and reduce duration of trials, pre-symptomatic patients
that are at high genetic risk of disease are increasingly
developed10. However, only carriers of causal mutations (APP,
PSEN1, and PSEN2) and the APOE ɛ4 allele are considered high
risk, while other common and rare genetic variants are ignored11. Despite that, the combined effects of all currently known variants
in a polygenic risk score (PRS) is associated with the conversion
of mild cognitive impairment to AD12,13, the neuropathological
hallmarks of AD, age at onset (AAO) of disease14–17 and lifetime
risk of AD18. In this work we aim to comprehend and expand the knowledge
of the genetic landscape underlying AD and provide additional
evidence that a PRS of variants can be a robust tool to select high
risk individuals with an earlier AAO. We first performed a meta-
GWAS integrating all currently published GWAS case-control Discovery. Common variants in Alzheimer’s disease and risk
stratification by polygenic risk scores
https://doi.org/10.1038/s41467-021-22491-8
OPEN Lastly, we test whether the PRS could be used to
identify individuals at the highest odds of having AD and we
compared AAO of the AD cases. This study describes the iden-
tification of six variants associated with AD risk and provides an
extended PRS tool to select individuals at high risk of AD. Results Meta-analysis Case-control + AD-by-proxy
Sensitivity analysis Case-control AD status
GR@ACEa
N=12,386
GR@ACEa
N= 12,386
IGAPb
N=82,771
IGAPb
N=82,771
UKBc
N=314,278
Global N= 409,435 participants
Global N= 58,190 (19,089 Cases and 39,101 Controls)
ADDN
N=637
2 cohorts
PGC-ALZ
N=17,537
3 cohorts
EADB
N=33,753
8 cohorts
AD and
GBCS
N=3,981
GR@ACEd
Replication
N=1,202
Follow-up. Analysis of genome-wide significant loci
Meta-GWAS analysis
Polygenic Risk Score
Pathological
dataset
Ncases=332e + 676f
Ncontrols=1,386
GR@ACEa
Ncases=6,331
Ncontrols=3,546g
Age at Onset
APOE stratification
Risk stratification
Effects of sub-threshold signal
Diagnostic
certainty
Concomitant
brain pathologies
Effect of sex
Age at Onset
NxC
N=1,078
Fig. 1 Flow chart of analysis steps. Discovery meta-analysis in GR@ACE, IGAP stage 1 + 2 and UKBiobank followed by a replication in 16 independen
cohorts. The genome-wide significant signals found in meta-GWAS were used to perform a Polygenic Risk Score in a clinical and pathological AD dataset
See Supplementary Methods to more information about the cohorts included and methods to the PRS generation. aExtended dataset (Moreno-Grau
et al.19), bStageI + StageII (Kunkle et al.20), cBy proxy AD: Meta-analysis of maternal and paternal history of dementia (Marioni et al.21), dExtra and
independent GR@ACE dataset incorporated only for replication purposes, ePathologically confirmed AD cases, fAD cases diagnosed based on clinical
criteria, gControls participants aged 55 years and younger. N = Total of individuals within specified data. 2
NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications Fig. 1 Flow chart of analysis steps. Discovery meta-analysis in GR@ACE, IGAP stage 1 + 2 and UKBiobank followed by a replication in 16 independent
cohorts. The genome-wide significant signals found in meta-GWAS were used to perform a Polygenic Risk Score in a clinical and pathological AD dataset. See Supplementary Methods to more information about the cohorts included and methods to the PRS generation. aExtended dataset (Moreno-Grau
et al.19), bStageI + StageII (Kunkle et al.20), cBy proxy AD: Meta-analysis of maternal and paternal history of dementia (Marioni et al.21), dExtra and
independent GR@ACE dataset incorporated only for replication purposes, ePathologically confirmed AD cases, fAD cases diagnosed based on clinical
criteria, gControls participants aged 55 years and younger. N = Total of individuals within specified data. Fig. 1 Flow chart of analysis steps. Discovery meta-analysis in GR@ACE, IGAP stage 1 + 2 and UKBiobank followed by a replication in 16 independent
cohorts. Results In APP, we identified a common (MAF = 0.46) intronic
variant associated with a reduced risk of AD (rs2154481, OR = 0.95
[0.94–0.96], p = 1.39 × 10−11, Fig. 2f). In SHARPIN (SHANK
Associated RH Domain Interactor) gene, we found two missense
mutations (rs34173062/p.Ser17Phe and rs34674752/p.Pro294Ser)
that are in linkage equilibrium (R2 = 1.3 × 10−6, D′ = 0.014,
p = 0.96). Both missense variants increased AD risk (p.Ser17Phe,
MAF = 0.085, OR = 1.14 [1.10–1.18], p = 9.6 × 10−13 and p. Pro294Ser, MAF = 0.052, OR = 1.13 [1.09–1.18], p = 1.0 × 10−9,
Fig. 2b). A variant close to the genes PRKD3 and NDUFAF7
(rs876461, MAF = 0.143) emerged as the most significant variant in
the region after the combined analysis (OR = 1.07 [1.05–1.09],
p = 1.3 × 10−9, Fig. 2c). In the 3’-UTR region of CHRNE
(Cholinergic Receptor Nicotinic Epsilon Subunit), rs72835061
(MAF = 0.085) was associated with a 1.09-fold increased risk of
AD (95% CI [1.06–1.11], p = 1.5 × 10−10, Fig. 2e). Our analysis also
strengthened the evidence of association with AD for three
additional genomic loci including an association with a variant in
PLCG2 (rs3935877, MAF = 0.13, OR = 0.92 [0.90–0.95], p = 6.9 ×
10−9, Fig. 2d), and confirmed another common variant in PLCG2, a
stop gain mutation in IL-34 and a variant near HS3ST1 (Table 1,
Supplementary Fig. 3 and Supplementary Data 2, 3). We were not Polygenic risk scores. In order to assess the robustness and
combined effect of the genetic landscape of AD (Fig. 3, Sup-
plementary Data 4), we constructed a weighted PRS based on
the 39 genetic variants (excluding APOE genotypes) that
showed GWS evidence of association with AD (see Methods,
Fig. 4 and Supplementary Data 5). We tested if the association
of the PRS with AD is independent of clinically important
factors that are considered in the selection of individuals for
clinical trials. First, we showed that the association of the PRS
with clinically diagnosed AD cases is similar to the association
with pathologically confirmed AD (OR = 1.30 vs. 1.38, per 1-SD
increase in the PRS). In this setting, adding variants below the
GWS threshold did not lead to a more significant association of
the PRS with AD (Fig. 4a). Next, we tested whether the PRS was
associated with AD in the presence of concomitant brain
pathologies (besides AD). Results The genome-wide significant signals found in meta-GWAS were used to perform a Polygenic Risk Score in a clinical and pathological AD dataset. See Supplementary Methods to more information about the cohorts included and methods to the PRS generation. aExtended dataset (Moreno-Grau
et al.19), bStageI + StageII (Kunkle et al.20), cBy proxy AD: Meta-analysis of maternal and paternal history of dementia (Marioni et al.21), dExtra and
independent GR@ACE dataset incorporated only for replication purposes, ePathologically confirmed AD cases, fAD cases diagnosed based on clinical
criteria, gControls participants aged 55 years and younger. N = Total of individuals within specified data. 2
NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 a a
c
b
d
e
f
Fig. 2 GWAS meta-analysis for AD risk (N = 467,623). a Manhattan plot of overall meta-analysis for genome-wide association in Alzheimer’s disease
highlighting in pink the loci associated with AD in this study (PRKD3/NDUFAF7, SHARPIN, CHRNE, PLCG2, and APP). b–f Locus plots for the signals
associated with AD in overall meta-analysis results. a
b
f a a b f f d c e c d e Fig. 2 GWAS meta-analysis for AD risk (N = 467,623). a Manhattan plot of overall meta-analysis for genome-wide association in Alzheimer’s disease
highlighting in pink the loci associated with AD in this study (PRKD3/NDUFAF7, SHARPIN, CHRNE, PLCG2, and APP). b–f Locus plots for the signals
associated with AD in overall meta-analysis results. able to replicate two loci (ELK2AP and SPPL2A regions) that
showed suggestive association with AD (p < 1 × 10−7 in discovery). approaches for the significant loci (R2 = 0.994, p = 8.1 × 10−37;
Supplementary Fig. 1e). Four genomic regions were not pre-
viously associated with AD (see Manhattan Plot, Fig. 2a). y
g
Next, we aimed at replicating the associated loci in 16 cohorts
(19,087 AD cases and 39,101 controls in total), many of them
collected and analyzed by the European Alzheimer’s Disease
Biobank (JPND-EADB) project. We tested all variants with
suggestive association (p < 10−5) located within a 200 kb region
from the sentinel SNP. Overall, 384 variants were tested in the
replication datasets (Supplementary Data 2). Discovery and
replication were combined, and we identified associations in six
variants comprising five genomic loci annotated using FUMA23
(Table 1, Fig. 2b–f, Supplementary Fig. 2 and Supplementary
Results). Results To identify people at the highest genetic
risk of AD based on the PRS, we used the validated 39-variants 3 NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Discovery meta-analysis
Follow-up datasets
Overall
Chr
Closest gene
SNP
BP
A1
A2
Freq A1
OR [CI 95%]
P
OR [CI 95%]
P
OR [CI 95%]
P
Variants showing novel genome-wide significant association with AD
2
PRKD3/NDUFAF7
rs876461
37515958
A
G
0.143
1.07 [1.04–1.09]
9.14 × 10−7
1.08 [1.04–1.13]
3.07 × 10−4
1.07 [1.05–1.09]
1.34 × 10−9
8
SHARPIN
rs34674752
145154222
A
G
0.052
1.11 [1.06–1.16]
4.02 × 10−6
1.20 [1.10–1.31]
1.65 × 10−5
1.13 [1.09–1.18]
1.00 × 10−9
8
SHARPIN
rs34173062
145158607
A
G
0.085
1.16 [1.11–1.21]
1.33 × 10−11
1.09 [1.02–1.17]
7.35 × 10−3
1.14 [1.10–1.18]
9.62 × 10−13
16
PLCG2
rs3935877
81900853
C
T
0.868
0.92 [0.90–0.95]
1.12 × 10−7
0.92 [0.85–0.99]
1.96 × 10−2
0.92 [0.90–0.95]
6.85 × 10−9
17
CHRNE
rs72835061
4805437
A
C
0.085
1.09 [1.06–1.12]
3.92 × 10−9
1.07 [1.02–1.12]
7.83 × 10−3
1.09 [1.06–1.11]
1.51 × 10−10
21
APP
rs2154481
27473875
C
T
0.483
0.95 [0.93–0.96]
9.26 × 10−10
0.96 [0.93–0.99]
3.31 × 10−3
0.95 [0.94–0.96]
1.39 × 10−11
Previously reported genome-wide significant hits replicating in the follow-up
4
HS3ST1
rs4351014
11027619
C
T
0.684
0.94 [0.92–0.96]
5.37 × 10−10
0.93 [0.88–0.98]
4.54 × 10−3
0.94 [0.92–0.95]
9.16 × 10−12
16
IL34
rs4985556
70694000
A
C
0.111
1.08 [1.05–1.11]
2.28 × 10−8
1.09 [1.03–1.16]
4.59 × 10−3
1.08 [1.06–1.11]
3.91 × 10−10
16
PLCG2
rs12444183
81773209
A
G
0.407
0.95 [0.93–0.97]
1.48 × 10−8
0.92 [0.88–0.96]
3.23 × 10−5
0.95 [0.93–0.96]
6.81 × 10−12
Suggestive signals (not replicating)
14
ELK2AP
rs7153315
106195719
C
G
0.750
0.94 [0.92–0.96]
9.80 × 10−8
1.16 [1.01–1.33]
0.0412
0.94 [0.92–0.97]
9.04 × 10−7
15
SPPL2A
rs76523702
51002342
C
T
0.802
1.06 [1.04–1.08]
6.86 × 10−8
1.02 [0.97–1.07]
0.3501
1.05 [1.03–1.08]
1.08 × 10−7
8
NATURE COMMUNICATIONS | (2021) 12:3417 | h PRS in the large GR@ACE dataset. The PRS was associated with a
1.27-fold (95% CI [1.23–1.32]) increased risk for every standard
deviation increase in the PRS (p = 7.3 × 10−39) and with a
gradual risk increase when we stratified the dataset into 2%
percentiles of the PRS (Fig. 5a, Supplementary Data 6). Next, we
stratified the dataset in APOE genotype risk groups. Results Among our autopsy-confirmed AD
patients (n = 332), 84% had at least one concomitant pathology,
and the PRS was associated with AD in the presence of all tested
concomitant pathologies (Fig. 4b). Moreover, the patients often
had more than one concomitant pathology (48.8%), but no
difference was observed in the effect estimate of the PRS when
more than one pathology was present (Fig. 4b). Last, we
investigated the effect of sex and AAO (Fig. 4c). Our analysis
revealed that the effect of the PRS was the same in both sexes
(Fig. 4c) and was consistent with both early-onset (onset before
65 years; OR = 1.58, 95% CI [1.22–2.05], p = 5.8 × 10−4) as well
as with late-onset AD (onset later than 85 years; OR = 1.29, 95%
CI [1.10–1.51], p = 1.5 × 10−3). PRSs has the potential to early identify subjects at risk of
complex diseases24. Results The PRS
percentiles were associated with AD within the APOE genotype
groups (Fig. 5b, Supplementary Data 7). Finally, we compared the
risk extremes and found a 16.2-fold (95% CI [8.84–29.5],
p = 1.5 × 10−19) increased risk for the highest-PRS group (APOE
ɛ4ɛ4) compared with the lowest-PRS group (APOE ɛ2ɛ2/ɛ2ɛ3;
Supplementary Data 8). When we compared the median AAO in
AD patients in these extreme risk groups we found a 9-year
difference in the median age (pWilcoxon = 1.7 × 10−6) (Fig. 5c). Lastly, we studied the effects on AAO of the PRS in the APOE
genotype groups. The PRS differentiated AAO only within APOE
ɛ4 carriers. In APOE ɛ4 heterozygotes the PRS determined a 4-
year difference in median AAO and in APOE ɛ4 homozygotes
(pWilcoxon = 6.9 × 10−5), where the PRS determined a median
AAO difference of 5.5 years (pWilcoxon = 4.6 × 10−5). For the
selection of high-risk individuals, it is important to note that we
found no difference in the odds and AAO for AD for APOE ɛ4
heterozygotes with the highest PRS compared to APOE ɛ4
homozygotes with the lowest PRS. The Cox regression also
showed an impact of APOE on AAO, mainly on APOE ε4ε4
(significant APOE-PRS interaction (p = 0.021), Fig. 5d, Supple-
mentary Data 9). NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 This figure
was constructed to our best knowledge of literature, but is not a systematic review of literature. For common variants, we selected only signals firmly
replicated in large meta-GWAS (Lambert et al.3, Kunkle et al.20, Jun et al.43, Sims et al.7, Jansen et al.38 and present study). For rare variants, we only
selected those variants widely replicated excluding those loci presenting conflicting results. Abbreviations and more information about the genes can be
found in Supplementary Data 4. The risk alleles associated with AD were represented in orange and the protective alleles in blue. GWAS Genome-Wide
Association Study, OR odds ratio. Variant frequency (%) Timeline Fig. 3 Genetic landscape for Alzheimer’s disease. This figure shows the history of genetic discoveries in AD research over the past 30 years. This figure
was constructed to our best knowledge of literature, but is not a systematic review of literature. For common variants, we selected only signals firmly
replicated in large meta-GWAS (Lambert et al.3, Kunkle et al.20, Jun et al.43, Sims et al.7, Jansen et al.38 and present study). For rare variants, we only
selected those variants widely replicated excluding those loci presenting conflicting results. Abbreviations and more information about the genes can be
found in Supplementary Data 4. The risk alleles associated with AD were represented in orange and the protective alleles in blue. GWAS Genome-Wide
Association Study, OR odds ratio. roles. We add to the growing body of evidence that the polygenic
scores of all genetic loci to date, in combination with APOE
genotypes, are robust tools that are associated with AD and its
AAO. These properties make PRS promising in selecting indivi-
duals at risk to apply preventative therapeutic strategies. (including rs2154481) increased the TFCP2 transcription factor
avidity to its binding site and increased the enhancer activity of
this specific intronic region26. Based on this evidence, we can
postulate that a life-long slightly higher APP gene expression
protects the brain from AD insults. Still, this seems counter-
intuitive as duplications of the gene lead to early-onset AD27. A
U-shaped effect, or hormesis effect of APP might help explain our
observations and it might also fit the accelerated cognitive dete-
rioration observed in AD patients treated with beta-secretase
inhibitors28,29 as these reduce beta-amyloid in their brain. An
alternative hypothesis is that mechanisms underlying the variant
are related to the overexpression of protective fragments of the
APP protein30. Methods
D t
P
i i Data. Participants in this study were obtained from multiple sources, including raw
data from case-control samples collected by GR@ACE/DEGESCO, summary sta-
tistics data from the case-control samples in the IGAP and the summary statistics
of AD-by-proxy phenotype from the UK Biobank. An additional case-control
samples from 16 independent cohorts (19,087 AD cases and 39,101 controls) was
used for replication, largely collected and analyzed by the European Alzheimer’s
Disease Biobank (JPND-EADB) project. Full descriptions of the samples and their
respective phenotyping and genotyping procedures are provided in the Supple-
mentary Methods. g
p
y
yp
Additionally, other three variants identified are altering protein
sequence or affecting regulatory motifs. Two independent mis-
sense mutations in SHARPIN increased the AD risk. SHARPIN
was previously proposed as an AD candidate gene31,32, and
functional analysis of a rare missense variant (NM_030974.3:p. Gly186Arg) resulted in the aberrant cellular localization of the
variant protein and attenuated the activation of NF-κB, a central
mediator of inflammatory and immune responses. Functional
analysis of the two identified missense variants will show if the
effect on immune reaction in AD is similar. The variant located in
the CHRNE which encodes a subunit of the cholinergic receptor
(AChR) is a strong modulator of CHRNE expression. The same
allele that increases AD risk increases the expression in the brain
and other tissues according to GTEx (p = 2.1 × 10−13) (Supple-
mentary Fig. 5). The detection of a potential hypermorph allele
linked to AD risk and affecting cholinergic function could rein-
troduce this neurotransmitter pathway into the search for pre-
ventative strategies. Further functional studies are needed to
consolidate this hypothesis. GR@ACE. The GR@ACE study19 recruited AD patients from Fundació ACE,
Institut Català de Neurociències Aplicades (Catalonia, Spain), and control indivi-
duals from three centers: Fundació ACE (Barcelona, Spain), Valme University
Hospital (Seville, Spain), and the Spanish National DNA Bank–Carlos III (Uni-
versity of Salamanca, Spain) (http://www.bancoadn.org). Additional cases and
controls were obtained from dementia cohorts included in the Dementia Genetics
Spanish Consortium (DEGESCO)33. At all sites, AD diagnosis was established by a
multidisciplinary working group—including neurologists, neuropsychologists, and
social workers—according to the DSM-IV criteria for dementia and the National
Institute on Aging and Alzheimer’s Association’s (NIA–AA) 2011 guidelines for
diagnosing AD. In our study, we considered as AD cases any individuals with
dementia diagnosed with probable or possible AD at any point in their clinical
course. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Family
studies
Candidate−gene
studies
GWAS studies
Goate et al. Shellenberg et al. Strittmatter et al. Chartier−Harlin et al. Levy−Lahad et al. Rogaeva et al. Bertram et al. Lambert et al. Harold et al. Seshadri et al. Jonsson et al. Hollingworth et al. Lambert et al. Jonsson et al. Benitez et al. Jun et al. Desikan et al. Nicolas et al. Sims et al. Marioni et al. Kunkle et al. Jansen et al. Broce et al. Current study
0
1
10
20
30
40
50
10
20000
100000
200000
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
Current study
1990s
Timeline
Variant frequency (%)
Study size
APP
PSEN1
APOE4
APOE2
PSEN2
CD33
CR1
APP − A673T
ABCA7
PILRA
TREM2 −R47H
ABCA7
SORL1
PLCG2 − P522R
TREM2 −R62H
ABI3
APH1B
ACE
SHARPIN S17F
SHARPIN P294S
IL34
SORL1
PICALM
CLU
BIN1
MS4A4A
FERMT2
HLA−DRB1
CASS4
EPHA1
INPP5D
SLC2A4
CD2AP
PTK2B
SPI1
TREML2
MAPT
HS3ST1
ECHDC3
PLCG2
SCIMP
IQCK
ADAM10
MINK1
PLCG2
PRKD3
APP
OR<1.1
OR=1.2
OR=2
OR>3
OR>0.95
OR=0.75
OR=0.5
Gene Culprit gene found
Gene Culprit gene not found Family
studies
Candidate−gene
studies
GWAS studies
Goate et al. Shellenberg et al. Strittmatter et al. Chartier−Harlin et al. Levy−Lahad et al. Rogaeva et al. Bertram et al. Lambert et al. Harold et al. Seshadri et al. Jonsson et al. Hollingworth et al. Lambert et al. Jonsson et al. Benitez et al. Jun et al. Desikan et al. Nicolas et al. Sims et al. Marioni et al. Kunkle et al. Jansen et al. Broce et al. Current study
0
1
10
20
30
40
50
10
20000
100000
200000
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
Current study
1990s
Timeline
Variant frequency (%)
Study size
APP
PSEN1
APOE4
APOE2
PSEN2
CD33
CR1
APP − A673T
ABCA7
PILRA
TREM2 −R47H
ABCA7
SORL1
PLCG2 − P522R
TREM2 −R62H
ABI3
APH1B
ACE
SHARPIN S17F
SHARPIN P294S
IL34
SORL1
PICALM
CLU
BIN1
MS4A4A
FERMT2
HLA−DRB1
CASS4
EPHA1
INPP5D
SLC2A4
CD2AP
PTK2B
SPI1
TREML2
MAPT
HS3ST1
ECHDC3
PLCG2
SCIMP
IQCK
ADAM10
MINK1
PLCG2
PRKD3
APP
OR<1.1
OR=1.2
OR=2
OR>3
OR>0.95
OR=0.75
OR=0.5
Gene Culprit gene found
Gene Culprit gene not found
Fig. 3 Genetic landscape for Alzheimer’s disease. This figure shows the history of genetic discoveries in AD research over the past 30 years. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Disentangling the molecular mechanism of our
finding will help refine and steer the amyloid hypothesis. Discussion
h
k Discussion
This work adds on the ongoing global effort to identify genetic
variants associated with AD (Fig. 3). In the present work, we
reported on the largest GWAS for AD risk to date, comprising
genetic information of 467,623 individuals of European ancestry. We identified six variants that were not previously associated with
the risk of AD and constructed a robust PRS for AD demon-
strating its potential value for selecting subjects at risk of AD,
especially within APOE ɛ4 carriers. This PRS was based on
European ancestries and may or may not generalize to other
ancestries. Validation in other populations will be required. We
also acknowledge that controls included in GR@ACE are younger
than cases and some of the controls might still develop AD later
in life. This fact does not invalidate the analysis although reported
estimates must be considered conservative. The differences in risk
and AAO determined by the PRS of AD are relevant for design
clinical trials that over-represent APOE ε4 carriers, as APOE ε4
heterozygous with highest-PRS values have a similar risk and
AAO to APOE ɛ4 homozygotes (Fig. 5b). These represents ~1%
of our control population, which is the same percentage as all
APOE ε4 homozygotes. A trial that aims to include APOE ɛ4
homozygotes, could consider widening the selection criteria and
in this way hasten the enrollment process. Also, our PRS could
aid at the interpretation of the results of clinical trials, as it
determines a relevant proportion of the AAO, which could either
mimic or obscure a treatment effect. The most interesting finding from our GWAS is the discovery
of a common protective (MAF (C-allele) = 0.483) intronic variant
in the APP gene. Our results directly support APP production or
processing as a causal pathway not only in familial AD but in
common sporadic AD. The SNP is in a DNase hypersensitive area
of 295 bp (chr21:27473781-27474075) possibly involved in the
transcriptional regulation of the APP gene. rs2154481 is an eQTL
for the APP mRNA and an antisense transcript of the APP gene
named AP001439.2 in public eQTL databases25 (Supplementary
Fig. 4). Functional
evidence
supports
a
modified
APP
transcription26 as an LD block of 13 SNPs within the APP locus URE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Quality control (QC) was
performed for samples and plates using the Affymetrix power tool (APT)
1.15.0 software following the Axiom data analysis workflow. The sample quality variations observed in the Spanish population. The DNA samples were genotyped
according to the manufacturer’s instructions (Axiom™2.0 Assay Manual
Workflow). The Axiom 2.0 assay interrogates biallelic SNPs and simple indels in a
single-assay workflow. Starting with 200 ng of genomic DNA, the samples were
processed through a manual target preparation protocol, followed by automated
processing of the array plates in the GeneTitan Multi-Channel (MC) instrument. Target preparation involved DNA amplification, fragmentation, purification, and
resuspension of the target in a hybridization cocktail. The hyb-ready targets were
then transferred to the GeneTitan MC instrument for automated, hands-free
processing, including hybridization, staining, washing, and imaging. The CEL files
were generated using the GeneTitan MC instrument. Quality control (QC) was
performed for samples and plates using the Affymetrix power tool (APT)
1.15.0 software following the Axiom data analysis workflow. The sample quality was determined based on the resolution of AT and GC channels in a group of non-
polymorphic SNPs (resolution > 0.82). Samples with a call rate greater than 97%
and plates with an average call rate above 98.5% were included for final SNP
calling. The samples were jointly called. Markers passing all the QC tests were used
in downstream analysis (NSNPs = 729,868; 95.4%) using the SNPolisher R package
(Thermo Fisher). To assess the sample genotyping concordance, we intentionally
resampled 200 samples and determined a concordance rate of 99.5%. was determined based on the resolution of AT and GC channels in a group of non-
polymorphic SNPs (resolution > 0.82). Samples with a call rate greater than 97%
and plates with an average call rate above 98.5% were included for final SNP
calling. The samples were jointly called. Markers passing all the QC tests were used
in downstream analysis (NSNPs = 729,868; 95.4%) using the SNPolisher R package
(Thermo Fisher). To assess the sample genotyping concordance, we intentionally
resampled 200 samples and determined a concordance rate of 99.5%. We also conducted previously described standard QC prior to imputation19. In
brief, individual QC includes genotype call rates >97%, sex checks, and no excess
heterozygosity; we removed population outliers as well (European cluster of 1000
Genomes). Methods
D t
P
i i For further details on the contribution of the sites, see Supplementary
Data 10. Written informed consent was obtained from all the participants. The
ethics and scientific committees have approved this research protocol (Acta 25/
2016, Ethics Committee H., Clinic I Provincial, Barcelona, Spain). Genotyping, quality control, and imputation. DNA was extracted from
peripheral blood according to standard procedures using the Chemagic system
(Perkin Elmer). Samples reaching DNA concentrations of >10 ng/μl and presenting
high integrity were included for genotyping. Cases and controls were randomized
across sample plates to avoid batch effects. Genotyping was conducted using the Axiom 815K Spanish biobank array
(Thermo Fisher) at the Spanish National Center for Genotyping (CeGEN, Santiago
de Compostela, Spain). The genotyping array not only is an adaptation of the
Axiom biobank genotyping array but also contains rare population-specific Altogether, we described six additional loci associated with
sporadic AD. These signals reinforce that AD is a complex disease
in which amyloid processing and immune response play key 5 variations observed in the Spanish population. The DNA samples were genotyped
according to the manufacturer’s instructions (Axiom™2.0 Assay Manual
was determined based on the resolution of AT and GC channels in a group of non-
polymorphic SNPs (resolution > 0.82). Samples with a call rate greater than 97%
a
b
c
Fig. 4 Polygenic risk scores for AD. a The 39-SNP PRS association with clinical (OR = 1.30, 95% CI [1.18–1.44], p = 1.1 × 10−7) and pathologically confirmed
AD cases (OR = 1.38, per 1-SD increase in the PRS, 95% CI [1.21–1.58], p = 1.5 × 10−6) from EADB–F.ACE/BBB dataset. b PRS association with AD in the
presence of concomitant brain pathologies (besides AD). c PRS association with AD stratified by sex and AAO. A similar association of the PRS with AD was
found in both sexes (ORmales = 1.33, [1.13–1.56], p = 5.8 × 10−4 vs. ORfemales = 1.32, [1.19–1.47], p = 2.5 × 10−7). In (a–c) data are presented as Odds Ratio
per 1-SD increase in PRS (95% CI). The generated PRS was validated using logistic regression adjusted by four principal components. ARTICLE
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 a b b b b c c Fig. 4 Polygenic risk scores for AD. a The 39-SNP PRS association with clinical (OR = 1.30, 95% CI [1.18–1.44], p = 1.1 × 10−7) and pathologically confirmed
AD cases (OR = 1.38, per 1-SD increase in the PRS, 95% CI [1.21–1.58], p = 1.5 × 10−6) from EADB–F.ACE/BBB dataset. b PRS association with AD in the
presence of concomitant brain pathologies (besides AD). c PRS association with AD stratified by sex and AAO. A similar association of the PRS with AD was
found in both sexes (ORmales = 1.33, [1.13–1.56], p = 5.8 × 10−4 vs. ORfemales = 1.32, [1.19–1.47], p = 2.5 × 10−7). In (a–c) data are presented as Odds Ratio
per 1-SD increase in PRS (95% CI). The generated PRS was validated using logistic regression adjusted by four principal components. Fig. 4 Polygenic risk scores for AD. a The 39-SNP PRS association with clinical (OR = 1.30, 95% CI [1.18–1.44], p = 1.1 × 10−7) and pathologically confirmed
AD cases (OR = 1.38, per 1-SD increase in the PRS, 95% CI [1.21–1.58], p = 1.5 × 10−6) from EADB–F.ACE/BBB dataset. b PRS association with AD in the
presence of concomitant brain pathologies (besides AD). c PRS association with AD stratified by sex and AAO. A similar association of the PRS with AD was
found in both sexes (ORmales = 1.33, [1.13–1.56], p = 5.8 × 10−4 vs. ORfemales = 1.32, [1.19–1.47], p = 2.5 × 10−7). In (a–c) data are presented as Odds Ratio
per 1-SD increase in PRS (95% CI). The generated PRS was validated using logistic regression adjusted by four principal components. variations observed in the Spanish population. The DNA samples were genotyped
according to the manufacturer’s instructions (Axiom™2.0 Assay Manual
Workflow). The Axiom 2.0 assay interrogates biallelic SNPs and simple indels in a
single-assay workflow. Starting with 200 ng of genomic DNA, the samples were
processed through a manual target preparation protocol, followed by automated
processing of the array plates in the GeneTitan Multi-Channel (MC) instrument. Target preparation involved DNA amplification, fragmentation, purification, and
resuspension of the target in a hybridization cocktail. The hyb-ready targets were
then transferred to the GeneTitan MC instrument for automated, hands-free
processing, including hybridization, staining, washing, and imaging. The CEL files
were generated using the GeneTitan MC instrument. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Likewise, the
14,338 participants whose fathers had dementia (paternal cases) were compared
with the 245,941 participants whose fathers did not have dementia21. The phe-
notype of the parents is independent, and therefore, the estimates could be meta-
analyzed. After analysis, the effect estimates were made comparable to a case-
control setting. Further information on the transformation of the effect sizes can be
found elsewhere21,39. The data available comprises summary statistics of 7,794,553
SNPs imputed to the HRC reference panel (full panel). IGAP summary statistics. The GWAS summary results from the IGAP were
downloaded from the National Institute on Aging Genetics of Alzheimer’s Disease
Data Storage Site (NIAGADS, https://www.niagads.org/)20. Details on data gen-
eration and analyses by the IGAP have been previously described20. In brief, the
IGAP is a large study based upon genome-wide association using individuals of
European ancestry. Stage 1 of the IGAP comprises 21,982 AD cases and 41,944
cognitively normal controls from four consortia: the Alzheimer Disease Genetics
Consortium (ADGC), the European Alzheimer’s Disease Initiative (EADI), the
Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE)
Consortium, and the Genetic and Environmental Risk in AD/Defining Genetic,
Polygenic, and Environmental Risk for Alzheimer’s Disease (GERAD/PERADES)
Consortium. Summary statistics are available for 11,480,632 variants, both geno-
typed and imputed (1000 Genomes phase 1, v3). In Stage 2, 11,632 SNPs were
genotyped in an independent set of 8362 AD cases and 10,483 controls. Meta-GWAS of AD. After study-specific variant filtering and quality-control
procedures, we performed a fixed effects inverse-variance–weighted meta-
analysis22 on the discovery and follow-up stages (Supplementary Data 1 and
Supplementary Data 12). To determine the lead SNPs (those with the strongest
association per genomic region), we performed clumping on SNPs with a GWS p
value (p < 5 × 10−8) (Plink v1.90, maximal linkage disequilibrium (LD) with R2 <
0.001 and physical distance 250 Kb). In the APOE region, we only considered the
APOE ɛ4 (rs429358) and APOE ɛ2 (rs7412) SNPs40. LD information was calculated
using the GR@ACE imputed genotypes as a reference. Polygenicity and con-
founding biases, such as cryptic relatedness and population stratification, can yield
an inflated distribution of test statistics in GWAS. To distinguish between inflation
from a true polygenic signal and bias we quantified the contribution of each by
examining the relationship between test statistics and linkage disequilibrium (LD)
using the LD Score regression intercept (LDSC software41). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 ARTICLE a
c
b
d
Fig. 5 Polygenic Risk Scores APOE stratification for AD in n = 12,386 biologically independent samples from GR@ACE/DEGESCO. a The AD risk of PRS
groups compared to those with the 2% lowest risk. The 2% highest risk had a 3.0-fold (95% CI [2.12–4.18], p = 3.2 × 10−10) increased risk compared with
those with the 2% lowest risk. No interaction was found between the PRS and APOE genotypes (p value = 0.76). b The AD risk stratified by PRS and APOE
risk groups compared to the lowest risk group (OR 95% CI). Association was found between highest and lowest-PRS percentiles within the APOE genotype
groups: ɛ2ɛ2/ɛ2ɛ3 carriers (OR = 2.48 [1.51–4.08], p = 3.4 × 10−4), ɛ3ɛ3 carriers (OR = 2.67 [1.93–3.69], p = 3.5 × 10−9), ɛ2ɛ4/ɛ3ɛ4 carriers (OR = 2.47
[1.67–3.66], p = 6.8 × 10−6), and ɛ4ɛ4 carriers (OR = 2.02 [1.05–3.85], p = 3.4 × 10−2). Comparisons of the highest and lowest-PRS percentiles with
respect to the APOE genotype groups: a difference was found between highest ɛ2ɛ2/ɛ2ɛ3 carriers vs. lowest ɛ3ɛ3 carriers (OR = 0.51 [0.34–0.75],
p = 7.8 × 10−4), but not between highest ɛ3ɛ3 carriers vs. lowest ɛ2ɛ4/ɛ3ɛ4 carriers (OR = 1.17 [0.82–1.66], p = 0.40) and highest ɛ2ɛ4/ɛ3ɛ4 carriers vs. lowest ɛ4ɛ4 carriers (OR = 0.89 [0.52–1.53], p = 0.68). c The AAO of AD stratified by PRS and APOE risk groups. No difference in odds for AD was found
between the PRS percentiles with AAO in APOE ɛ2ɛ2/ɛ2ɛ3 (lowest = 82 years, highest = 83 years, pWilcoxon = 0.39) and APOE ɛ3ɛ3 (lowest = 82 years,
highest = 81 years, p = 0.16). However, a 4-year difference was found between APOE ɛ4 heterozygotes (pWilcoxon = 6.9 × 10−5, 81 years compared with 77
years) and 5.5 years difference (pWilcoxon = 4.6 × 10−5, 78.5 years compared with 73 years) in APOE ɛ4 homozygotes. Data are represented as boxplots as
described in the manual of ggplot2 package in R. a–c Logistic regression models adjusted for four population ancestry components were used as statistical
test. d Cox regression model on AAO. The determinants are the PRS and the APOE categories, a PRS*APOE interaction term and population substructure as
covariates. The curve shows the probability a case in one of the eight groups has developed AD by a certain age (x-axis). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 We included variants with a call rate of >95%, with a minor allele
frequency (MAF) of >0.01, in Hardy–Weinberg equilibrium (p < 1 × 10−4 in
controls) and without differential missingness between cases and controls p
p
We also conducted previously described standard QC prior to imputation19. In
brief, individual QC includes genotype call rates >97%, sex checks, and no excess
heterozygosity; we removed population outliers as well (European cluster of 1000
Genomes). We included variants with a call rate of >95%, with a minor allele
frequency (MAF) of >0.01, in Hardy–Weinberg equilibrium (p < 1 × 10−4 in
controls) and without differential missingness between cases and controls NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 s APOE stratification for AD in n = 12,386 biologically independent samples from GR@ACE/DEGESCO. a The AD ris (2006–2010) (http://www.ukbiobank.ac.uk)36. Several groups have demonstrated
the utility of self-report of parental history of AD for case ascertainment in GWAS
(proxy–AD approach)21,37,38. For this study, we used the published summary
statistics of Marioni et al.21. They included, after stringent QC, 314,278 unrelated
individuals for whom AD information was available on at least one parent in the
UK Biobank (https://datashare.is.ed.ac.uk/handle/10283/3364). In brief, the 27,696
participants whose mothers had dementia (maternal cases) were compared with
the 260,980 participants whose mothers did not have dementia. Likewise, the
14,338 participants whose fathers had dementia (paternal cases) were compared
with the 245,941 participants whose fathers did not have dementia21. The phe-
notype of the parents is independent, and therefore, the estimates could be meta-
analyzed. After analysis, the effect estimates were made comparable to a case-
control setting. Further information on the transformation of the effect sizes can be
found elsewhere21,39. The data available comprises summary statistics of 7,794,553
SNPs imputed to the HRC reference panel (full panel). (Supplementary Data 11, Supplementary Fig. 1). Imputation was carried out using
the Haplotype reference consortium34 (HRC, full panel) and the 1000 Genomes
reference panel35 (for indels only) on the Michigan Imputation Server (https://
imputationserver.sph.umich.edu). Rare variants (MAF < 0.001) and variants with
low imputation quality (R2 < 0.30) were excluded. Logistic regression models,
adjusted for the first four ancestry principal components19, were fitted using Plink
(v2.00a). Population-based controls were used; therefore, age was not included as a
covariate. Age and gender statistically behave like phenotype proxies (for AD status
in this case). Therefore, adjusting for co-variation with age and gender could result
in an over-adjustment of GWAS results. After QC steps, we included 6,331 AD
cases and 6,055 control individuals and tested 14,542,816 genetic variants for
association with AD. (2006–2010) (http://www.ukbiobank.ac.uk)36. Several groups have demonstrated
the utility of self-report of parental history of AD for case ascertainment in GWAS
(proxy–AD approach)21,37,38. For this study, we used the published summary
statistics of Marioni et al.21. They included, after stringent QC, 314,278 unrelated
individuals for whom AD information was available on at least one parent in the
UK Biobank (https://datashare.is.ed.ac.uk/handle/10283/3364). In brief, the 27,696
participants whose mothers had dementia (maternal cases) were compared with
the 260,980 participants whose mothers did not have dementia. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 a b b b a d c d c Fig. 5 Polygenic Risk Scores APOE stratification for AD in n = 12,386 biologically independent samples from GR@ACE/DEGESCO. a The AD risk of PRS
groups compared to those with the 2% lowest risk. The 2% highest risk had a 3.0-fold (95% CI [2.12–4.18], p = 3.2 × 10−10) increased risk compared with
those with the 2% lowest risk. No interaction was found between the PRS and APOE genotypes (p value = 0.76). b The AD risk stratified by PRS and APOE
risk groups compared to the lowest risk group (OR 95% CI). Association was found between highest and lowest-PRS percentiles within the APOE genotype
groups: ɛ2ɛ2/ɛ2ɛ3 carriers (OR = 2.48 [1.51–4.08], p = 3.4 × 10−4), ɛ3ɛ3 carriers (OR = 2.67 [1.93–3.69], p = 3.5 × 10−9), ɛ2ɛ4/ɛ3ɛ4 carriers (OR = 2.47
[1.67–3.66], p = 6.8 × 10−6), and ɛ4ɛ4 carriers (OR = 2.02 [1.05–3.85], p = 3.4 × 10−2). Comparisons of the highest and lowest-PRS percentiles with
respect to the APOE genotype groups: a difference was found between highest ɛ2ɛ2/ɛ2ɛ3 carriers vs. lowest ɛ3ɛ3 carriers (OR = 0.51 [0.34–0.75],
p = 7.8 × 10−4), but not between highest ɛ3ɛ3 carriers vs. lowest ɛ2ɛ4/ɛ3ɛ4 carriers (OR = 1.17 [0.82–1.66], p = 0.40) and highest ɛ2ɛ4/ɛ3ɛ4 carriers vs. lowest ɛ4ɛ4 carriers (OR = 0.89 [0.52–1.53], p = 0.68). c The AAO of AD stratified by PRS and APOE risk groups. No difference in odds for AD was found
between the PRS percentiles with AAO in APOE ɛ2ɛ2/ɛ2ɛ3 (lowest = 82 years, highest = 83 years, pWilcoxon = 0.39) and APOE ɛ3ɛ3 (lowest = 82 years,
highest = 81 years, p = 0.16). However, a 4-year difference was found between APOE ɛ4 heterozygotes (pWilcoxon = 6.9 × 10−5, 81 years compared with 77
years) and 5.5 years difference (pWilcoxon = 4.6 × 10−5, 78.5 years compared with 73 years) in APOE ɛ4 homozygotes. Data are represented as boxplots as
described in the manual of ggplot2 package in R. a–c Logistic regression models adjusted for four population ancestry components were used as statistical
test. d Cox regression model on AAO. The determinants are the PRS and the APOE categories, a PRS*APOE interaction term and population substructure as
covariates. The curve shows the probability a case in one of the eight groups has developed AD by a certain age (x-axis). References 1. Pericak-Vance, M. A. et al. Linkage studies in familial Alzheimer disease:
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2. Seshadri, S. et al. Genome-wide analysis of genetic loci associated with
Alzheimer disease. JAMA J. Am. Med. Assoc. 303, 1832–1840 (2010). Polygenic risk scores. We calculated a weighted individual PRS based on the 39
genetic variants that showed GWS evidence of association with AD in the present
study, excluding APOE to check the impact of PRS modulating APOE risk (Table 1
and Supplementary Data 3). The selected variants were directly genotyped or
imputed with high quality (median imputation score R² = 0.93). The PRSs were
generated by multiplying the genotype dosage of each risk allele for each variant by
its respective weight and then summing across all variants. We weighted this by the
effect size from previous IGAP studies [Kunkle et al.42 (36 variants), Sims et al.7 (2
variants), Jun et al.43 (MAPT locus), Supplementary Data 5]. The generated PRS
was validated using logistic regression adjusted by four principal components in a
sample of 676 AD cases diagnosed based on clinical criteria and 332 pathologically
confirmed AD cases from the European Alzheimer’s Disease Biobank–Fundació
ACE/Barcelona Brain Bank dataset (EADB–F.ACE/BBB, Supplementary Infor-
mation). This dataset was not used in prior genetic studies. In this dataset, all
pathologically confirmed cases were scored for the presence or absence of con-
comitant pathologies. In all analyses, we compared the AD patients to the same
control dataset (n = 1386). We performed analyses to test the robustness of the
PRS. We tested the effect of adding variants below the genome-wide significance
threshold using a pruning and thresholding approach. For this, we used the
summary statistics of the IGAP42 study, and we selected independent variants
using the clump_data() function from the TwoSampleMR package (v0.4.25). We
used strict settings for clumping (R2 = 0.001 and window = 1 MB) and increasing p
value thresholds (>1 × 10−7, >1 × 10−6, >1 × 10−5, >1 × 10−4, >1 × 10−3, and >1 ×
10−2). We tested the association of the results with clinically diagnosed and
pathologically confirmed AD patients. To evaluate the effect of diagnostic certainty,
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Risk stratification of the validated PRSs. We searched for the groups at the
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stratified the population into PRS percentiles, taking into account survival bias
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the percentiles in the control participants aged 55 years and younger (n = 3546). Based on the boundaries from this population, the rest of the controls and all AD
cases were then assigned into their appropriate percentiles. We first explored risk
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percentiles) and compared all groups with that which had the lowest PRS. Second,
we explored risk stratification considering both the APOE genotypes and the PRSs. The APOE genotypes were pooled in the analyses as APOE ɛ2ɛ2/ɛ2ɛ3 (n = 998,
split into 7 PRS groups), APOE ɛ3ɛ3 (n = 7611, split into 25 PRS groups), APOE
ɛ2ɛ4/ɛ3ɛ4 (n = 3399, split into 15 PRS groups), and APOE ɛ4ɛ4 (n = 382, split into
3 PRS groups). We studied the effect of PRS across groups of individuals stratified
by the APOE genotypes with the lowest-PRS group (APOE as the reference group
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18534-1 (2020). 18. van der Lee, S. J. et al. The effect of APOE and other common genetic variants
on the onset of Alzheimer’s disease and dementia: a community-based cohort
study. Lancet Neurol. https://doi.org/10.1016/S1474-4422(18)30053-X (2018). 19. Moreno-Grau, S. et al. Genome-wide association analysis of dementia and its
clinical endophenotypes reveal novel loci associated with Alzheimer’s disease
and three causality networks: The GR@ACE project. Alzheimer’s Dement. 0,
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)
y
p
g
We implemented a Cox regression model on AAO in the GR@ACE/DEGESCO
dataset case-only adjusted for covariates as APOE group, the interaction between
the PRS and APOE and four population ancestry components. All analyses were
done in R (v3.4.2). 20. Kunkle, B. W. et al. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 et al.3, Kunkle et al.42, and Jansen et al.38). We tested all variants with suggestive
association (p < 10−5) located in proximity (200 kb) of genomic regions selected for
follow-up to allow for the potential refinement of the top associated variant. et al.3, Kunkle et al.42, and Jansen et al.38). We tested all variants with suggestive
association (p < 10−5) located in proximity (200 kb) of genomic regions selected for
follow-up to allow for the potential refinement of the top associated variant. Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. p
p
p
Conditional analyses were performed in regions where multiple variants were
associated with AD using logistic regression models, adjusting for the genetic
variants in the region (Supplementary Data 13, 14). Received: 17 July 2020; Accepted: 17 March 2021; Received: 17 July 2020; Accepted: 17 March 2021; Received: 17 July 2020; Accepted: 17 March 2021; elements). As a reference genome, we used GRCh37. Quantile–quantile plots,
Manhattan plots, and the exploration of genomic inflation factors were performed
using the R package qqman. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Chromosomal regions
associated with AD in previous studies were excluded from follow-up (Lambert UK Biobank summary statistics. UK Biobank data—including health, cognitive, and
genetic data—was collected on over 500000 individuals aged 37–73 years from
across Great Britain (England, Wales, and Scotland) at the study baseline 7 7 TURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/ Data availability The discovery summary statistics of this study are publicly available in Fundació ACE
server [https://fundacioace-my.sharepoint.com/:u:/g/personal/iderojas_fundacioace_org/
EaTwlPg9cRJHn7Kos4h39OUBaxajsjJHL_C110fC89bc8w?e=ZdcEUy]. Regional plots were generated with a mixture of homemade Python (v2.7) and
R (v3.6.0) scripts. Briefly, given an input variant, we calculated the LD between the
input variant and all the surrounding variants within a window of length defined
by the user. The LD was calculated in the 1000 Genomes samples of European
ancestry. We used gene positions from RefSeq (release 93); in the case of multiple
gene models for a given gene, we reported the model with the largest number of
exons. We used recombination rates from HapMap II and chromatin states from
ENCODE/Broad (15 states were grouped to highlight the predicted functional
elements). As a reference genome, we used GRCh37. Quantile–quantile plots,
Manhattan plots, and the exploration of genomic inflation factors were performed
using the R package qqman. Acknowledgements I.d.R. Study supervision/management: A.C., L.Ta, M.Bo, M.Me, J.C., and A.Ru. IGAP. Critical revision: A.C.N., B.W.K., L.A.F., J.L.H., L.S.W., M.A.P.V., R.May, M.A.I., J.C.B.,
A.L.D.S., C.L.S., E.B., M.F., Q.Y., X.J., R.S., C.H., K.M., S.Mea, V.E.P., A.Meg, P.A.H., R. Mar, P.A., G.Sc, J.Will, and S.Se. EADB: Sample contribution. N.T., I.E.J., N.St, K.A.M.,
C.D., G.N., G.C., G.Sp, K.S., M.I., M.K., R.F.S., J.C.L., A.Ra, D.G., J.S.V., D.R., E.Gr, H.Ha,
I.G., J.K., L.Fa, L.Fr, A.M.H., J.V., L.H., G.H., N.Sca, M.H.K., M.Y., H.Ho, W.M.v.d..F, M. H.u.l., N.M.v.S., A.T.H., B.G.N., C.V.B., E.S., R.V., S.E., T.N., F.K., J.V.D., V.G., A.U., A. Benu, A.K.S., B.B., C.Mas, C.F., E.C., F.Ma, G.B., I.Ap, J.Q.T., L.Ki, L.K.l., L.P., L.Tr, L.B.,
M.L., M.Ar, R.G., S.F., F.J., J.D.S., O.G., T.G., M.J.H., T.P., K.Bu, M.E.si, S.R.H., E.Dur, A. Ru, I.H., S.M.G., I.d.R., Y.A.L..P, A.d.M., C.C.l., J.P., S.J.v.d.L., C.G., N.B., O.H., P.B., A. H., T.K., M.E.w., O.A.S., R.N.K., J.Wilf, P.F., P.R., P.Sc, P.Sa, N.Sch, D.W., E.R., G.R., H. S., I.R., A.Sc, A.Sp, A.Sq, C.Cha, C.Chi, C.P., A.P., B.A., B.N., C.M.F., D.S., E.Da, E.düz, E. F., F.T., F.P., F.S.G., G.Gi, G.Gra, G.P., H.B., J.H., J.L., M.C.D., M.T., M.T.H., M.Schm, M. W., M.S., O.Q., O.L., P.C., P.D., R.C., S.So, S.He, S.A., S.B., S.C., T.L., V.B., V.D., P.G.K.,
M M N M C D N O P W M A W L I Ap C B F A A L K G B M Sca M Sp M V g
We would like to thank patients and controls who participated in this project. The
present work has been performed as part of the doctoral program of I. de Rojas at the
Universitat de Barcelona (Barcelona, Spain) supported by national grant from the
Instituto de Salud Carlos III FI20/00215. The Genome Research @ Fundació ACE project
(GR@ACE) is supported by Grifols SA, Fundación bancaria “La Caixa”, Fundació ACE,
and CIBERNED. A.R. and M.B. receive support from the European Union/EFPIA
Innovative Medicines Initiative Joint undertaking ADAPTED and MOPEAD projects
(grant numbers 115975 and 115985, respectively). M.B. and A.R. are also supported by
national grants PI13/02434, PI16/01861, PI17/01474, PI19/01240 and PI19/01301. Acción Estratégica en Salud is integrated into the Spanish National R + D + I Plan and
funded by ISCIII (Instituto de Salud Carlos III)—Subdirección General de Evaluación
and the Fondo Europeo de Desarrollo Regional (FEDER—“Una manera de hacer
Europa”). Author contributions N.T., I.E.J., N.St, K.A.M.,
C.D., G.N., G.C., G.Sp, K.S., M.I., M.K., R.F.S., J.C.L., A.Ra, D.G., J.S.V., D.R., E.Gr, H.Ha,
I.G., J.K., L.Fa, L.Fr, A.M.H., J.V., L.H., G.H., N.Sca, M.H.K., M.Y., H.Ho, W.M.v.d..F, M. H.u.l., N.M.v.S., A.T.H., B.G.N., C.V.B., E.S., R.V., S.E., T.N., F.K., J.V.D., V.G., A.U., A. Benu, A.K.S., B.B., C.Mas, C.F., E.C., F.Ma, G.B., I.Ap, J.Q.T., L.Ki, L.K.l., L.P., L.Tr, L.B.,
M.L., M.Ar, R.G., S.F., F.J., J.D.S., O.G., T.G., M.J.H., T.P., K.Bu, M.E.si, S.R.H., E.Dur, A. Ru, I.H., S.M.G., I.d.R., Y.A.L..P, A.d.M., C.C.l., J.P., S.J.v.d.L., C.G., N.B., O.H., P.B., A. H., T.K., M.E.w., O.A.S., R.N.K., J.Wilf, P.F., P.R., P.Sc, P.Sa, N.Sch, D.W., E.R., G.R., H. S., I.R., A.Sc, A.Sp, A.Sq, C.Cha, C.Chi, C.P., A.P., B.A., B.N., C.M.F., D.S., E.Da, E.düz, E. F., F.T., F.P., F.S.G., G.Gi, G.Gra, G.P., H.B., J.H., J.L., M.C.D., M.T., M.T.H., M.Schm, M. W., M.S., O.Q., O.L., P.C., P.D., R.C., S.So, S.He, S.A., S.B., S.C., T.L., V.B., V.D., P.G.K.,
M.M.N., M.C.D.N., O.P., W.M., A.W.L., I.Ap, C.B.F., A.A.L.K., G.B., M.Sca, M.Sp, M.V.,
M.Hi, K.F., L.W., M.D., P.H., and A.Ra. Analysis: N.J.A., R.M.T., V.An, N.T., I.E.J., N.St,
S.J.v.d.L., I.d.R., S.M.G., B.G.B., and C.B. Studies supervision/management: S.H.H., K.A. M., C.D., G.N., G.C., G.S., K.S., M.I., M.K., R.F.S., D.G., J.S.V., D.R., E.Gr, H.Ha, I.G., J.K.,
L.Fr, A.M.H., J.V., L.H., G.H., N.Sca, M.H.K., M.Y., H.Ho, W.M.v.d.F., M.Hui, N.M.v.S.,
N.J.A., J.D., M.Sche, A.K.S., C.G., N.B., O.H., P.B., A.H., T.K., J.Wilf, P.F., P.R., P.Sa, P.Sc,
M.Hul, N.T., I.E.J., A.T.H., B.G.N., C.V.B., E.S., R.V., S.E., P.A., A.Ru, and J.C.L. PGC-
ALZ: Sample contribution. I.E.J., A.Ro, I.Sa, D.Aa, G.Se, S.B.S., S.D., D.P., S.H., I.K.K., N. L.P., C.A.R., and O.A.A. Analysis: S.Ha, I.K.K., and I.E.J. Study supervision/management:
O.A.A., C.M.v.D., and D.P. AD and GBCS: Sample contribution. H.Z., S.K., I.S., and K.B. Analysis: N.M.S. and A.Z. Study supervision/management: I.S.k., A.Z., and K.B.l. NxC:
S
pl
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d C A 40. Jun, G. et al. Comprehensive search for Alzheimer disease susceptibility loci in
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grant from the Netherlands Ministry of Health, Welfare and Sports, Directorate of Long-
Term Care. The authors are grateful to all LASA participants, the fieldwork team and all
researchers for their ongoing commitment to the study. This work was supported by a
grant (European Alzheimer DNA BioBank, EADB) from the EU Joint Program—Neu-
rodegenerative Disease Research (JPND) and also funded by Inserm, Institut Pasteur de
Lille, the Lille Métropole Communauté Urbaine, the French government’s LABEX
DISTALZ program (development of innovative strategies for a transdisciplinary
approach to AD). Genotyping of the German case-control samples was performed in the
context of EADB (European Alzheimer DNA biobank) funded by the JPco-fuND
(German Federal Ministry of Education and Research, BMBF: 01ED1619A). Full
acknowledgments for the studies that contributed data can be found in the Supple-
mentary Note. We thank the numerous participants, researchers, and staff from many
studies who collected and contributed to the data. We thank the International Genomics
of Alzheimer’s Project (IGAP) for providing summary results data for these analyses. The
investigators within IGAP contributed to the design and implementation of IGAP and/or
provided data but did not participate in analysis or writing of this report. IGAP was made
possible by the generous participation of the control subjects, the patients, and their
families. The i–Select chips was funded by the French National Foundation on AD and
related disorders. EADI was supported by the LABEX (laboratory of excellence program
investment for the future) DISTALZ grant, Inserm, Institut Pasteur de Lille, Université
de Lille 2 and the Lille University Hospital. GERAD was supported by the Medical
Research Council (Grant n° 503480), Alzheimer’s Research UK (Grant n° 503176), the
Wellcome Trust (Grant n° 082604/2/07/Z) and German Federal Ministry of Education
and Research (BMBF): Competence Network Dementia (CND) grant n° 01GI0102,
01GI0711, 01GI0420. CHARGE was partly supported by the NIA/NHLBI grants
AG049505, AG058589, HL105756 and AGES contract N01–AG–12100, the Icelandic
Heart Association, and the Erasmus Medical Center and Erasmus University. ADGC was
supported by the NIH/NIA grants: U01 AG032984, U24 AG021886, U01 AG016976, and
the Alzheimer’s Association grant ADGC–10–196728. This research has been conducted
using the UK Biobank public resource obtained through the University of Edinburg Data
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404–413 (2019). Author contributions A novel Alzheimer disease locus located near the gene encoding
tau protein. Mol. Psychiatry 21, 108–117 (2016). Acknowledgements Some control samples and data from patients included in this study were
provided in part by the National DNA Bank Carlos III (www.bancoadn.org, University of
Salamanca, Spain) and Hospital Universitario Virgen de Valme (Sevilla, Spain); they
were processed following standard operating procedures with the appropriate approval of
the Ethical and Scientific Committee. Amsterdam dementia Cohort (ADC): Research of
the Alzheimer center Amsterdam is part of the neurodegeneration research program of
Amsterdam Neuroscience. The Alzheimer Center Amsterdam is supported by Stichting
Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was
developed with funding from Stichting Dioraphte. Genotyping of the Dutch case-control
samples was performed in the context of EADB (European Alzheimer DNA biobank)
funded by the JPco-fuND FP-829-029 (ZonMW project number 733051061). 100-Plus
study: We are grateful for the collaborative efforts of all participating centenarians and
their family members and/or relations. This work was supported by Stichting Alzheimer
Nederland (WE09.2014-03), Stichting Diorapthe, horstingstuit foundation, Memorabel
(ZonMW project number 733050814, 733050512) and Stichting VUmc Fonds. Geno-
typing of the 100-Plus Study was performed in the context of EADB (European Alz-
heimer DNA biobank) funded by the JPco-fuND FP-829-029 (ZonMW project number H., T.K., M.E.w., O.A.S., R.N.K., J.Wilf, P.F., P.R., P.Sc, P.Sa, N.Sch, D.W., E.R., G.R., H. Author contributions 39. Lloyd-Jones, L. R., Robinson, M. R., Yang, J. & Visscher, P. M. Transformation
of summary statistics from linear mixed model association on all-or-none
traits to odds ratio. Genetics 208, 1397–1408 (2018). A.Ru and S.v.d.L. designed and conceptualized the study, interpreted the data and drafted
the paper. I.d.R. contributed to data acquisition, the analysis, interpreted the data, and
co-wrote the paper. S.M.G. and N.T. contributed to the analysis and interpreted the data. S.J.v.d.L. and I.d.R. performed polygenic score analyses. L.C.C. and J.C. conducted the
functional analysis of APP. H.Ho, W.v.d.F., S.J.v.d.L., and A.Ru supervised the study. All
authors critically revised the paper for important intellectual content and approved the
final paper. GR@ACE/DEGESCO: Study design or conception: M.Me, J.C., and A.Ru. Data generation: L.M., L.C.C., A.G.P., M.E.S., S.M.G., I.d.R., I.Q., and A.C. Sample
contribution: M.Bo, J.M.G.A., M.Me, M.Ma, M.C., T.S., S.G.M., G.G.R., A.L.M., J.M., L. M.R., G.P.R., M.M.G., C.M.R., I.R.A., V.A.l., C.L., E.R.R., P.S.J., D.A.l., P.P., M.D.F., I.A.l.,
J.P.T., A.C., L.Ta, A.M.M., M.J.B., A.F.G., I.Q., I.H., L.M., P.G.G., E.A.M., S.V., O.S.G., A. Bena, A.P.C., A.E., A.Sa, C.Ab, G.O., M.R.R., M.A.l., N.R., S.G., A.O., A.Rab, A.Bel, F.Mo,
M.Z., A.C.G., J.A.P., M.F.F., E.F.M., D.B.R., M.B.S.A., P.M., R.H.V., A.A.P., A.A., L.M.P.,
R.S.V., E.Ge, A.L., R.B., J.F., J.L.R., S.Men, M.Ba, I.d.R., and S.M.G. Analysis: S.M.G. and
I.d.R. Study supervision/management: A.C., L.Ta, M.Bo, M.Me, J.C., and A.Ru. IGAP. Critical revision: A.C.N., B.W.K., L.A.F., J.L.H., L.S.W., M.A.P.V., R.May, M.A.I., J.C.B.,
A.L.D.S., C.L.S., E.B., M.F., Q.Y., X.J., R.S., C.H., K.M., S.Mea, V.E.P., A.Meg, P.A.H., R. Mar, P.A., G.Sc, J.Will, and S.Se. EADB: Sample contribution. N.T., I.E.J., N.St, K.A.M.,
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I.G., J.K., L.Fa, L.Fr, A.M.H., J.V., L.H., G.H., N.Sca, M.H.K., M.Y., H.Ho, W.M.v.d..F, M. H.u.l., N.M.v.S., A.T.H., B.G.N., C.V.B., E.S., R.V., S.E., T.N., F.K., J.V.D., V.G., A.U., A. Benu, A.K.S., B.B., C.Mas, C.F., E.C., F.Ma, G.B., I.Ap, J.Q.T., L.Ki, L.K.l., L.P., L.Tr, L.B.,
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(see Supplementary Methods and Supplementary Data 15–18). FUMA is an online
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and genes using information from 18 biological data repositories and tools. As
input, we used the summary statistics of our meta-GWAS. Gene prioritization is
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Identify Individuals With Risk Equivalent to Monogenic Mutations. Nat. Genet. 50, 1219–1224 (2018). NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunicatio Author contributions W., M.S., O.Q., O.L., P.C., P.D., R.C., S.So, S.He, S.A., S.B., S.C., T.L., V.B., V.D., P.G.K.,
M.M.N., M.C.D.N., O.P., W.M., A.W.L., I.Ap, C.B.F., A.A.L.K., G.B., M.Sca, M.Sp, M.V.,
M.Hi, K.F., L.W., M.D., P.H., and A.Ra. Analysis: N.J.A., R.M.T., V.An, N.T., I.E.J., N.St,
S.J.v.d.L., I.d.R., S.M.G., B.G.B., and C.B. Studies supervision/management: S.H.H., K.A. M., C.D., G.N., G.C., G.S., K.S., M.I., M.K., R.F.S., D.G., J.S.V., D.R., E.Gr, H.Ha, I.G., J.K.,
L.Fr, A.M.H., J.V., L.H., G.H., N.Sca, M.H.K., M.Y., H.Ho, W.M.v.d.F., M.Hui, N.M.v.S.,
N.J.A., J.D., M.Sche, A.K.S., C.G., N.B., O.H., P.B., A.H., T.K., J.Wilf, P.F., P.R., P.Sa, P.Sc,
M.Hul, N.T., I.E.J., A.T.H., B.G.N., C.V.B., E.S., R.V., S.E., P.A., A.Ru, and J.C.L. PGC-
ALZ: Sample contribution. I.E.J., A.Ro, I.Sa, D.Aa, G.Se, S.B.S., S.D., D.P., S.H., I.K.K., N. L.P., C.A.R., and O.A.A. Analysis: S.Ha, I.K.K., and I.E.J. Study supervision/management:
O.A.A., C.M.v.D., and D.P. AD and GBCS: Sample contribution. H.Z., S.K., I.S., and K.B. Analysis: N.M.S. and A.Z. Study supervision/management: I.S.k., A.Z., and K.B.l. NxC:
Sample contribution. A.C.A., M.T.M., M.S.R., and C.An. A.Ru and S.v.d.L. designed and conceptualized the study, interpreted the data and drafted
the paper. I.d.R. contributed to data acquisition, the analysis, interpreted the data, and
co-wrote the paper. S.M.G. and N.T. contributed to the analysis and interpreted the data. S.J.v.d.L. and I.d.R. performed polygenic score analyses. L.C.C. and J.C. conducted the
functional analysis of APP. H.Ho, W.v.d.F., S.J.v.d.L., and A.Ru supervised the study. All
authors critically revised the paper for important intellectual content and approved the
final paper. GR@ACE/DEGESCO: Study design or conception: M.Me, J.C., and A.Ru. Data generation: L.M., L.C.C., A.G.P., M.E.S., S.M.G., I.d.R., I.Q., and A.C. Sample
contribution: M.Bo, J.M.G.A., M.Me, M.Ma, M.C., T.S., S.G.M., G.G.R., A.L.M., J.M., L. M.R., G.P.R., M.M.G., C.M.R., I.R.A., V.A.l., C.L., E.R.R., P.S.J., D.A.l., P.P., M.D.F., I.A.l.,
J.P.T., A.C., L.Ta, A.M.M., M.J.B., A.F.G., I.Q., I.H., L.M., P.G.G., E.A.M., S.V., O.S.G., A. Bena, A.P.C., A.E., A.Sa, C.Ab, G.O., M.R.R., M.A.l., N.R., S.G., A.O., A.Rab, A.Bel, F.Mo,
M.Z., A.C.G., J.A.P., M.F.F., E.F.M., D.B.R., M.B.S.A., P.M., R.H.V., A.A.P., A.A., L.M.P.,
R.S.V., E.Ge, A.L., R.B., J.F., J.L.R., S.Men, M.Ba, I.d.R., and S.M.G. Analysis: S.M.G. and
I.d.R. Study supervision/management: A.C., L.Ta, M.Bo, M.Me, J.C., and A.Ru. IGAP. Critical revision: A.C.N., B.W.K., L.A.F., J.L.H., L.S.W., M.A.P.V., R.May, M.A.I., J.C.B.,
A.L.D.S., C.L.S., E.B., M.F., Q.Y., X.J., R.S., C.H., K.M., S.Mea, V.E.P., A.Meg, P.A.H., R. Mar, P.A., G.Sc, J.Will, and S.Se. EADB: Sample contribution. © The Author(s) 2021 Additional information Open Access This article is licensed under a Creative Commons Attri-
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1,2,306, Sonia Moreno-Grau1,2,306, Niccolo Tesi3,4,5,306, Benjamin Grenier-Boley6,306,
Victor Andrade7,8,306, Iris E. Jansen3,9,306, Nancy L. Pedersen10, Najada Stringa
11, Anna Zettergren
12,
Isabel Hernández1,2, Laura Montrreal1, Carmen Antúnez13, Anna Antonell14, Rick M. Tankard
15,
Joshua C. Bis16, Rebecca Sims
17, Céline Bellenguez
6, Inés Quintela18, Antonio González-Perez19,
Miguel Calero
2,20,21, Emilio Franco-Macías22, Juan Macías23, Rafael Blesa2,24, Laura Cervera-Carles
2,24,
Manuel Menéndez-González
25,26,27, Ana Frank-García2,28,29,30, Jose Luís Royo31, Fermin Moreno2,32,33,
Raquel Huerto Vilas34,35, Miquel Baquero36, Mónica Diez-Fairen
37,38, Carmen Lage
2,39,
Sebastián García-Madrona40, Pablo García-González
1, Emilio Alarcón-Martín1,31, Sergi Valero1,2,
Oscar Sotolongo-Grau1, Abbe Ullgren41,42, Adam C. Naj43,44, Afina W. Lemstra3, Alba Benaque1,
Alba Pérez-Cordón
1, Alberto Benussi
45, Alberto Rábano2,21,46, Alessandro Padovani45,
Alessio Squassina
47, Alexandre de Mendonça48, Alfonso Arias Pastor34,35, Almar A. L. Kok11,49,
Alun Meggy50, Ana Belén Pastor
21,46, Ana Espinosa1,2, Anaïs Corma-Gómez23, Angel Martín Montes
2,29,51,
Ángela Sanabria1,2, Anita L. DeStefano52,53, Anja Schneider8,54, Annakaisa Haapasalo
55,
Anne Kinhult Ståhlbom41,42, Anne Tybjærg-Hansen56,57, Annette M. Hartmann58, Annika Spottke54,59,
Arturo Corbatón-Anchuelo60,61, Arvid Rongve
62,63, Barbara Borroni45, Beatrice Arosio64,65,
Benedetta Nacmias
66,67, Børge G. Nordestgaard
57,68, Brian W. Kunkle69,70, Camille Charbonnier71,
Carla Abdelnour1,2, Carlo Masullo72, Carmen Martínez Rodríguez26,73, Carmen Muñoz-Fernandez74,
Carole Dufouil75,76, Caroline Graff41,42, Catarina B. Competing interests
h
h
d
l p
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The authors declare no competing interests. The authors declare no competing inte 9 TURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications Additional information Ferreira77, Caterina Chillotti78, Chandra A. Reynolds
79,
Chiara Fenoglio80, Christine Van Broeckhoven
81,82,83, Christopher Clark
84, Claudia Pisanu
47,
Claudia L. Satizabal
52,85,86, Clive Holmes87, Dolores Buiza-Rueda2,88, Dag Aarsland
89,90, Dan Rujescu58,
Daniel Alcolea
2,24, Daniela Galimberti
80,91, David Wallon
92, Davide Seripa93, Edna Grünblatt
94,95,96,
Efthimios Dardiotis97, Emrah Düzel98,99, Elio Scarpini80,91, Elisa Conti100, Elisa Rubino101, Ellen Gelpi102,103,
Eloy Rodriguez-Rodriguez
2,39, Emmanuelle Duron104,105,106, Eric Boerwinkle107,108, Evelyn Ferri65,
Fabrizio Tagliavini109, Fahri Küçükali
81,82,83, Florence Pasquier
110,111, Florentino Sanchez-Garcia112,
Francesca Mangialasche113, Frank Jessen54,114,115, Gaël Nicolas
73, Geir Selbæk
116,117,118, Gemma Ortega1,2,
Geneviève Chêne75,76, Georgios Hadjigeorgiou119, Giacomina Rossi109, Gianfranco Spalletta120,121,
Giorgio Giaccone109, Giulia Grande122, Giuliano Binetti123,124, Goran Papenberg122, Harald Hampel125,
Henri Bailly106,126, Henrik Zetterberg
127,128,129,130, Hilkka Soininen
131,132, Ida K. Karlsson10,133,
Ignacio Alvarez
37,38, Ildebrando Appollonio100,134, Ina Giegling58, Ingmar Skoog12, Ingvild Saltvedt
135,136,
Innocenzo Rainero137, Irene Rosas Allende26,138, Jakub Hort139,140, Janine Diehl-Schmid141,
Jasper Van Dongen
81,82, Jean-Sebastien Vidal
106,126, Jenni Lehtisalo131,142, Jens Wiltfang143,144,145, Itziar de Rojas
1,2,306, Sonia Moreno-Grau1,2,306, Niccolo Tesi3,4,5,306, Benjamin Grenier-Boley6,306,
Victor Andrade7,8,306, Iris E. Jansen3,9,306, Nancy L. Pedersen10, Najada Stringa
11, Anna Zettergren
12,
Isabel Hernández1,2, Laura Montrreal1, Carmen Antúnez13, Anna Antonell14, Rick M. Tankard
15,
Joshua C. Bis16, Rebecca Sims
17, Céline Bellenguez
6, Inés Quintela18, Antonio González-Perez19,
Miguel Calero
2,20,21, Emilio Franco-Macías22, Juan Macías23, Rafael Blesa2,24, Laura Cervera-Carles
2,24,
Manuel Menéndez-González
25,26,27, Ana Frank-García2,28,29,30, Jose Luís Royo31, Fermin Moreno2,32,33,
Raquel Huerto Vilas34,35, Miquel Baquero36, Mónica Diez-Fairen
37,38, Carmen Lage
2,39,
Sebastián García-Madrona40, Pablo García-González
1, Emilio Alarcón-Martín1,31, Sergi Valero1,2,
Oscar Sotolongo-Grau1, Abbe Ullgren41,42, Adam C. Naj43,44, Afina W. Lemstra3, Alba Benaque1,
Alba Pérez-Cordón
1, Alberto Benussi
45, Alberto Rábano2,21,46, Alessandro Padovani45,
Alessio Squassina
47, Alexandre de Mendonça48, Alfonso Arias Pastor34,35, Almar A. L. Kok11,49,
Alun Meggy50, Ana Belén Pastor
21,46, Ana Espinosa1,2, Anaïs Corma-Gómez23, Angel Martín Montes
2,29,51,
Ángela Sanabria1,2, Anita L. DeStefano52,53, Anja Schneider8,54, Annakaisa Haapasalo
55,
Anne Kinhult Ståhlbom41,42, Anne Tybjærg-Hansen56,57, Annette M. Hartmann58, Annika Spottke54,59,
Arturo Corbatón-Anchuelo60,61, Arvid Rongve
62,63, Barbara Borroni45, Beatrice Arosio64,65,
Benedetta Nacmias
66,67, Børge G. Nordestgaard
57,68, Brian W. Kunkle69,70, Camille Charbonnier71,
Carla Abdelnour1,2, Carlo Masullo72, Carmen Martínez Rodríguez26,73, Carmen Muñoz-Fernandez74,
Carole Dufouil75,76, Caroline Graff41,42, Catarina B. Ferreira77, Caterina Chillotti78, Chandra A. Reynolds
79,
Chiara Fenoglio80, Christine Van Broeckhoven
81,82,83, Christopher Clark
84, Claudia Pisanu
47,
Claudia L. 1Research Center and Memory clinic Fundació ACE, Institut Català de Neurociències Aplicades, Universitat Internacional de Catalunya, Barcelona,
Spain. 2CIBERNED, Network Center for Biomedical Research in Neurodegenerative Diseases, National Institute of Health Carlos III, Madrid, Spain.
3Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC,
Amsterdam, The Netherlands. 4Section Genomics of Neurodegenerative Diseases and Aging, Department of Clinical Genetics, Vrije Universiteit
Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands. 5Delft Bioinformatics Lab, Delft Univeristy of Technology, Delft, The Netherlands.
6Univ. Lille, Inserm, Institut Pasteur de Lille, CHU Lille, U1167-Labex DISTALZ-RID-AGE-Risk Factors and Molecular Determinants of Aging-Related
Diseases, Lille, France. 7Division of Neurogenetics and Molecular Psychiatry, Department of Psychiatry and Psychotherapy, University of Cologne,
Medical Faculty, Cologne, Germany. 8Department of Neurodegenerative diseases and Geriatric Psychiatry, University Clinic Bonn, Bonn, Germany.
9Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University,
Amsterdam, The Netherlands. 10Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 11Amsterdam NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications ARTICLE ARTICLE Additional information Satizabal
52,85,86, Clive Holmes87, Dolores Buiza-Rueda2,88, Dag Aarsland
89,90, Dan Rujescu58,
Daniel Alcolea
2,24, Daniela Galimberti
80,91, David Wallon
92, Davide Seripa93, Edna Grünblatt
94,95,96,
Efthimios Dardiotis97, Emrah Düzel98,99, Elio Scarpini80,91, Elisa Conti100, Elisa Rubino101, Ellen Gelpi102,103,
Eloy Rodriguez-Rodriguez
2,39, Emmanuelle Duron104,105,106, Eric Boerwinkle107,108, Evelyn Ferri65,
Fabrizio Tagliavini109, Fahri Küçükali
81,82,83, Florence Pasquier
110,111, Florentino Sanchez-Garcia112,
Francesca Mangialasche113, Frank Jessen54,114,115, Gaël Nicolas
73, Geir Selbæk
116,117,118, Gemma Ortega1,2,
Geneviève Chêne75,76, Georgios Hadjigeorgiou119, Giacomina Rossi109, Gianfranco Spalletta120,121,
Giorgio Giaccone109, Giulia Grande122, Giuliano Binetti123,124, Goran Papenberg122, Harald Hampel125,
Henri Bailly106,126, Henrik Zetterberg
127,128,129,130, Hilkka Soininen
131,132, Ida K. Karlsson10,133,
Ignacio Alvarez
37,38, Ildebrando Appollonio100,134, Ina Giegling58, Ingmar Skoog12, Ingvild Saltvedt
135,136,
Innocenzo Rainero137, Irene Rosas Allende26,138, Jakub Hort139,140, Janine Diehl-Schmid141,
Jasper Van Dongen
81,82, Jean-Sebastien Vidal
106,126, Jenni Lehtisalo131,142, Jens Wiltfang143,144,145, NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Margaret A. Pericak-Vance
70, Margaret Esiri165, María Bernal Sánchez-Arjona22, Maria Carolina Dalmasso7 TURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 11 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 74Servicio de Neurología, Hospital Universitario de Gran Canaria Dr.Negrín, Las Palmas, Spain. 75Inserm, Bordeaux Population Health Research
Center, UMR 1219, Univ. Bordeaux, ISPED, CIC 1401-EC, Univ Bordeaux, Bordeaux, France. 76CHU de Bordeaux, Pole de Santé Publique, Bordeaux,
France. 77Instituto de Medicina Molecular João lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. 78Unit of Clinical
Pharmacology, University Hospital of Cagliari, Cagliari, Italy. 79Department of Psychology, University of California—Riverside, Riverside, CA, USA. 80University of Milan, Dino Ferrari Center, Milan, Italy. 81VIB Center for Molecular Neurology, Antwerp, Belgium. 82Laboratory of Neurogenetics,
Institute Born-Bunge, Antwerp, Belgium. 83Department of Biomedical Sciences, University of Antwerp., Antwerp, Belgium. 84Insititute for
Regenerative Medicine, University of Zürich, Zürich, Switzerland. 85Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, San
Antonio, TX, USA. 86Department of Population Health Sciences, UT Health San Antonio, San Antonio, TX, USA. 87Division of Clinical
Neurosciences, School of Medicine, University of Southampton, Southampton, UK. 88Unidad de Trastornos del Movimiento, Servicio de Neurología
y Neurofisiología, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain. 89Department of Old Age Psychiatry, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK. 90Centre of Age-
Related Medicine, Stavanger University Hospital, Stavanger, Norway. 91Fondazione IRCCS Ca’ Granda, Ospedale Policlinico, Milan, Italy. 92Normandie Univ, UNIROUEN, Inserm U1245, CHU Rouen, Department of Neurology and CNR-MAJ, FHU G4 Génomique, F-76000 Rouen,
France. 93Complex Structure of Geriatrics, Department of Medical Sciences Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni
Rotondo (FG), Italy. 94Department of Child and Adolescent Psychiatry and Psychotherapy, Psychiatric University Hospital Zurich (PUK),
University of Zurich, Zurich, Switzerland. 95Neuroscience Center Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland. 96Zurich Center
for Integrative Human Physiology, University of Zurich, Zurich, Switzerland. 97School of Medicine, University of Thessaly, Larissa, Greece. 98German Center for Neurodegenerative Diseases (DZNE) Magdeburg Germany 99Institute of Cognitive Neurology and Dementia Research UMC-Vrije Universiteit Amsterdam, Department of Epidemiology and Data Science, Amsterdam Public Health Research Institute, Amsterdam,
The Netherlands. 12Neuropsychiatric Epidemiology Unit, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology,
Sahlgrenska Academy, Centre for Ageing and Health (AgeCap), University of Gothenburg, Gothenburg, Sweden. 13Unidad de Demencias, Hospital
Clínico Universitario Virgen de la Arrixaca, Murcia, Spain. 14Alzheimer’s disease and other cognitive disorders unit. Service of Neurology, Hospital
Clínic of Barcelona. Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain. 15Mathematics and
Statistics, Murdoch University, Perth, WA, Australia. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 UMC-Vrije Universiteit Amsterdam, Department of Epidemiology and Data Science, Amsterdam Public Health Research Institute, Amsterdam,
The Netherlands. 12Neuropsychiatric Epidemiology Unit, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology,
Sahlgrenska Academy, Centre for Ageing and Health (AgeCap), University of Gothenburg, Gothenburg, Sweden. 13Unidad de Demencias, Hospital
Clínico Universitario Virgen de la Arrixaca, Murcia, Spain. 14Alzheimer’s disease and other cognitive disorders unit. Service of Neurology, Hospital
Clínic of Barcelona. Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain. 15Mathematics and
Statistics, Murdoch University, Perth, WA, Australia. 16Cardiovascular Health Research Unit, Department of Medicine, University of Washington,
Seattle, WA, USA. 17Division of Psychological Medicine and Clinial Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics,
Cardiff University, Cardiff, UK. 18Grupo de Medicina Xenómica, Centro Nacional de Genotipado (CEGEN-PRB3-ISCIII), Universidade de Santiago de
Compostela, Santiago de Compostela, Spain. 19CAEBI, Centro Andaluz de Estudios Bioinformáticos, Sevilla, Spain. 20UFIEC, Instituto de Salud
Carlos III, Madrid, Spain. 21CIEN Foundation/Queen Sofia Foundation Alzheimer Center, Madrid, Spain. 22Unidad de Demencias, Servicio de
Neurología y Neurofisiología, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla,
Spain. 23Unidad Clínica de Enfermedades Infecciosas y Microbiología, Hospital Universitario de Valme, Sevilla, Spain. 24Department of Neurology,
II B Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain. 25Servicio de Neurología, Hospital
Universitario Central de Asturias, Oviedo, Spain. 26Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain. 27Departamento de Medicina, Universidad de Oviedo, Oviedo, Spain. 28Department of Neurology, La Paz University Hospital, Instituto de
Investigación Sanitaria del Hospital Universitario La Paz, IdiPAZ, Madrid, Spain. 29Hospital La Paz Institute for Health Research, IdiPAZ, Madrid,
Spain. 30Universidad Autónoma de Madrid, Madrid, Spain. 31Departamento de Especialidades Quirúrgicas, Bioquímicas e Inmunología, School of
Medicine, University of Málaga, Málaga, Spain. 32Department of Neurology, Hospital Universitario Donostia, San Sebastian, Spain. 33Neurosciences
Area, Instituto Biodonostia, San Sebastian, Spain. 34Unitat Trastorns Cognitius, Hospital Universitari Santa Maria de Lleida, Lleida, Spain. 35Institut
de Recerca Biomedica de Lleida (IRBLLeida), Lleida, Spain. 36Servei de Neurologia, Hospital Universitari i Politècnic La Fe, Valencia, Spain. 37Fundació Docència i Recerca MútuaTerrassa, Terrassa, Barcelona, Spain. 38Memory Disorders Unit, Department of Neurology, Hospital
Universitari Mutua de Terrassa, Terrassa, Barcelona, Spain. 39Neurology Service, Marqués de Valdecilla University Hospital (University of
Cantabria and IDIVAL), Santander, Spain. 40Hospital Universitario Ramon y Cajal, IRYCIS, Madrid, Spain. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 41Karolinska Institutet, Center for
Alzheimer Research, Department NVS, Division of Neurogeriatrics, Stockholm, Sweden. 42Unit for Hereditary Dementias, Theme Aging, Karolinska
University Hospital-Solna, Stockholm, Sweden. 43Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman
School of Medicine, Philadelphia, PA, USA. 44Penn Neurodegeneration Genomics Center, Department of Pathology and Laboratory Medicine,
University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. 45Centre for Neurodegenerative Disorders, Department of Clinical
and Experimental Sciences, University of Brescia, Brescia, Italy. 46BT-CIEN, Madrid, Spain. 47Department of Biomedical Sciences, Section of
Neuroscience and Clinical Pharmacology, University of Cagliari, Cagliari, Italy. 48Faculty of Medicine, University of Lisbon, Lisbon, Portugal. 49Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Psychiatry, Amsterdam Public Health Research Institute, Amsterdam, The
Netherlands. 50UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK. 51Department of Neurology, La Paz University Hospital,
Madrid, Spain. 52Department of Neurology, Boston University School of Medicine, Boston, MA, USA. 53Department of Biostatistics, Boston
University School of Public Health, Boston, MA, USA. 54German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 55A.I Virtanen
Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. 56Department of Clinical Biochemistry, Rigshospitalet, Copenhagen,
Denmark. 57Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 58Martin-Luther-University Halle-Wittenberg, University Clinic and Outpatient Clinic for Psychiatry, Psychotherapy and Psychosomatics, Halle
(Saale), Germany. 59Department of Neurology, University of Bonn, Bonn, Germany. 60Instituto de Investigación Sanitaria, Hospital Clínico San
Carlos (IdISSC), Madrid, Spain. 61Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid,
Spain. 62Haugesund Hospital, Helse Fonna, Department of Research and Innovation, Haugesund, Norway. 63University of Bergen, Institute of
Clinical Medicine (K1), Bergen, Norway. 64Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. 65Geriatic Unit,
Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy. 66Department of Neuroscience, Psychology, Drug Research and Child
Health University of Florence, Florence, Italy. 67IRCCS Fondazione Don Carlo Gnocchi, Florence, Italy. 68Department of Clinical Biochemistry,
Herlev Gentofte Hospital, Herlev, Denmark. 69Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller
School of Medicine, Miami, FL, USA. 70John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL,
USA. 71Normandie Univ, UNIROUEN, Inserm U1245, CHU Rouen, Department of Genetics and CNR-MAJ, FHU G4 Génomique, F-76000 Rouen,
France. 72Institute of Neurology, Catholic University of the Sacred Heart, School of Medicine, Milan, Italy. 73Hospital de Cabueñes, Gijón, Spain. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 16Cardiovascular Health Research Unit, Department of Medicine, University of Washington,
Seattle, WA, USA. 17Division of Psychological Medicine and Clinial Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics,
Cardiff University, Cardiff, UK. 18Grupo de Medicina Xenómica, Centro Nacional de Genotipado (CEGEN-PRB3-ISCIII), Universidade de Santiago de
Compostela, Santiago de Compostela, Spain. 19CAEBI, Centro Andaluz de Estudios Bioinformáticos, Sevilla, Spain. 20UFIEC, Instituto de Salud
Carlos III, Madrid, Spain. 21CIEN Foundation/Queen Sofia Foundation Alzheimer Center, Madrid, Spain. 22Unidad de Demencias, Servicio de
Neurología y Neurofisiología, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla,
Spain. 23Unidad Clínica de Enfermedades Infecciosas y Microbiología, Hospital Universitario de Valme, Sevilla, Spain. 24Department of Neurology,
II B Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain. 25Servicio de Neurología, Hospital
Universitario Central de Asturias, Oviedo, Spain. 26Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain. 27
28 12 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Paris, France. 106APHP, Hôpital Broca, Paris, France. 107School of Public Health, Human Genetics Center, University of Texas Health Science Center
at Houston, Houston, TX, USA. 108Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA. 109Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milan, Italy. 110Inserm U1172, CHU, DISTAlz, LiCEND, Univ Lille, Lille, France. 111CHU CNR-MAJ, Lille, France. 112S
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113Di i i
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i t i Paris, France. APHP, Hôpital Broca, Paris, France. School of Public Health, Human Genetics Center, University of Texas Health Science Center
at Houston, Houston, TX, USA. 108Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA. 109Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milan, Italy. 110Inserm U1172, CHU, DISTAlz, LiCEND, Univ Lille, Lille, France. 111CHU CNR-MAJ, Lille, France. 112Servicio de Inmunología, Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Spain. 113Division of Clinical Geriatrics,
Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Stockholm, Sweden. 114Department of Psychiatry and Psychotherapy, University of Cologne, Medical Faculty, Cologne, Germany. 115Excellence Cluster on Cellular Stress
Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. 116Norwegian National Advisory Unit on Ageing and
Health, Vestfold Hospital Trust, Tønsberg, Norway. 117Department of Geriatric Medicine, Oslo University Hospital, Oslo, Norway. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 118Institute of
Clinical Medicine, University of Oslo, Oslo, Norway. 119Department of Neurology, Medical School, University of Cyprus, Nicosia, Cyprus. 120Laboratory of Neuropsychiatry, IRCCS Santa Lucia Foundation, Rome, Italy. 121Beth K. and Stuart C. Yudofsky Division of Neuropsychiatry,
Department of Psychiatry and Behavioral Sciences, Baylor College of Medicine, Houston, TX, USA. 122Aging Research Center, Department of
Neurobiology, Care Sciences and Society, Karolinska Institutet and Stockholm University, Stockholm, Sweden. 123MAC—Memory Clinic, IRCCS
Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy. 124Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio
Fatebenefratelli, Brescia, Italy. 125Sorbonne University, GRC n° 21, Alzheimer Precision Medicine (APM), AP-HP, Pitié-Salpêtrière Hospital, Paris,
France. 126EA 4468, Sorbonne Paris Cité, Université Paris Descartes, Paris, France. 127Clinical Neurochemistry Laboratory, Sahlgrenska University
Hospital, Mölndal, Sweden. 128Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy at
the University of Gothenburg, Gothenburg, Sweden. 129Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK. 130UK
Dementia Research Institute at UCL, London, UK. 131Institute of Clinical Medicine Neurology, University of Eastern Finland, Kuopio, Finland. 132Neurocenter, neurology, Kuopio University Hospital, Kuopio, Finland. 133Institute for Gerontology and Aging Research Network—Jönköping
(ARN-J), School of Health and Welfare, Jönköping University, Jönköping, Sweden. 134Neurology Unit, ‘San Gerardo’ hospital, Monza, Italy. 135Department of Geriatrics, Clinic of Medicine, St Olavs Hospital, University Hospital of Trondheim, Trondheim, Norway. 136Department of
Neuromedicine and Movement Science, Norwegian University of Science and Technhology (NTNU), Trondheim, Norway. 137Department of
Neuroscience “Rita Levi Montalcini”, University of Torino, Torino, Italy. 138Laboratorio de Genética, Hospital Universitario Central de Asturias,
Oviedo, Spain. 139Memory Clinic, Department of Neurology, 2nd Faculty of Medicine and Motol University Hospital, Charles University, Prague,
Czech Republic. 140International Clinical Research Center, St. Anne’s University Hospital Brno, Brno, Czech Republic. 141Department of Psychiatry
and Psychotherapy, School of Medicine Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. 142Population Health Unit,
Finnish Institute for Health and Welfare, Helsinki, Finland. 143Department of Psychiatry and Psychotherapy, University Medical Center Goettingen,
Goettingen, Germany. 144German Center for Neurodegenerative Diseases (DZNE), Goettingen, Germany. 145Neurosciences and Signaling Group,
Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Aveiro, Portugal. 146Department of Psychiatry and
Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany. 147Department of
Population & Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH, USA. 148Cleveland Institute for Computational
Biology, Case Western Reserve University, Cleveland, OH, USA. 149Translational Neuroscience Laboratory, McLean Hospital, Harvard Medical
School, Belmont, MA, USA. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 150Department of Geriatric Psychiatry, University Hospital of Psychiatry Zürich, Zürich, Switzerland. 151University of
Zürich, Zürich, Switzerland. 152Old age Psychiatry, University Hospital of Lausanne, Lausanne, Switzerland. 153Department of Psychiatry,
Psychosomatics and Psychotherapy, Center of Mental Health, University Hospital, Wuerzburg, Germany. 154Institute for Stroke and Dementia
Research, Klinikum der Universität München, Ludwig-Maximilians-Universität LMU, Munich, Germany. 155German Center for Neurodegenerative
Diseases (DZNE), Munich, Germany. 156Schools of Life Sciences and Medicine, University of Nottingham, Nottingham, UK. 157Department of
Public Health and Caring Sciences/Geriatrics, Uppsala, Sweden. 158Institute of Medical Biometry, Informatics and Epidemiology, University
Hospital of Bonn, Bonn, Germany. 159Departments of Medicine (Biomedical Genetics), Neurology, Ophthalmology, Epidemiology, and Biostatistics,
Boston University Schools of Medicine and Public Health, Boston, MA, USA. 160Centre for Memory Disturbances, Lab of Clinical Neurochemistry,
Section of Neurology, University of Perugia, Perugia, Italy. 161Department of Geriatric Psychiatry, Central Institute for Mental Health Mannheim,
Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany. 162Department of Epidemiology, Erasmus Medical Center, Rotterdam,
The Netherlands. 1631st Department of Neurology Aristotle University of Thessaloniki, Thessaloniki, Greece. 164Azienda Ospedaliero-Universitaria,
Parma, Italy. 165Nuffield Department of Clinical Neurosciences, Oxford, UK. 166Institute of Human Genetics, University of Bonn, School of Medicine
& University Hospital Bonn, Bonn, Germany. 167Munich Cluster for Systems Neurology (SyNergy), Munich, Germany. 168Department of Primary
Medical Care, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. 169Laboratory of Cognitive Neuroscience, School of
Psychology, Aristotle University of Thessaloniki, Thessaloniki, Greece. 170Department of Nutrition and Dietetics, Harokopio University, Athens,
Greece. 171Institute of Gerontology and Geriatrics, Department of Medicine, University of Perugia, Perugia, Italy. 172Institute of Public Health and
Clinical Nutrition, University of Eastern Finland, Kuopio, Finland. 173Neuroepidemiology and Ageing Research Unit, School of Public Health, Imperial
College London, London, UK. 174Stockholms Sjukhem, Research & Development Unit, Stockholm, Sweden. 175Institute of Biomedicine, University of
Eastern Finland, Kuopio, Finland. 176Brown Foundation Institute of Molecular Medicine, University of Texas Health Sciences Center at Houston,
Houston, TX, USA. 1771st Department of Neurology, Aiginition Hospital, National and Kapodistrian University of Athens, Medical School, Athens,
Greece. 178Taub Institute for Research in Alzheimer’s Disease and the Aging Brain, The Gertrude H. Sergievsky Center, Depatment of Neurology,
Columbia University, New York, NY, USA. 179LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Medical Faculty, University of
Duisburg-Essen, Essen, Germany. 180Department of Psychiatry and Psychotherapy and Experimental and Clinical Research Center (ECRC),
Charité-Universitätsmedizin Berlin, Berlin, Germany. 181German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany. 182Bristol
Medical School (THS), University of Bristol, Southmead Hospital, Bristol, UK. NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications Paris, France. 106APHP, Hôpital Broca, Paris, France. 107School of Public Health, Human Genetics Center, University of Texas Health Science Center
at Houston, Houston, TX, USA. 108Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA. 109Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milan, Italy. 110Inserm U1172, CHU, DISTAlz, LiCEND, Univ Lille, Lille, France. 111CHU CNR-MAJ, Lille, France.
112Servicio de Inmunología, Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Spain. 113Division of Clinical Geriatrics,
Center for Alzheimer Research Department of Neurobiology Care Sciences and Society (NVS) Karolinska Institutet Stockholm Sweden NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 Trondheim, Trondheim, Norway. 202Department of Neuromedicine and Movement Science, Faculty of Medicine and Health Sciences, Norwegian
University of Science and Technology, Trondheim, Norway. 203Department of Laboratory Diagnostics, III Laboratory of Analysis, Brescia Hospital,
Brescia, Italy. 204Alzheimer Research Center & Memory Clinic, Andalusian Institute for Neuroscience, Málaga, Spain. 205MRC Prion Unit at UCL,
Institute of Prion Diseases, London, UK. 206Department of Medical Genetics, Oslo University Hospital, Oslo, Norway. 207NORMENT, Department
of Clinical Science, University of Bergen, Bergen, Norway. 208Institute of Social Medicine, Occupational Health and Public Health, University of
Leipzig, Leipzig, Germany. 209Department of Neurology/CIEN Foundation/Queen Sofia Foundation Alzheimer Center, Madrid, Spain. 210Azienda
Ospedaliero-Universitaria Careggi Largo Brambilla, Florence, Italy. 211UKDRI Cardiff, Cardiff University, Cardiff, UK. 212Unitat de Genètica Molecular,
Institut de Biomedicina de València-CSIC, Valencia, Spain. 213Unidad Mixta de Neurologia Genètica, Instituto de Investigación Sanitaria La Fe,
Valencia, Spain. 214Department of Neurosciences, Faculty of Medicine and Nursery, University of the Basque Country, San Sebastián, Spain. 215Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Madrid, Spain. 216Instituto de Investigacion Sanitaria ‘Hospital la Paz’ (IdIPaz),
Madrid, Spain. 217Departamento de Especialidades Quirúrgicas, Bioquímica e Inmunología. Facultad de Medicina, Universidad de Málaga, Málaga,
Spain. 218Fundación Pública Galega de Medicina Xenómica-CIBERER-IDIS, Santiago de Compostela, Spain. 219Framingham Heart Study,
Framingham, MA, USA. 220Nuffield Department of Population Health, University of Oxford, Oxford, UK. 221Centre for Healthy Brain Ageing
(CHeBA), School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. 222Neuroscience Research Australia,
Sydney, NSW, Australia. 223Department of Sociology, VU University, Amsterdam, The Netherlands. 224NORMENT Centre, Institute of Clinical
Medicine, University of Oslo, Oslo, Norway. 225Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway. 226Department of
Psychiatry, Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, San Antonio, TX, USA. 306These authors contributed equally:
Itziar de Rojas, Sonia Moreno-Grau, Niccolo Tesi, Benjamin Grenier-Boley, Victor Andrade, Iris E. Jansen. 307These authors jointly supervised this
work: Jordi Clarimón, Mercè Boada, Wiesje M. van der Flier, Alfredo Ramirez, Jean-Charles Lambert, Sven J. van der Lee, Agustín Ruiz. *Lists of
authors and their affiliations appears at the end of the paper. ✉email: s.j.vanderlee@amsterdamumc.nl; aruiz@fundacioace.org EADB contributors Vardy259, Fabrizio Piras120, Fausto Roveta137,
Federica Piras120, Federico Ariel Prestia245,257, Francesca Assogna120, Francesca Salani183, Gessica Sala100,
Giordano Lacidogna236, Gisela Novack257,258, Gordon Wilcock260, Håkan Thonberg41,42, Heike Kölsch261,
Heike Weber153, Henning Boecker54,262, Ignacio Etchepareborda250,251, Irene Piaceri66,
Jaakko Tuomilehto142,263,264,265, Jaana Lindström142, Jan Laczo139,140, Janet Johnston238,
Jean-François Deleuze233, Jenny Harris231, Jonathan M. Schott266, Josef Priller181,267, Juan Ignacio Bacha247,
Julie Snowden231,232, Julieta Lisso268, Kalina Yonkova Mihova269, Latchezar Traykov255, Laura Morelli257,258,
Luis Ignacio Brusco240,241, Malik Rainer253, Mari Takalo175, Maria Bjerke196,270, Maria Del Zompo47,271,
Maria Serpente80, Mariana Sanchez Abalos
272, Mario Rios229, Markku Peltonen142, Martin J. Herrman153,
Mary H. Kosmidis169, Matias Kohler250,256, Matias Rojo240, Matthew Jones231,273, Michela Orsini236,
Nancy Medel268, Natividad Olivar240, Nick C. Fox266,274, Nicola Salvadori160, Nigel M. Hooper232,
Pablo Galeano244,257,258, Patricia Solis268, Patrizia Bastiani171, Patrizia Mecocci171, Peter Passmore238,
Reinhard Heun261, Riitta Antikainen275,276,277, Robert Olaso233, Robert Perneczky167,278,279,280,
Sandra Germani240, Sara López-García2,39, Seth Love182,281, Shima Mehrabian255, Silvia Bagnoli66,
Silvia Kochen268, Simona Andreoni100, Stefan Teipel282,283, Stephen Todd238, Stuart Pickering-Brown254,
Teemu Natunen175, Thomas Tegos163, Tiina Laatikainen172,284,285, Timo Strandberg275,286,
Tuomo M. Polvikoski189,190, Vaclav Matoska287, Valentina Ciullo120, Valeria Cores244,245,252,
Vincenzo Solfrizzi239, Viviana Lisetti160 & Zulma Sevillano268 A. David Smith227,228, Aldo Saenz229, Alessandra Bizzarro230, Alessandra Lauria230, Alessandro Vacca137,
Alina Solomon113,131, Anna Anastasiou163, Anna Richardson231,232, Anne Boland233, Anne Koivisto234,235,
Antonio Daniele236, Antonio Greco237, Arnaoutoglou Marianthi163, Bernadette McGuinness238, Bertrand Fin233,
Camilla Ferrari66, Carlo Custodero239, Carlo Ferrarese100,134, Carlos Ingino240,241, Carlos Mangone240,242,
Carlos Reyes Toso240, Carmen Martínez26,243, Carolina Cuesta244,245, Carolina Muchnik240,
Catharine Joachim246, Cecilia Ortiz247, Céline Besse233, Charlotte Johansson41,42, Chiara Paola Zoia100,
Christoph Laske248,249, Costas Anastasiou170, Dana Lis Palacio250,251, Daniel G. Politis244,245,252,
Daniel Janowitz253, David Craig238, David M. Mann254, David Neary231, Deckert Jürgen153, Delphine Daian233,
Diyana Belezhanska255, Eduardo Kohler256, Eduardo M. Castaño257,258, Effrosyni Koutsouraki163, Elena Chipi160,
Ellen De Roeck196, Emanuele Costantini236, Emma R. L. C. Vardy259, Fabrizio Piras120, Fausto Roveta137,
Federica Piras120, Federico Ariel Prestia245,257, Francesca Assogna120, Francesca Salani183, Gessica Sala100,
Giordano Lacidogna236, Gisela Novack257,258, Gordon Wilcock260, Håkan Thonberg41,42, Heike Kölsch261,
Heike Weber153, Henning Boecker54,262, Ignacio Etchepareborda250,251, Irene Piaceri66,
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238 A. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 183Experimental Neuro-psychobiology Laboratory, Department of
Clinical and Behavioral Neurology, IRCCS Santa Lucia Foundation, Rome, Italy. 184Unit of Neuroscience, DIMEC, University of Parma, Parma, Italy. 185Athens Association of Alzheimer’s disease and Related Disorders, Athens, Greece. 186Institute of Medical Genetics and Pathology, University
Hospital Basel, Basel, Switzerland. 187Department of Psychiatry, Social Medicine Center East- Donauspital, Vienna, Austria. 188Center of Mental
Health, Clinic and Policlinic of Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Würzburg, Würzburg, Germany. 189Translational and Clincial Research Institute, Newcastle University, Newcastle upon Tyne, UK. 190Campus for Ageing anf Vitality, Newcastle
upon Tyne UK 191Taub Institute on Alzheimer’s Disease and the Aging Brain Department of Neurology Columbia University New York NY USA 13 EADB contributors EADB contributors
A. David Smith227,228, Aldo Saenz229, Alessandra Bizzarro230, Alessandra Lauria230, Alessandro Vacca137,
Alina Solomon113,131, Anna Anastasiou163, Anna Richardson231,232, Anne Boland233, Anne Koivisto234,235,
Antonio Daniele236, Antonio Greco237, Arnaoutoglou Marianthi163, Bernadette McGuinness238, Bertrand Fin233,
Camilla Ferrari66, Carlo Custodero239, Carlo Ferrarese100,134, Carlos Ingino240,241, Carlos Mangone240,242,
Carlos Reyes Toso240, Carmen Martínez26,243, Carolina Cuesta244,245, Carolina Muchnik240,
Catharine Joachim246, Cecilia Ortiz247, Céline Besse233, Charlotte Johansson41,42, Chiara Paola Zoia100,
Christoph Laske248,249, Costas Anastasiou170, Dana Lis Palacio250,251, Daniel G. Politis244,245,252,
Daniel Janowitz253, David Craig238, David M. Mann254, David Neary231, Deckert Jürgen153, Delphine Daian233,
Diyana Belezhanska255, Eduardo Kohler256, Eduardo M. Castaño257,258, Effrosyni Koutsouraki163, Elena Chipi160,
Ellen De Roeck196, Emanuele Costantini236, Emma R. L. C. Vardy259, Fabrizio Piras120, Fausto Roveta137,
Federica Piras120, Federico Ariel Prestia245,257, Francesca Assogna120, Francesca Salani183, Gessica Sala100,
Giordano Lacidogna236, Gisela Novack257,258, Gordon Wilcock260, Håkan Thonberg41,42, Heike Kölsch261,
Heike Weber153, Henning Boecker54,262, Ignacio Etchepareborda250,251, Irene Piaceri66,
Jaakko Tuomilehto142,263,264,265, Jaana Lindström142, Jan Laczo139,140, Janet Johnston238,
Jean-François Deleuze233, Jenny Harris231, Jonathan M. Schott266, Josef Priller181,267, Juan Ignacio Bacha247,
Julie Snowden231,232, Julieta Lisso268, Kalina Yonkova Mihova269, Latchezar Traykov255, Laura Morelli257,258,
Luis Ignacio Brusco240,241, Malik Rainer253, Mari Takalo175, Maria Bjerke196,270, Maria Del Zompo47,271,
Maria Serpente80, Mariana Sanchez Abalos
272, Mario Rios229, Markku Peltonen142, Martin J. Herrman153,
Mary H. Kosmidis169, Matias Kohler250,256, Matias Rojo240, Matthew Jones231,273, Michela Orsini236,
Nancy Medel268, Natividad Olivar240, Nick C. Fox266,274, Nicola Salvadori160, Nigel M. Hooper232,
Pablo Galeano244,257,258, Patricia Solis268, Patrizia Bastiani171, Patrizia Mecocci171, Peter Passmore238,
Reinhard Heun261, Riitta Antikainen275,276,277, Robert Olaso233, Robert Perneczky167,278,279,280,
Sandra Germani240, Sara López-García2,39, Seth Love182,281, Shima Mehrabian255, Silvia Bagnoli66,
Silvia Kochen268, Simona Andreoni100, Stefan Teipel282,283, Stephen Todd238, Stuart Pickering-Brown254,
Teemu Natunen175, Thomas Tegos163, Tiina Laatikainen172,284,285, Timo Strandberg275,286,
Tuomo M. Polvikoski189,190, Vaclav Matoska287, Valentina Ciullo120, Valeria Cores244,245,252,
Vincenzo Solfrizzi239, Viviana Lisetti160 & Zulma Sevillano268 A. David Smith227,228, Aldo Saenz229, Alessandra Bizzarro230, Alessandra Lauria230, Alessandro Vacca137,
Alina Solomon113,131, Anna Anastasiou163, Anna Richardson231,232, Anne Boland233, Anne Koivisto234,235,
Antonio Daniele236, Antonio Greco237, Arnaoutoglou Marianthi163, Bernadette McGuinness238, Bertrand Fin233,
Camilla Ferrari66, Carlo Custodero239, Carlo Ferrarese100,134, Carlos Ingino240,241, Carlos Mangone240,242,
Carlos Reyes Toso240, Carmen Martínez26,243, Carolina Cuesta244,245, Carolina Muchnik240,
Catharine Joachim246, Cecilia Ortiz247, Céline Besse233, Charlotte Johansson41,42, Chiara Paola Zoia100,
Christoph Laske248,249, Costas Anastasiou170, Dana Lis Palacio250,251, Daniel G. Politis244,245,252,
Daniel Janowitz253, David Craig238, David M. Mann254, David Neary231, Deckert Jürgen153, Delphine Daian233,
Diyana Belezhanska255, Eduardo Kohler256, Eduardo M. Castaño257,258, Effrosyni Koutsouraki163, Elena Chipi160,
Ellen De Roeck196, Emanuele Costantini236, Emma R. L. C. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 267Department of Neuropsychiatry, Charité, Berlin, Germany. 268ENYS (Estudio en Neurociencias y Sistemas Complejos) CONICET-Hospital El
Cruce ‘Nestor Kirchner’-UNAJ, Buenos Aires, Argentina. 269Molecular Medicine Center, Department of Medical chemistry and biochemistry,
Medical University of Sofia, Sofia, Bulgaria. 270Laboratory of Neurochemistry, UZ Brussel, Brussel, Belgium. 271Unit of Clinical Pharmacology,
Teaching Hospital of Cagliari, AOUCA, Cagliari, Italy. 272Programa del Adulto Mayor-Min, Salud de la Pcia. de Jujuy, San Salvador de Jujuy,
Argentina. 273Division of Neuroscience and Experimental Psychology, University of Manchester, Manchester, UK. 274UK Dementia Research
Institute at UCL, UCL Queen Square Institute of Neurology, University College London, London, UK. 275Center for Life Course Health Research,
University of Oulu, Oulu, Finland. 276Medical Research Center Oulu, Oulu University Hospital, Oulu, Finland. 277Oulu City Hospital, Oulu, Finland. 278German Center for Neurodegenerative Diseases (DZNE, Munich), Munich, Germany. 279Department of Psychiatry and Psychotherapy,
University Hospital LMU Munich, Munich, Germany. 280Ageing Epidemiology Research Unit (AGE), School of Public Health, Imperial College
London, London, UK. 281University of Bristol Institute of Clinical Neurosciences, School of Clinical Sciences, Frenchay Hospital, Bristol, UK. 282German Center for Neurodegenerative Diseases (DZNE), Rostock, Germany. 283Department of Psychosomatic Medicine, Rostock University
Medical Center, Rostock, Germany. 284Finnish Institute for Health and Welfare, Helsinki, Finland. 285Joint Municipal Authority for North Karelia
Social and Health Services (Siun Sote), Joensuu, Finland. 286Helsinki University Hospital, University of Helsinki, Helsinki, Finland. 287Department of
Clinical Biochemistry, Hematology and Immunology, Na Homolce Hospital, Prague, Czech Republic. The GR@ACE study group C. Abdelnour1,2, N. Aguilera1, E. Alarcon1,31, M. Alegret1,2, A. Benaque1, M. Boada1,2, M. Buendia1, P. Cañabate1,2,
A. Carracedo18,218, A. Corbatón-Anchuelo6, I. de Rojas1, S. Diego1, A. Espinosa1,2, A. Gailhajenet1,
P. García-González1, S. Gil1, M. Guitart1, A. González-Pérez19, I. Hernández1,2, M. Ibarria1, A. Lafuente1,
J. Macias23, O. Maroñas18, E. Martín1, M. T. Martínez62, M. Marquié1, A. Mauleón1, L. Montrreal1,
S. Moreno-Grau1,2, M. Moreno1, A. Orellana1, G. Ortega1,2, A. Pancho1, E. Pelejá1, A. Pérez-Cordon1,
J. A. Pineda23, S. Preckler1, I. Quintela31, L. M. Real8,31, M. Rosende-Roca1, A. Ruiz1,2, M. E. Sáez19, A. Sanabria1,2,
M. Serrano-Rios61, O. Sotolongo-Grau1, L. Tárraga1,2, S. Valero1,2 & L. Vargas1 EADB contributors David Smith227,228, Aldo Saenz229, Alessandra Bizzarro230, Alessandra Lauria230, Alessandro Vacca137,
Alina Solomon113,131, Anna Anastasiou163, Anna Richardson231,232, Anne Boland233, Anne Koivisto234,235,
Antonio Daniele236, Antonio Greco237, Arnaoutoglou Marianthi163, Bernadette McGuinness238, Bertrand Fin233,
Camilla Ferrari66, Carlo Custodero239, Carlo Ferrarese100,134, Carlos Ingino240,241, Carlos Mangone240,242,
Carlos Reyes Toso240, Carmen Martínez26,243, Carolina Cuesta244,245, Carolina Muchnik240,
Catharine Joachim246, Cecilia Ortiz247, Céline Besse233, Charlotte Johansson41,42, Chiara Paola Zoia100,
Christoph Laske248,249, Costas Anastasiou170, Dana Lis Palacio250,251, Daniel G. Politis244,245,252,
Daniel Janowitz253, David Craig238, David M. Mann254, David Neary231, Deckert Jürgen153, Delphine Daian233,
Diyana Belezhanska255, Eduardo Kohler256, Eduardo M. Castaño257,258, Effrosyni Koutsouraki163, Elena Chipi160,
Ellen De Roeck196, Emanuele Costantini236, Emma R. L. C. Vardy259, Fabrizio Piras120, Fausto Roveta137,
Federica Piras120, Federico Ariel Prestia245,257, Francesca Assogna120, Francesca Salani183, Gessica Sala100,
Giordano Lacidogna236, Gisela Novack257,258, Gordon Wilcock260, Håkan Thonberg41,42, Heike Kölsch261, o Tuomilehto142,263,264,265, Jaana Lindström142, Jan Laczo139,140, Janet Johnston238, ,
,
,
,
Jean-François Deleuze233, Jenny Harris231, Jonathan M. Schott266, Josef Priller181,267, Juan Ignacio Bacha247,
Julie Snowden231,232, Julieta Lisso268, Kalina Yonkova Mihova269, Latchezar Traykov255, Laura Morelli257,258,
240 241
253
175
196 270
47 271 Jean-François Deleuze233, Jenny Harris231, Jonathan M. Schott266, Josef Priller181,267, Juan Ignacio Bacha247,
Julie Snowden231,232, Julieta Lisso268, Kalina Yonkova Mihova269, Latchezar Traykov255, Laura Morelli257,258, 14 NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 227University of Oxford (OPTIMA), Oxford, UK. 228OPTIMA, Department of Pharmacology, University of Oxford, Oxford, UK. 229Dirección de
Atención de Adultos Mayores del Min., Salud Desarrollo Social y Deportes de la Pcia. de Mendoza, Mendoza, Argentina. 230Geriatrics Unit
Fondazione Policlinico A. Gemelli IRCCS, Rome, Italy. 231Cerebral Function Unit, Greater Manchester Neurosciences Centre, Salford Royal Hospital,
Salford, UK. 232Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health,
University of Manchester, Manchester, UK. 233Centre National de Recherche en Génomique Humaine (CNRGH), Institut de Biologie François
Jacob, CEA, Université Paris-Saclay, F-91057 Evry, France. 234Department of Neurology, Kuopio University Hospital, Kuopio, Finland. 235Insitute of
Clinical Medicine-Neurology, University of Eastenr Finland, Kuopio, Finland. 236Institute of Neurology, Catholic University of Sacred Heart,
Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy. 237Research Laboratory, Complex Structure of Geriatrics, San Giovanni Rotondo,
Fondazione IRCCS Casa Sollievo della Sofferenza, Foggia, Italy. 238Centre for Public Health, School of Medicine, Queen’s University Belfast, Belfast,
UK. 239Clinica Medica “Frugoni” and Geriatric Medicine-Memory Unit, University of Bari Aldo Moro, Bari, Italy. 240CENECON-FMED-UBA, Buenos
Aires, Argentina. 241ENERI, Vienna, Austria. 242Htal Santojani, Buenos Aires, Argentina. 243Servicio de Neurología, Hospital de Cabueñes, Gijón,
Spain. 244UBA, Buenos Aires, Argentina. 245HIGA Eva Perón, Billinghurst, Argentina. 246University of Oxford, Oxford, UK. 247Neurología Clinica,
Madrid, Spain. 248German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany. 249Section for Dementia Research, Hertie
Institute for Clinical Brain Research and Department of Psychiatry and Psychotherapy, University of Tübingen, Tübingen, Germany. 250Hospital Dr. Lucio Molas, Santa Rosa, Argentina. 251Fundacion Ayuda Enfermo Renal y Alta Complejidad (FERNAC), Santa Rosa, Argentina. 252CONICET,
Buenos Aires, Argentina. 253Institute for Stroke and Dementia Research (ISD), University Hospital, Ludwig-Maximilian University Munich, Munich,
Germany. 254Faculty of Medical and Human Sciences, Institute of Brain, Behaviour and Mental Health, Manchester, UK. 255Clinic of Neurology UH
‘Alexandrovska’, Medical University—Sofia, Sofia, Bulgaria. 256Fundacion Sinapsis, Santa Rosa, Argentina. 257Laboratory of Brain Aging and
Neurodegeneration- FIL, Buenos Aires, Argentina. 258IIBBA (CONICET), Buenos Aires, Argentina. 259Salford Royal NHS Foundation Trust, Salford,
England. 260Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK. 261Department of Psychiatry, University of Bonn,
Bonn, Germany. 262Department of Radiology, University Hospital Bonn, Bonn, Germany. 263Department of Public Health, University of Helsinki,
Helsinki, Finland. 264National School of Public Health, Madrid, Spain. 265South Ostrobothnia Central Hospital, Seinäjoki, Finland. 266Dementia
Research Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK. NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications DEGESCO consortium DEGESCO consortium
A. D. Adarmes-Gómez2,22, E. Alarcón-Martín1,31, M. D. Alonso288, I. Álvarez37,38, V. Álvarez26,138,
G. Amer-Ferrer289, M. Antequera13, C. Antúnez13, M. Baquero36, M. Bernal22, R. Blesa2,24, M. Boada1,2,
D. Buiza-Rueda2,22, M. J. Bullido2,215,216, J. A. Burguera36, M. Calero2,21,40, F. Carrillo2,22, M. Carrión-Claro2,22,
M. J. Casajeros290, J. Clarimón2,24, J. M. Cruz-Gamero31, M. M. de Pancorbo291, I. de Rojas1,2, T. del Ser216,
M. Diez-Fairen37,38, R. Escuela2,22, L. Garrote-Espina2,22, J. Fortea2,24, E. Franco-Macías22,
A. Frank-García2,29,216, J. M. García-Alberca204, S. Garcia Madrona40, G. Garcia-Ribas40, P. Gómez-Garre2,22,
I. Hernández1,2, S. Hevilla204, S. Jesús2,22, M. A. Labrador Espinosa2,22, C. Lage2,39, A. Legaz13, A. Lleó2,24,
A. Lopez de Munain32,33, S. López-García2,39, D. Macias-García2,22, S. Manzanares289,292, M. Marín22,
J. Marín-Muñoz13, T. Marín204, M. Marquié1,2, A. Martín Montes2,29,215, B. Martínez13, C. Martínez26,243, A. D. Adarmes-Gómez2,22, E. Alarcón-Martín1,31, M. D. Alonso288, I. Álvarez37,38, V. Álvarez26,138,
G. Amer-Ferrer289, M. Antequera13, C. Antúnez13, M. Baquero36, M. Bernal22, R. Blesa2,24, M. Boada1,2,
D. Buiza-Rueda2,22, M. J. Bullido2,215,216, J. A. Burguera36, M. Calero2,21,40, F. Carrillo2,22, M. Carrión-Claro2,22,
M. J. Casajeros290, J. Clarimón2,24, J. M. Cruz-Gamero31, M. M. de Pancorbo291, I. de Rojas1,2, T. del Ser216,
M. Diez-Fairen37,38, R. Escuela2,22, L. Garrote-Espina2,22, J. Fortea2,24, E. Franco-Macías22,
A. Frank-García2,29,216, J. M. García-Alberca204, S. Garcia Madrona40, G. Garcia-Ribas40, P. Gómez-Garre2,22,
I. Hernández1,2, S. Hevilla204, S. Jesús2,22, M. A. Labrador Espinosa2,22, C. Lage2,39, A. Legaz13, A. Lleó2,24,
A. Lopez de Munain32,33, S. López-García2,39, D. Macias-García2,22, S. Manzanares289,292, M. Marín22,
J. Marín-Muñoz13, T. Marín204, M. Marquié1,2, A. Martín Montes2,29,215, B. Martínez13, C. Martínez26,243, COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunications 15 ARTICLE L. Vivancos13 L. Vivancos13 288Servei de Neurologia, Hospital Clínic Universitari de València, València, Spain. 289Department of Neurology, Hospital Universitario Son Espases,
Palma, Spain. 290Hospital Universitario Ramón y Cajal, Madrid, Spain. 291BIOMICs, País Vasco, Centro de Investigación Lascaray, Universidad del
País Vasco UPV/EHU, Leioa, Spain. 292Fundación para la Formación e Investigación Sanitarias de la Región de Murcia, El Palmar, Spain. 293Fundación CITA-alzheimer, Centro de Investigacio´n y Terapias Avanzadas, San Sebastián, Spain. 294Navarrabiomed, Pamplona, Spain. 295Barcelona beta Brain Research Center–Fundació Pasqual Maragall, Barcelona, Spain. 296Hospital Universitario La Princesa, Madrid, Spain. IGAP (ADGC, CHARGE, EADI, GERAD) C. Macleod297, C. McCracken298, Carol Brayne299, Catherine Bresner17, Detelina Grozeva17, Eftychia Bellou17,
Ewen W. Sommerville17, F. Matthews300, Ganna Leonenko17, Georgina Menzies50, Gill Windle297,
Janet Harwood17, Judith Phillips301, K. Bennett302, Lauren Luckuck17, Linda Clare303, Robert Woods304,
Salha Saad17 & Vanessa Burholt305 297School of Health Sciences, Bangor University, Bangor, UK. 298Institute of Psychology, Health and Society, University of Liverpool, Liverpool,
England. 299School of Clinical Medicine, Cambridge Institute of Public Health, University of Cambridge, Cambridge, England. 300Faculty of
Medicine, Institute of Health and Society, University of Newcastle, Callaghan, NSW, Australia. 301Dementia Studies, University of Stirling, Stirling,
Scotland. 302School of Psychology, University of Liverpool, Liverpool, England. 303School of Psychology, Centre for Research in Ageing and
Cognitive Health (REACH), University of Exeter, Exeter, England. 304Dementia Services Development Centre, Bangor University, Wales, UK. 305Swansea University, School of Human Sciences, Centre for Innovative Ageing, Swansea, UK. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22491-8 V. Martínez13, P. Martínez-Lage Álvarez293, M. Medina2,216, M. Mendioroz Iriarte294,
M. Menéndez-González25,26, P. Mir2,22, J. L. Molinuevo295, L. Montrreal1, A. Orellana1, P. Pastor37,38,
J. Pérez Tur2,212,213, T. Periñán-Tocino2,22, R. Pineda-Sanchez2,22, G. Piñol-Ripoll2,34,35, A. Rábano2,21,46,
D. Real de Asúa296, S. Rodrigo22, E. Rodríguez-Rodríguez2,39, J. L. Royo31, A. Ruiz1,2, R. Sanchez del Valle Díaz14,
P. Sánchez-Juan2,39, I. Sastre2,215, O. Sotolongo-Grau1, S. Valero1,2, M. P. Vicente13, R. Vigo-Ortega2,22 &
L. Vivancos13 PGC-ALZ consortia PGC ALZ consortia
Iris E. Jansen3,9, Nancy L. Pedersen10, Arvid Rongve62, Chandra A. Reynolds
79, Dag Aarsland
89,90,
Geir Selbæk
116,117,118, Ida K. Karlsson10,133, Ingvild Saltvedt
135,136, Sara Hägg
10, Sigrid Botne Sando201,202,
Srdjan Djurovic
206,207, Ole A. Andreassen
224,225, Danielle Posthuma9 Iris E. Jansen3,9, Nancy L. Pedersen10, Arvid Rongve62, Chandra A. Reynolds
79, Dag Aarsland
89,90,
Geir Selbæk
116,117,118, Ida K. Karlsson10,133, Ingvild Saltvedt
135,136, Sara Hägg
10, Sigrid Botne Sando201,202,
Srdjan Djurovic
206,207, Ole A. Andreassen
224,225, Danielle Posthuma9 16 NATURE COMMUNICATIONS | (2021) 12:3417 | https://doi.org/10.1038/s41467-021-22491-8 | www.nature.com/naturecommunicatio
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English
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Closing the Gaps in Our Knowledge of the Hydrological Cycle over Land: Conceptual Problems
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Surveys in geophysics
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Surv Geophys
DOI 10.1007/s10712-013-9221-7 Surv Geophys
DOI 10.1007/s10712-013-9221-7 Closing the Gaps in Our Knowledge of the Hydrological
Cycle over Land: Conceptual Problems William A. Lahoz • Gabrie¨lle J. M. De Lannoy Received: 9 October 2012 / Accepted: 25 January 2013 Received: 9 October 2012 / Accepted: 25 January 2013
The Author(s) 2013. This article is published with open access at Springerlink.com p
y
The Author(s) 2013. This article is published with open access at Springerlink.com Abstract
This paper reviews the conceptual problems limiting our current knowledge of
the hydrological cycle over land. We start from the premise that to understand the
hydrological cycle we need to make observations and develop dynamic models that
encapsulate our understanding. Yet, neither the observations nor the models could give a
complete picture of the hydrological cycle. Data assimilation combines observational and
model information and adds value to both the model and the observations, yielding
increasingly consistent and complete estimates of hydrological components. In this review
paper we provide a historical perspective of conceptual problems and discuss state-of-the-
art hydrological observing, modelling and data assimilation systems. Keywords
Hydrological cycle Earth observation Land surface models
Data assimilation G. J. M. De Lannoy
Global Modeling and Assimilation Office (Code 610.1), NASA/GSFC, Greenbelt, MD, USA
e-mail: Gabrielle.Delannoy@nasa.gov W. A. Lahoz (&)
NILU, Instituttveien 18, PO Box 100, 2027 Kjeller, Norway
e-mail: wal@nilu.no 1 Introduction The water stored on land is a key variable controlling numerous processes and feedback
loops within the climate system (see, e.g., Dirmeyer 2000; Koster et al. 2004a, b; Sene-
viratne et al. 2010). It constrains plant transpiration and photosynthesis and thus is of major
relevance for the Earth’s water and energy cycles and impacts the exchanges of trace gases
on land, including carbon dioxide. Figure 1, from IPCC (2007), provides an overview of
the main terrestrial components and exchanges within the climate system. This shows the
complexity of land processes and feedbacks, to a large extent owing to the high spatial W. A. Lahoz (&)
NILU, Instituttveien 18, PO Box 100, 2027 Kjeller, Norway
e-mail: wal@nilu.no G. J. M. De Lannoy
Global Modeling and Assimilation Office (Code 610.1), NASA/GSFC, Greenbelt, MD, USA
e-mail: Gabrielle.Delannoy@nasa.gov 123 Surv Geophys Fig. 1 Global climate system. Figure from IPCC (2007) Fig. 1 Global climate system. Figure from IPCC (2007) variability in soils, vegetation and topography (ranging from metres to kilometres). Pro-
cesses affecting the amount of water stored on land, for example, precipitation and radi-
ation, have spatial scales of kilometres (e.g., associated with weather fronts) and have high
temporal variability (hours). The amount of water stored in the unsaturated soil zone is generally referred to as soil
moisture, although the exact definition can vary depending on the context. Soil moisture is
one of the key geophysical variables for understanding the Earth’s hydrological cycle. It is
classed as an essential climate variable of the Global Climate Observing System (GCOS)
(GCOS-107, 2006). Soil moisture determines the partitioning of incoming water into infiltration and run-off. It directly affects plant growth and other organic processes and thus connects the water
cycle to the carbon cycle. Run-off and base flow from the soil profile determine river flows
and flooding, which connects hydrology with hydraulics. Soil moisture also has a signif-
icant impact on the partitioning of water and heat fluxes (latent and sensible heat), thereby
connecting the hydrological (i.e. water) cycle with the energy cycle. Soil moisture is a source of water for the atmosphere through evapotranspiration from
land. Evapotranspiration is a major component of the continental water cycle, as it returns
as much as 60 % of the whole land precipitation back to the atmosphere (e.g., Oki and
Kanae 2006). Furthermore, evapotranspiration is also an important energy flux (Trenberth
et al. 123 1 Introduction 2009) and is connected to the surface skin and soil temperature, which make up other
important state variables of the land surface system. Together, soil moisture, temperature
and their impacts on the water, energy and carbon cycle play a major role in climate-
change projections (IPCC 2007; Seneviratne et al. 2010). Snow on land is another
important variable affecting the global energy and water budgets, because of its high
albedo, low thermal conductivity, considerable spatial and temporal variation and medium-
term capacity for water storage. 123 123 Surv Geophys Quantifying the land state and fluxes and understanding soil moisture–temperature and
soil moisture–precipitation couplings allow a better representation of hydrological pro-
cesses in climate models and significantly help to reduce uncertainties in future climate
scenarios, in particular regarding changes in climate variability and extreme events, and
ecosystem/agricultural impacts (Seneviratne et al. 2010). This understanding is also cru-
cially important for improving short-range numerical weather prediction (NWP) capabil-
ities, in particularly regarding prediction of convective precipitation (Sherwood 1999;
Adams et al. 2011, and references therein). Hydrological observations are prone to errors and are discrete in space and time with
the result that the information provided by these observations has gaps. Figure 2 shows an
example of gaps in satellite observations. It is desirable to fill gaps in the observed
information using additional information and computational techniques. Algorithms or
models to fill in information gaps should organize, summarize and propagate the infor-
mation from observations in an objective and consistent way. A simple approach such as
linear interpolation could be a reasonably accurate ‘‘model’’, when observations are dense
enough. However, linear interpolation may not be consistent with our advanced under-
standing of how the land surface behaves. A more realistic approach would be to fill in the
gaps using a land surface model (LSM). While observations give an instantaneous view of
the land surface, LSMs provide continuous estimates, based on physical laws that are
derived from historical observations. These models are not perfect, and gaps in their
structure, parametrization or initialization can be filled in with observations. Fig. 2 Plot representing retrieved soil moisture data from the Soil Moisture Ocean Salinity (SMOS, Kerr
et al. 1 Introduction 2010) mission for August 3, 2012 (top left panel), August 10, 2012 (top right panel), August 17, 2012
(bottom left panel), and August 25, 2012 (bottom right panel), based on the observational geometry from
ascending orbits from SMOS (units of m3 m-3). Blue denotes relatively wet values; red denotes relatively
dry values. The uncoloured (i.e. grey) areas over land represent gaps between the satellite orbits. Noteworthy are the sparse SMOS observations over Scandinavia, where retrievals from remotely sensed
observations are particularly difficult, when the land is covered with snow, ice, forest, water bodies or rocks
1 Fig. 2 Plot representing retrieved soil moisture data from the Soil Moisture Ocean Salinity (SMOS, Kerr
et al. 2010) mission for August 3, 2012 (top left panel), August 10, 2012 (top right panel), August 17, 2012
(bottom left panel), and August 25, 2012 (bottom right panel), based on the observational geometry from
ascending orbits from SMOS (units of m3 m-3). Blue denotes relatively wet values; red denotes relatively
dry values. The uncoloured (i.e. grey) areas over land represent gaps between the satellite orbits. Noteworthy are the sparse SMOS observations over Scandinavia, where retrievals from remotely sensed
observations are particularly difficult, when the land is covered with snow, ice, forest, water bodies or rocks 12 Surv Geophys Data assimilation (Kalnay 2003) provides an intelligent method to fill in the observa-
tional gaps using a model or to steer models using observations. By intelligent, it is meant
an ‘‘objective’’ way which makes use of quantitative concepts (e.g., mathematical) for
combining imperfect information. By combining observational and model information,
data assimilation can be used to test the self-consistency and error characteristics of this
information (Talagrand 2010b). In this paper we focus on off-line land data assimilation, where the LSM is uncoupled
from an atmospheric model. By using an uncoupled LSM, it can be forced with more
observation-based forcings, rather than often inaccurate atmospheric analyses, and less
computational resources are needed. The uncoupled approach can be regarded as a first
step towards the land data assimilation goal of coupling an LSM to an atmospheric model
to improve predictions at weather, seasonal and climate timescales (Palmer et al. 2008). In this paper we discuss observations (Sect. 2), models (Sect. 3) and data assimilation
methods (Sect. 4) used in the studies of the hydrological cycle and provide illustrative
examples, with a focus on soil moisture. 1 Introduction We pay special attention to the conceptual
problems and key challenges associated with making use of observational and model
information of the land surface in data assimilation systems (Sect. 5). We finish by pro-
viding conclusions (Sect. 6). 2 Observations of the Hydrological Cycle Observations of the hydrological cycle are commonly divided into conventional obser-
vations (e.g., in situ ground-based measurements such as screen-level relative humidity)
and remotely sensed observations (e.g., satellite or aircraft microwave observations). These
data sets are complementary: conventional observations have relatively high spatio-tem-
poral resolution (order metres and minutes) but only have local coverage, so have poor
representativity for a large area; satellite observations have relatively low spatio-temporal
resolution but have global coverage, so have good representativity for a large area. In situ
observations are typically used as ground truth for calibration and validation of remote
sensing products, and model and assimilation results. Table 1 gives an overview of satellite sensors and missions that contribute to our current
understanding of the hydrological cycle or may potentially contribute to this understanding
in the near future. Depending on the observed wavelengths, the orbit altitude and design
details, there are large differences in horizontal, vertical and temporal resolution of each
observation type. For example, satellite-based observations of soil moisture are made using
passive and active microwave instruments. The horizontal resolution of these sensors
ranges from 50 to 10 km; the temporal resolution is about one observation every 2–3 days,
depending on the location on Earth. These instruments typically penetrate the first few
millimetres to centimetres of the soil: a few millimetres for the X-band (8–12 GHz, e.g.,
Advanced Microwave Sounding Radiometer for EOS, AMSR-E; Njoku and Chan 2006);
*1 cm for the C-band (4–8 GHz, e.g., AMSR-E; Advanced SCATterometer, ASCAT;
Bartalis et al. 2007); and *5 cm for the L-band (1–2 GHz, e.g., Soil Moisture Ocean
Salinity, SMOS; Kerr et al. 2010). An immediate conceptual problem is to estimate soil
moisture of actual interest in the root zone (1 m) at a finer resolution. For this, observa-
tional information needs to be transferred from the surface layer to the root zone (e.g.,
Calvet et al. 1998; Sabater et al. 2007; De Lannoy et al. 2007a; Draper et al. 2012) and
downscaled from the coarse scale to finer scales (Reichle et al. 2001a; Pan et al. 2 Observations of the Hydrological Cycle In November 2009, the
ESA Earth Explorer mission SMOS was launched followed by another L-band mission,
NASA/CONAE Aquarius (Le Vine et al. 2006), in June 2011. Aquarius measures various
elements of the hydrological cycle, and its coarse resolution makes it less attractive for soil
moisture estimation. The NASA mission SMAP is focused on soil moisture and freeze–
thaw detection and is scheduled for launch in 2014 (Entekhabi et al. 2010a). To illustrate
the importance of soil moisture information, Table 2 identifies key benefits from satellite
soil moisture measurements. A special issue on soil moisture from the SMOS mission has recently appeared in the IEEE
Transactions on Geoscience and Remote Sensing (Kerr et al. 2012a). The papers in this
special issue describe the SMOS mission (Mecklenburg et al. 2012); the radiometric per-
formance (Kainulainen et al. 2012); the SMOS soil moisture retrieval algorithm (Kerr et al. 2012b; Mattar et al. 2012); the impact of radio frequency interference (RFI) on the SMOS
soil moisture measurements (Castro et al. 2012; Misra and Ruf 2012; Oliva et al. 2012); Table 2 Key benefits expected from satellite soil moisture observations
Area
Products
Comment
Meteorology
NWP models
Soil moisture plays a fundamental role in the transfer of water and
energy between the surface and the atmosphere. Introduction of
this variable in current NWP models will allow improving
predictions, especially important under adverse meteorological
conditions
Climatology
Models
Variability of the soil moisture time series with a long integration
period may provide relevant information for the study of climate
change
Risk
Management
Flooding risk map
The soil’s risk of flooding is significantly conditioned by the
amount of water stored in the vadose zone. The generation of this
type of products will require the inclusion of soil moisture data in
hydrological and NWP models (precipitation predictions)
Fire risk map
The risk of fire is determined by several factors, including
meteorological, geophysical and biophysical factors. 2 Observations of the Hydrological Cycle 2009; 123 12 Surv Geophys Table 1 Characteristics of hydrological observations potentially available within the next decade
(see ‘‘Appendix’’ for details of sensor acronyms)
Hydrological
quantity
Remote
sensing
technique
Timescale
Spatial
scale
Accuracy considerations
Examples of
sensors
Precipitation
Thermal
infrared
Hourly
4 km
Tropical convective clouds
only
GOES, MODIS,
AVHRR,
Landsat, ASTER
1 day
1 km
15 days
60 m
Passive
microwave
3 h
10 km
Land calibration problems
TRMM, SSMI,
AMSR-E, GPM
Active
microwave
Daily
10 m
Land calibration problems
TRMM, GPM
Surface soil
moisture
Passive
microwave
1–3 days
25–50 km
Limited to sparse
vegetation, low
topographic relief
AMSR-E, SMOS,
Aquarius, SMAP
Active
microwave
3 days
3 km
Significant noise from
vegetation and roughness
ERS, JERS,
Radarsat,
ASCAT
30 days
10 m
Surface skin
temperature
Thermal
infrared
1 h
4 km
Soil/vegetation average,
cloud contamination
GOES, MODIS,
AVHRR,
Landsat, ASTER
1 day
1 km
15 days
60 m
Snow cover
Visible/
thermal
infrared
1 h
4 km
Cloud contamination,
vegetation masking,
bright soil problems
GOES, MODIS,
AVHRR,
Landsat, ASTER
1 day
500 m–
1 km
15 days
30–60 m
Snow water
equivalent
(SWE)
Passive
microwave
1–3 days
10 km
Limited depth penetration
AMSR-E
Active
microwave
30 days
100 m
Limited spatial coverage
SnoSat, SCLP,
Cryosat-2,
CoreH2O
Water level/
velocity
Laser
10 days
100 m
Cloud penetration problems
ICESAT,
ICESAT2,
SWOT,
DESDynl
Radar
30 days
1 km
Limited to large rivers
TOPEX/
POSEIDON
Total water
storage
changes
Gravity
changes
30 days
1,000 km
Bulk water storage change
GRACE, GOCS,
GRACEII
Evaporation
1 h
4 km
Significant assumptions
GOES, MODIS,
AVHRR,
Landsat, ASTER
1 day
1 km
15 days
60 m
Table updated from Houser et al. (2010) Table 1 Characteristics of hydrological observations potentially available within the next dec
(see ‘‘Appendix’’ for details of sensor acronyms) Table updated from Houser et al. (2010) De Lannoy et al. 2010; Sahoo et al. 2013) typically using a land surface model (discussed
in Sect. 3) and/or land data assimilation (discussed in Sect. 4). With the design of new sensors, one aims to gain resolution, increase the sensitivity to
the variables of interest and reduce instrument errors (USGEO 2010). Examples of new
missions for soil moisture observations are the SMOS and SMAP (Soil Moisture Active
and Passive) missions, both using L-band sensors and designed with a target uncertainty 23 Surv Geophys lower than that of earlier missions, like AMSR-E and ASCAT. Table adapted from http://www.cp34-smos.icm.csic.es/index.htm 2 Observations of the Hydrological Cycle 2004) for its ability to measure an
integrated water quantity of soil moisture and snow, as well as water in deeper layers. Satellite instruments do not measure directly hydrological parameters. What they
measure is photon counts (level 0 data). Algorithms then transform the level 0 data into
radiances (level 1 data). Subsequently, using retrieval techniques (Rodgers 2000), retri-
evals of layer quantities (e.g., of soil moisture) or integrated amounts (e.g., total water
storage) are derived (level 2 data). Fields derived from manipulation of level 2 data, for
example, by interpolation to a common grid are termed level 3 data. Analyses derived from
the assimilation of level 1 and/or 2 data are termed level 4 data. Satellite observations (from level 0 and up) have associated with them a number of
errors, including random and systematic errors in the measurement, and the error of rep-
resentativeness (or representativity). Random errors (sometimes termed precision) have the
property that averaging the data can reduce them. This is not the case of the systematic
error or bias (sometimes termed accuracy). The error of representativeness is associated
with the extent to which the measurement represents a point or volume in space. In land
surface measurements, the error of representativeness is important to consider in com-
parisons of coarse-scale satellite data with point data. Satellite-based hydrological data are becoming increasingly available, although little
progress has been made in understanding their observational errors. Evaluation of the
accuracy of land surface satellite data is a challenge, and novel methods to characterize
their errors are being applied. Examples include triple collocation (e.g., Scipal et al. 2008;
Dorigo et al. 2010; Parinussa et al. 2011); the R-metrics approach (Crow 2007; Crow and
Zhan 2007; Crow et al. 2010); and data assimilation (Houser et al. 2010, and references
therein). A number of in situ network and airborne hydrological studies have been set up in the
last decade for evaluation of satellite data. Examples of in situ networks include
SMOSMANIA in France (Calvet et al. 2007; Albergel et al. 2009); NVE (Norges vassd-
rags-og energidirektorat, Norwegian Water Resources and Energy Directorate) in Norway
(http://www.nve.no/en/); several large-scale (larger than 10,000 km2) networks in the USA
and elsewhere (see Table 1 in Crow et al. 2012); and several local- to regional-scale (larger
than 100 km2, smaller than 10,000 km2) networks in the USA and elsewhere (see Table 2
in Crow et al. 2012). 2 Observations of the Hydrological Cycle The
information on soil moisture may be directly assimilated in
drawing up fire risk maps as they provide direct information on
evapotranspiration, water content assimilated by vegetation and
quality of vegetation
Famine risk map
The merging of geopolitical, meteorological/climatological
information and data in the quality and estimates of agricultural
and/or marine products (derived with the help of soil moisture
data) may be of great use in early prediction of famine episodes
in areas of Earth where resources are scarce
Drought risk model
Analysing soil moisture trends in large areas may serve to generate
drought models, along with data from other sensors
Agriculture
Agricultural
production
estimate
On the basis of soil moisture data and by means of the application
of hydrological models, it is possible to determine the amount of
water assimilated by the vegetation, a value that is very useful for
estimating agricultural production
Hydrology
Models
The content of water stored in the soil is an important parameter to
be taken into consideration in any hydrological model, as it is an
indispensable variable in understanding the water cycle Table 2 Key benefits expected from satellite soil moisture observations On the basis of soil moisture data and by means of the application
of hydrological models, it is possible to determine the amount of
water assimilated by the vegetation, a value that is very useful for
estimating agricultural production The content of water stored in the soil is an important parameter to
be taken into consideration in any hydrological model, as it is an
indispensable variable in understanding the water cycle 12 Surv Geophys soil processes in boreal regions (Rautiainen et al. 2012); disaggregation of SMOS data
(Merlin et al. 2012); and various aspects of the validation of SMOS soil moisture data (Al
Bitar et al. 2012; Bircher et al. 2012; dall’Amico et al. 2012; Jackson et al. 2012; Lacava et al. 2012; Mialon et al. 2012; Peischl et al. 2012b; Rowlandson et al. 2012; Sanchez et al. 2012;
Schlenz et al. 2012; Schwank et al. 2012). In view of the applications discussed later in this paper, we briefly mention that snow
measurements are often provided by AMSR-E to measure snow water equivalent (SWE),
and MODIS (MODerate resolution Imaging Spectroradiometer, Morisette et al. 2002) to
give a picture of the snow-covered area. Finally, it is worth to mention GRACE (Gravity
Recovery And Climate Experiment, Tapley et al. 2 Observations of the Hydrological Cycle In situ soil moisture data from various networks across the world are
consolidated in the International Soil Moisture Network (ISMN; http://www.ipf.tuwien.ac. at/insitu). As of January 2013, the ISMN includes data from 37 networks—Table 3 pro-
vides details. An example of an airborne study on evaluation of satellite data is the
Australian Airborne Cal/Val Experiments for SMOS (AACE, Peischl et al. 2012a). An
example of in situ ground-based station data used to evaluate satellite data is SMOSREX
(de Rosnay et al. 2006). The temporal scale of in situ platforms ranges from minutes to
hours; the spatial scale of in situ platforms ranges from tens of metres (individual stations)
to thousands of kilometres (regional-scale networks). Along with the availability of dense 123 Surv Geophys Table 3 Contributing networks to the International Soil Moisture Network (ISMN)
Name
Country
Stations
Website
AACES
Australia
49
http://www.moisturemap.monash. edu.au/
AMMA
Benin, Niger, Mali
7
http://amma-international.org/
ARM
USA
25
http://www.arm.gov
AWDN
USA
50
http://www.hprcc.unl.edu/awdn/
CALABRIA
Italy
5
http://www.cfcalabria.it
CAMPANIA
Italy
2
http://www.regione.campania.it/
CHINA
China
40
COSMOS
USA, Germany,
Switzerland,
France, Brasil, Kenya,
UK, Mexico
67
http://cosmos.hwr.arizona.edu/
FLUXNET-AMERIFLUX
USA
2
http://www.fluxnet.ornl.gov/
fluxnet/index.cfm
FMI
Finland
1
http://fmiarc.fmi.fi/
GTK
Finland
7
HOBE
Denmark
30
http://www.hobe.dk/
HSC_SELMACHEON
Korea
1
http://www.hsc.re.kr
HYDROL-NET_PERUGIA
Italy
1
http://www.dica.unipg.it/DICA
HYU_CHEONGMICHEON
Korea
1
http://wrrsl.hanyang.ac.kr/html/
introduction.htm
ICN
USA
19
http://www.isws.illinois.edu/warm
IIT_KANPUR
India
1
http://www.iitk.ac.in
IOWA
USA
6
MAQU
China
20
MetEROBS
Italy
1
http://mistrals.sedoo.fr/HyMeX/
Plateform-search?datsId=532
MOL-RAO
Germany
2
http://www.dwd.de/mol
MONGOLIA
Mongolia
44
OZNET
Australia
52
http://www.oznet.org.au/
REMEDHUS
Spain
23
http://campus.usal.es/*hidrus/
RUSWET-AGRO
Former Soviet Union
78
RUSWET-GRASS
Former Soviet Union
122
RUSWET-VALDAI
Former Soviet Union
3
SCAN
USA
182
http://www.wcc.nrcs.usda.gov/scan/
SMOSMANIA
France
21
http://www.hymex.org/
SNOTEL
USA
374
http://www.wcc.nrcs.usda.gov/snow/
SWEX_POLAND
Poland
6
UDC_SMOS
Germany
11
http://www.geographie.uni-
muenchen.de/department/fiona/
forschung/projekte/index.php? projekt_id=103
UMBRIA
Italy
7
http://www.cfumbria.it/
http://hydrology.irpi.cnr.it/
UMSUOL
Italy
1 Table 3 Contributing networks to the International Soil Moisture Network (ISMN) Table 3 continued
Name
Country
Stations
Website
USCRN
USA
114
http://www.ncdc.noaa.gov/crn/
USDA-ARS
USA
4
VAS
Spain
3
http://nimbus.uv.es/
Table adapted from http://www.ipf.tuwien.ac.at/insitu/index.php/insitu-networks.html
Surv Geophys Surv Geophys Surv Geophys Table adapted from http://www.ipf.tuwien.ac.at/insitu/index.php/insitu-networks.html Table adapted from http://www.ipf.tuwien.ac.at/insitu/index.php/insitu-networks.html in situ data for validation, it is also important to select appropriate validation measures
(Entekhabi et al. 2010b). The assimilation of satellite data for land surface applications has only gained signifi-
cance in the last decade; it started later than atmospheric and oceanographic data assim-
ilation (see various chapters in Lahoz et al. 2010a). 2 Observations of the Hydrological Cycle This can be attributed to: (1) a lack of
dedicated land surface state (water and energy) remote sensing instruments; (2) inadequate
retrieval algorithms for deriving global land surface information from remote sensing
observations; and (3) a lack of mature techniques to objectively improve and constrain land
surface model predictions using remote sensing data. 3 Models of the Hydrological Cycle 2009); the
SURFEX model (Le Moigne 2009); the Interaction between Soil Biosphere and Atmo-
sphere (ISBA) model (Noilhan and Mahfouf 1996); and the Joint UK Land Environment
Simulator (JULES) model (Best et al. 2011; Clark et al. 2011a). An example of an inte-
grated system is the NASA Land Information System (LIS), which offers the capability to
simulate with different models, observations and data assimilation techniques (Kumar et al. 2008). An LSM has several elements, including a soil moisture scheme, a snow scheme, a
rainfall–run-off scheme and a routing/hydraulic scheme. The soil moisture scheme can take
several forms, such as explicit numerical solutions of Richards’ equations over multiple
discretized layers (e.g., in CLM), or using a force-restore method (e.g., Deardorff 1977,
used in SURFEX), or other more non-traditional approaches, such as a soil moisture
calculation as a deviation from the equilibrium soil moisture profile between the surface
and the water table (Catchment LSM). The different profile structures involve different
state variables, for example, describing soil moisture at the surface (superficial volumetric
water content) or describing soil moisture over the root zone (mean volumetric content of
the root zone). The coupling strength between the surface and deeper soil layers is a
sensitive point for successful propagation of surface observations to deeper layers (Kumar
et al. 2009). The presence of snow covering the ground and vegetation can greatly influence the
energy and mass transfers between the land surface and the atmosphere. Notably, the snow
layer modifies the radiative balance by increasing the albedo. Furthermore, the amount of
water stored in the snowpack has an important impact on water availability in the spring
time. The prognostic variables in most snow schemes include variables related to snow
water equivalent (SWE), including snow depth and density, and the snow heat content. These variables most often determine the diagnostics such as snow area extent and albedo. The snow scheme can have one layer, or several layers. In a one-layer scheme, the
evolution of the snow water equivalent of the snow reservoir depends on the precipitation
of snow (a source) and the snow sublimation from the snow surface (a sink). Multi-layer
schemes are often designed to have intermediate complexity, having simplified physical
parametrizations based on those of highly detailed internal-process snow models, while
having computational requirements resembling those of single-layer schemes (Loth et al. 1993; Lynch-Stieglitz 1994; Sun et al. 1999). 3 Models of the Hydrological Cycle As discussed above, observational information has gaps in space and time. It is desirable to
fill in these observational gaps using a model. Such models can range from simple linear
interpolation to full land surface models (LSMs). Land surface processes are part of the
global processes controlling the Earth, which are typically represented in global general
circulation models (GCMs). The land component in these models is represented in (largely
physically based) LSMs, which simulate the water and energy balance over land using
simple algebraic equations or more complex systems of partial differential equations. The
main state variables of these models include the water content and temperature of soil
moisture, snow and vegetation. These variables are referred to as prognostic state vari-
ables. Changes in these state variables account for fluxes, for example, evapotranspiration
and run-off, which are referred to as diagnostic state variables. Most land surface models used in GCMs view the soil column as the fundamental hydro-
logical unit, ignoring the role of, for example, topography on spatially variable processes
(Stieglitz et al. 1997) to limit the complexity and computations for these coupled models. During the last decades, LSMs have become increasingly complex to accommodate for better
understood processes, like snow and vegetation. Along with a more complex structure often
comes a more complex parametrization, and several authors (Beven 1989; Duan et al. 1992)
have stated that LSMs are over-parametrized given the data typically available for calibration. Atlargerscales,thesemodelsoftenrelyonsatellite-observedparameters,suchasgreennessand
LAI (leaf area index). For field-scale studies, the LSMs are usually calibrated to specific
circumstances to limit systematic prediction errors. Model calibration or parameter estimation
reliesonobserveddataand can bedefined asa specific typeofdata assimilation(Nichols2010). Many LSMs have been developed and enhanced since the mid-1990s, with varying
features, such as sub-grid variability, community-wide input, advanced physical repre-
sentations and compatibility with atmospheric models (Houser et al. 2010). Some examples
of widely used LSMs are the NCAR Community Land Model (CLM) (Oleson et al. 2010);
the Variable Infiltration Capacity (VIC) Model (Liang et al. 1994); the Noah Model (Ek 123 Surv Geophys et al. 2003); the Catchment LSM (Koster et al. 2000); the TOPMODEL-based Land
Atmosphere Transfer Scheme (TOPLATS) model (Famiglietti and Wood 1994); the
Hydrology-Tiled European Centre for Medium-range Weather Forecasts (ECMWF)
Scheme for Surface Exchange over Land (H-TESSEL) model (Balsamo et al. 123 3 Models of the Hydrological Cycle A number of approaches have been implemented for rainfall–run-off schemes. Water
that cannot be stored in the soil profile either runs off over land (Horton run-off, e.g.,
Decharme and Douville 2006) or gravitationally drains out of the profile (Mahfouf and
Noilhan 1996; Boone 1999). The TOPMODEL run-off approach combines key distributed
effects of channel network topology and dynamic contributing areas for run-off generation
(Beven and Kirkby 1979; Silvapalan et al. 1987). This formalism takes explicit account of
topographic heterogeneities (Decharme et al. 2006; Decharme and Douville 2006, 2007). Run-off and drainage exiting from hydrological models can be used as a boundary to
hydraulic models that predict river flow and potential flooding (Matgen et al. 2010). A hydraulic flood routing scheme uses numerical methods to solve simultaneously the
equations of continuity and momentum for a fluid (see, e.g., Guo 2006). It is often applied
to a river network, typically in a hierarchy including hillslope routing, sub-network routing
and main channel routing. An example of a routing scheme is the river transport model 123 123 Surv Geophys developed for the NCAR Community Land Model (CLM) (Branstetter and Erickson III
2003, and references therein). A river transport model is also useful because it can be used
to evaluate the performance of an LSM against gauge station data. developed for the NCAR Community Land Model (CLM) (Branstetter and Erickson III
2003, and references therein). A river transport model is also useful because it can be used
to evaluate the performance of an LSM against gauge station data. Most LSMs are soil–vegetation–atmosphere transfer (SVAT) models, where the veg-
etation is not a truly dynamic component. Recently, coupling of hydrological or SVAT
models with vegetation models has received some attention, to serve more specific eco-
logical, biochemical or agricultural purposes. Dynamic vegetation models are used to
simulate the evolution of vegetation cover, photosynthesis, carbon and nutrient inventories
and the fluxes of water, CO2, CH4, N2O, volatile organic carbon and fire-related emissions
between the land surface and atmosphere. Illustrative examples of vegetation models are
the Land biosphere Process and eXchange (LPX) model (Wania 2007; Spahni et al. 2010)
and the CoupModel (Gustafsson et al. 2004; Jansson and Karlberg 2004; Jansson et al. 2005, 2008; Karlberg et al. 2006, 2007; Klemedtsson et al. 2008; Norman et al. 2008;
Svensson et al. 2008). There are a number of potential problems with LSMs that can cause errors in the
forecast. 3 Models of the Hydrological Cycle These include components that cause ‘‘model error’’ and components that cause
‘‘predictability error’’. Components that cause ‘‘model error’’ are as follows: incomplete
description of physical processes perhaps done for computational efficiency, perhaps a
reflection of incomplete knowledge; inaccurate parameters; and inaccurate forcings. Components that cause ‘‘predictability error’’ are inaccurate initial states and boundaries. All these problems are the subject of research in the land surface modelling and assimi-
lation community. 4 Data Assimilation of the Hydrological Cycle 4.1 Introduction 4.1 Introduction The only practical way to observe the land surface on continental to global scales is by
satellite remote sensing. However, this cannot provide information on the entire system,
and measurements only represent a snapshot in time. Land surface models can predict
spatial/temporal land system variations, but these predictions are often poor, due to model
initialization, parameter and forcing errors and inadequate model physics and/or resolution. A way forward is to merge the observational and model information through data
assimilation (Kalnay 2003). Mathematics provides rules for combining information objectively, based on principles
which aim to maximize (or minimize) a quantity (e.g., a ‘‘penalty function’’) or on
established statistical concepts (e.g., Bayesian methods) that relate prior information
(understanding, which comes from prior combination of observations and models), with
new information (e.g., an extra observation). The merged product, termed the posterior
estimate or an analysis, adds value to both observational and model information. The data
assimilation methodology takes account of the different nature (e.g., spatio-temporal res-
olution) of the observational and model information, using an observation operator (see,
e.g., Talagrand 2010a). Assimilation of land surface observations is at an earlier stage than, for example,
assimilation of atmospheric observations (see various chapters in Lahoz et al. 2010a). However, during the past decade, land data assimilation has been a very active field of
research. Land data assimilation considers both ground-based in situ data and satellite data. Often, satellite land surface data are assimilated and the process validated using in situ 3 123 Surv Geophys measurements. Assimilated satellite observations include retrievals of land surface tem-
perature, soil moisture, snow water equivalent (SWE) and snow cover area (e.g., Van den
Hurk et al. 2002; Andreadis and Lettenmaier 2006; Slater and Clark 2006; Bosilovich et al. 2007; Dong et al. 2007; Drusch 2007; Ni-Meister 2008; Reichle et al. 2008; Houser et al. 2010). Houser (2003) discusses the assimilation of land surface retrieved quantities and
radiances. Early reviews of land data assimilation have been provided by McLaughlin
(2002), Reichle (2008), Moradkhani (2008) and Houser et al. (2010). Land data assimilation uses observations to constrain the physical parametrizations and
initialization of land surface states critical for seasonal-to-interannual prediction. These
constraints can be imposed in four ways: (1) by forcing the land surface primarily by
observations (such as precipitation and radiation), often severe atmospheric NWP land
surface forcing biases can be avoided (e.g., Saha et al. 2010; Reichle et al. 4.1 Introduction 2011); (2) by
employing innovative land surface data assimilation techniques, observations of land
surface storages (such as snow, soil temperature and moisture) can be used to constrain
unrealistic simulated storages (e.g., Houser et al. 2010; Reichle et al. 2013); (3) by tuning
adjustable parameters (e.g., Pauwels et al. 2009; Vrugt et al. 2012); and (4) the land surface
physical structure itself can be improved through the data assimilation process when the
constant confrontation of model states against observations returns useful information
about structural deficits. Integration of soil moisture information from satellite instruments,
and ground-based and in situ observations of the land surface, using land data assimilation,
provides a comprehensive picture of the state and variability of the land surface. 4.2 Data Assimilation Methods Three methods are commonly used for land data assimilation (Houser et al. 2010): vari-
ational (3- and 4-dimensional, 3D-Var and 4D-Var); sequential (Kalman filter (KF) and
Extended Kalman filter (EKF)); and ensemble (Ensemble Kalman filter, EnKF). Bouttier
and Courtier (1999) provide details of these methods. Talagrand (2010a) and Kalnay
(2010) discuss more recent developments in variational methods and ensemble methods,
respectively. In the 3-D variational (3D-Var) method, a minimization algorithm is used to find a
model state, x, that minimizes the misfit between x and the background state xb, and also
between the observation predictions H(x) and the observations y. The observation operator
H maps the model state x to the measurement space, where y resides. In 3D-Var, we seek
the minimum with respect to x of the penalty function, J, given by Eq. (1). The first term on
the right hand side (Jb) quantifies the misfit to the background term, and the second term
(Jo) is the misfit to the observations. If the observation operator is linear (written H), the
penalty function, J, is quadratic and is guaranteed to have a unique minimum. J ¼ 1
2 ½x xbTB1½x xb þ 1
2 ½y HðxÞTR1½y HðxÞ
ð1Þ ð1Þ 4-D variational (4D-Var) assimilation is an extension of 3D-Var in which the temporal
dimension is included, that is, 4D-Var is a smoother. In 4D-Var, observations are used at
their correct time. 4D-Var has two new features compared to 3D-Var. First, it includes a
model operator, M, that carries out the evolution forward in time. The first derivative, or
differential, of M, M, is the tangent linear model (if M is linear, represented by M, its
derivative is M). The transpose of the tangent linear model operator, MT, integrates the
adjoint variables backward in time. The tangent linear model is only defined under the
condition that the function J defined by Eq. (1) be differentiable—this is the tangent linear 4-D variational (4D-Var) assimilation is an extension of 3D-Var in which the temporal
dimension is included, that is, 4D-Var is a smoother. In 4D-Var, observations are used at
their correct time. 4D-Var has two new features compared to 3D-Var. First, it includes a
model operator, M, that carries out the evolution forward in time. The first derivative, or
differential, of M, M, is the tangent linear model (if M is linear, represented by M, its
derivative is M). 4.2 Data Assimilation Methods The transpose of the tangent linear model operator, MT, integrates the
adjoint variables backward in time. The tangent linear model is only defined under the
condition that the function J defined by Eq. (1) be differentiable—this is the tangent linear Surv Geophys hypothesis. Second, J can include an extra term in which the model errors associated with
the model’s temporal evolution are accounted for. In the formulation of Zupanski (1997),
an analogous term involving Q-1 is included in J, where Q is the model error covariance. Examples for the land surface using variational methods include Calvet et al. (1998) and
Reichle et al. (2001a). Note that variational methods are very common for parameter
estimation (e.g., Dumont et al. 2012), but with replacement of the misfit to the background
with a misfit to prior parameter guesses. In the Kalman filter (KF), a recursive sequential algorithm is applied to evolve a
forecast, xf, and an analysis, xa, as well as their respective error covariance matrices, Pf and
Pa. The KF equations are (subscripts denote the time step) as follows: xf
n ¼ Mn1xa
n1;
ð2aÞ
Pf
n ¼ Mn1Pa
n1MT
n1 þ Qn1;
ð2bÞ
xa
n ¼ xf
n þ Kn½yn Hnxf
n;
ð2cÞ
Kn ¼ Pf
nHT
n½Rn þ HnPf
nHT
n1;
ð2dÞ
Pa
n ¼ ½I KnHnPf
n:
ð2eÞ xf
n ¼ Mn1xa
n1;
ð2aÞ
Pf
n ¼ Mn1Pa
n1MT
n1 þ Qn1;
ð2bÞ
xa
n ¼ xf
n þ Kn½yn Hnxf
n;
ð2cÞ
Kn ¼ Pf
nHT
n½Rn þ HnPf
nHT
n1;
ð2dÞ
Pa
n ¼ ½I KnHnPf
n:
ð2eÞ ð2aÞ ð2eÞ Equation (2a) represents the forecast of the model fields from time step n - 1 to n,
while Eq. (2b) calculates the forecast error covariance from the analysis error covariance
Pa and the model error covariance Q. Equations (2c) and (2e) are the analysis steps, using
the Kalman gain defined in Eq. (2d). Q and Pa are assumed to be uncorrelated. For
optimality, all errors must be uncorrelated in time. The KF can be generalized to nonlinear H and M operators, although in this case neither
the optimality of the analysis nor the equivalence with 4D-Var holds. The resulting
equations are known as the Extended Kalman filter (EKF) as, for example, used for the
land surface by Boulet et al. (2002), Reichle et al. (2002b), Matgen et al. (2010), Ru¨diger
et al. 4.2 Data Assimilation Methods (2010) and de Rosnay et al. (2012b). The Ensemble Kalman filter, EnKF, uses a Monte Carlo ensemble of short-range
forecasts to estimate Pf. The estimation becomes more accurate as the ensemble size
increases. The EnKF is more general than the EKF to the extent that it does not require
validity of the tangent linear hypothesis. Evensen (2003) provides a comprehensive review
of the theory and numerical implementation of the EnKF. Examples for the land surface
are identified in Table 4 (see below). The Particle Filter (PF) is also an ensemble method. It does not require a specific form
for the state distribution but, typically, a re-sampling algorithm needs to be applied (van
Leeuwen 2009). Because PF methods typically make no assumptions of linearity in the
model equations or that model and observational errors are Gaussian, they are well suited
to deal with the land surface where model evolution is highly nonlinear, and model and
observational errors can be non-Gaussian. The PF has been applied in hydrology to esti-
mate model parameters and state variables (e.g., Moradkhani et al. 2005a; Weerts and El
Serafy 2006; Plaza et al. 2012; Vrugt et al. 2012). 4.3 Representation of Errors Representation of errors is fundamental to data assimilation. One needs to consider errors
in observations, background information and model (see Eqs. (1, 2a–e) above for identi-
fication of the error covariance matrices mentioned in the following). R, the observational 3 3 Surv Geophys Table 4 Selected studies on land surface data assimilation, sorted by assimilated observation type
Observation
State
Parameter/model
EnKF/EnKS
Other
Soil moisture,
retrievals
Reichle and Koster
(2005), Ni-Meister et al. (2006), Reichle et al. (2007, 2008), Kumar
et al. (2009), Pan and
Wood (2010), Liu et al. (2011), Han et al. (2012a), Draper et al. (2012), Sahoo et al. (2013)
Houser et al. (1998),
Pauwels et al. (2002),
Paniconi et al. (2003),
Francois et al. (2003),
Hurkmans et al. (2006),
Parajka et al. (2006),
Crow (2007), Crow and
Bolten (2007), Parada
and Liang (2008), Crow
and van den Berg
(2010), Draper et al. (2009), Mahfouf
(2010), Dharssi et al. (2011), de Rosnay et al. (2012a, b)
Santanello et al. (2007),
Ines and Mohanty
(2009), Pauwels et al. (2009)
Soil moisture, in situ
Sabater et al. (2007), De
Lannoy et al. (2007a,
2009), Camporese et al. (2009), Monsivais-
Huerteroet et al. (2010),
Han et al. (2012a)
Calvet et al. (1998),
Wingeron et al. (1999),
Walker et al. (2001b,
2002)
Boulet et al. (2002), De
Lannoy et al. (2006),
Vereecken et al. (2008),
Loew and Mauser
(2008), Nagarajanar
et al. (2011)
Snow cover or
albedo, retrievals
Clark et al. (2006), Su
et al. (2008, 2010), De
Lannoy et al. (2012),
Arsenault et al. (2013)
Rodell and Houser
(2004), Zaitchik and
Rodell (2009), Saha
et al. (2010), de Rosnay
et al. (2012a, b)
Essery and Pomeroy
(2004), De´ry et al. (2005), Kolberg and
Gottschalk (2010),
Dumont et al. (2012)
Snow water
equivalent,
retrievals or in situ
Andreadis and
Lettenmaier (2006),
Slater and Clark (2006),
Dong et al. (2007),
De Lannoy et al. (2010,
2012), He et al. (2012)
Brasnett (1999), Sun et al. (2004), Drusch et al. (2004)
Clark and Vrugt (2006),
Clark et al. (2011b),
Su et al. (2011)
Backscatter, from soil
or vegetation
Flores et al. (2012)
Hoeben and Troch
(2000), Zhan et al. (2006)
Marzahn and Ludwig
(2009), Nearing et al. (2010)
Brightness
temperature, for
soil or vegetation
Margulis et al. (2002),
Reichle et al. (2002a),
Crow (2003), Crow and
Wood (2003), Dunne
and Entekhabi (2006)
Entekhabi et al. (1994),
Galantowicz et al. (1999), Crosson et al. 4.3 Representation of Errors (2002), Reichle et al. (2001a, b), Jones et al. (2003), Wilker et al. (2006), Balsamo et al. (2006), Loew et al. (2009), Dumedah et al. (2011)
Zhang et al. (2011),
Montzka et al. (2012),
De Lannoy et al. (2013)
Brightness
temperature, for
snow
Durand and Margulis
(2007), Durand et al. (2009)
DeChant and Moradkhani
(2010)
Tedesco et al. (2010),
Vachon et al. (2010),
Forman et al. (2012a) 123 12 Surv Geophys Table 4 continued
Observation
State
Parameter/model
EnKF/EnKS
Other
Surface soil or skin
temperature,
evapotranspiration,
retrievals or in situ
Pipunic et al. (2008),
Ghent et al. (2010),
Reichle et al. (2010),
Xu et al. (2011)
Castelli et al. (1999),
Lakshmi (2000),
Boni et al. (2001),
Schuurmans et al. (2003), Bosilovich et al. (2007), Renzullo et al. (2008), Sini et al. (2008), Meng et al. (2009), Barrett and
Renzullo (2009),
Mackaro et al. (2011)
Caparrini et al. (2004),
Kalma et al. (2008),
Gutmann and Small
(2010)
Water stage,
retrievals
Andreadis et al. (2007),
Durand et al. (2008),
Biancamaria et al. (2010)
Matgen et al. (2010),
Giustarini et al. (2011)
Montanari et al. (2009)
Terrestrial water
storage, retrievals
Zaitchik et al. (2008), Su
et al. (2010), Li et al. (2012), Forman et al. (2012b)
–
Gu¨nter (2008), Lo et al. (2010)
Discharge, gauge
Weerts and El Serafy
(2006), Vrugt et al. (2006), Pauwels and De
Lannoy (2006, 2009)
Aubert et al. (2003),
Moradkhani et al. (2005a), Seo et al. (2009), Lee et al. (2011), Vrugt et al. (2012)
Madsen (2003),
Moradkhani et al. (2005b), Montanari and
Toth (2007), Vrugt
et al. (2008), Quets
et al. (2010)
Leaf area index,
remotely sensed
Pauwels et al. (2006),
Nearing et al. (2012)
Jarlan et al. (2008),
Albergel et al. (2010),
Ru¨diger et al. (2010)
Lewis et al. (2012)
Screen-level
observations
–
Balsamo et al. (2004),
Seuffert et al. (2004),
Drusch and Viterbo
(2007), Mahfouf et al. (2009), Draper et al. (2011), Mahfouf and
Bliznak (2011)
–
Synthetic observation studies are classified by the observation type that is mirrored. For land surface
(-coupled) state updating, the studies are divided into sets using either the EnKF or EnKS (Ensemble
Kalman Smoother) and those using any other assimilation technique. For parameter and model structure
updating, examples relate to either forward models or land surface(-coupled) models Synthetic observation studies are classified by the observation type that is mirrored. 4.3 Representation of Errors For NWP many
assimilation schemes now incorporate a bias correction, and various techniques have been
developed to correct observations to remove biases (e.g., Dee 2005); these methods are
now being applied to land data assimilation (De Lannoy et al. 2007a, b). 4.4 Advantages and Disadvantages of Assimilation Methods 4.4 Advantages and Disadvantages of Assimilation Methods The feasibility of 4D-Var has been demonstrated in NWP systems (see, e.g., Simmons and
Hollingsworth 2002). Its main advantage is that it considers observations over a time
window that is generally much longer than the model time step, that is, it is a smoothing
algorithm. This allows more observations to constrain the system and, considering satellite
coverage, increases the geographical area influenced by the data. For nonlinear systems (as
is generally the case for the land surface), this feature of 4D-Var, together with the non-
diagonal nature of the adjoint operator which transfers information from observed regions
to unobserved regions, reduces the weight of the background error covariance matrix in the
final 4D-Var analysis compared to the KF analysis (for linear systems, the general
equivalence between 4D-Var and the KF implies that the same weight is given to all data in
both systems). In contrast to the above advantages of 4D-Var, three weaknesses must be mentioned. First, its numerical cost is very high compared to approximate versions of the KF or
ensemble methods. Second, its formalism cannot determine the analysis error directly;
rather, it has to be computed from the inverse of the Hessian matrix (again, this procedure
is prohibitive in both computation time and memory). Finally, its formalism requires the
calculation of the adjoint model, which is time-consuming and may be difficult for a
system such as the land surface which exhibits nonlinearities and on–off processes (e.g.,
presence or lack of snow). The EKF is capable of handling some departure from Gaussian distributions of model
errors and nonlinearity of the model operator. However, if the model becomes too non-
linear or the errors become highly skewed or non-Gaussian, the trajectories computed by
the EKF will become inaccurate. The EnKF is attractive as, for example, it requires no derivation of a tangent linear
operator or adjoint equations and no integrations backward in time, as for 4D-Var (see
Evensen 2003). The EnKF also provides a cost-effective representation of the background
error covariance matrix, Pf. 4.3 Representation of Errors For land surface
(-coupled) state updating, the studies are divided into sets using either the EnKF or EnKS (Ensemble
Kalman Smoother) and those using any other assimilation technique. For parameter and model structure
updating, examples relate to either forward models or land surface(-coupled) models error covariance matrix, is typically assumed to be diagonal, although this is not always
justified. R includes errors of the measurements themselves, E, and errors of representa-
tiveness, F; R = E ? F. B is the background error covariance matrix in variational
methods (the analogue in the KF and ensemble methods is Pf); its diagonal elements
determine the relative weight of the forecasts, and its off-diagonal elements determine how
information is spread spatially. Estimating B or Pf is a key part of the data assimilation
method (Bannister 2008a, b). Estimating model error Q is a research topic. In the EnKF, the background (or forecast) errors are represented by the spread of the
ensemble. This simplifies the computation of Pf, implicitly accounts for the model error
Q and avoids the calculation of Eq. (2b). For land data assimilation, the relative fraction of 123 Surv Geophys the observation error R and the model error Q (associated with the temporal evolution of
the model) is often tuned or adaptively updated (e.g., Desroziers et al. 2005; Reichle et al. 2008). the observation error R and the model error Q (associated with the temporal evolution of
the model) is often tuned or adaptively updated (e.g., Desroziers et al. 2005; Reichle et al. 2008). In general, in data assimilation, errors are assumed to be Gaussian. The most funda-
mental justification for assuming Gaussian errors, which is entirely pragmatic, is the rel-
ative simplicity and ease of implementation of statistical linear estimation under these
conditions. Because Gaussian probability distribution functions are fully determined by
their mean and variance, the solution of the data assimilation problem becomes compu-
tationally practical. Note that the assumption of a Gaussian distribution is often not jus-
tified in land data assimilation applications. Typically, there are biases between different observations, and between observations
and model (see, e.g., Me´nard 2010). These biases are spatially and temporally varying, and
it is a major challenge to estimate and correct them. Despite this, and mainly for pragmatic
reasons, in data assimilation it is often assumed that errors are unbiased. 4.3 Representation of Errors Several issues need to be considered in developing the EnKF:
(1) ensemble size; (2) ensemble collapse; (3) correlation model for Pf, including locali-
zation (see, e.g., Kalnay 2010); and (4) specification of model errors. 123 Surv Geophys The major drawback of the above techniques is the underlying assumption that the
model states have a Gaussian distribution. The PF does not require a specific form for the
state distribution, but its major drawback is that distribution of particle weights quickly
becomes skewed, and a re-sampling algorithm needs to be applied. The EnKF and PF are complementary. This complementarity makes a hybrid EnKF/PF
version highly attractive for systems that can exhibit nonlinear and non-Gaussian features,
an example being the land surface. For example, the EnKF could be used as an efficient
sampling tool to create an ensemble of particles with optimal characteristics with respect to
observations. The PF methodology could then be applied on that ensemble afterwards to
resolve nonlinearity and non-Gaussianity in the system. This method is getting increased
attention (see, e.g., Kotecha and Djuric´ 2003). 4.5 Example of a Land Data Assimilation System 4.5 Example of a Land Data Assimilation System 4.5 Example of a Land Data Assimilation System 4.5 Example of a Land Data Assimilation System For illustrative purposes, we describe the elements of the NILU SURFEX-EnKF land data
assimilation system (Lahoz et al. 2010b). These elements are the following: (1) a data
assimilation scheme (mainly variants of the EnKF, but also variants of the PF, and the
EKF); (2) a land surface model (SURFEX model developed at Me´te´o-France, Le Moigne
2009); (3) observations; (4) the observation operator; and (5) error characteristics for the
model and the observations. The SURFEX model used at NILU (and at Me´te´o-France) can be run in uncoupled or
coupled mode. It includes the following elements: •
A soil and vegetation scheme: ISBA and ISBA-A-gs; •
A water surface scheme: COARE/ECUME (Coupled Ocean–Atmosphere Response
Experiment/Exchange Coefficients from Unified Multi-campaign Estimates) for the
sea; FLAKE for inland water; •
A water surface scheme: COARE/ECUME (Coupled Ocean–Atmosphere Response
Experiment/Exchange Coefficients from Unified Multi-campaign Estimates) for the
sea; FLAKE for inland water; •
Urban and artificial areas: Town Energy Balance—TEB model; •
A surface boundary layer (SBL) scheme; •
Chemistry and aerosols; •
A land use database: ECOCLIMAP. Figure 3 illustrates how SURFEX works. During a model time step, each surface grid
box receives from the atmosphere the following information: upper air temperature, spe-
cific humidity, horizontal wind components, pressure, total precipitation, long-wave
radiation, short-wave direct and diffuse radiation and, possibly, concentrations of chemical
species and dust. In return, SURFEX computes averaged fluxes of momentum, sensible and
latent heat, and, possibly, chemical species and dust fluxes. These fluxes are then sent back
to the atmosphere with the addition of radiative terms like surface temperature, surface
direct and diffuse albedo, and surface emissivity. The above information transferred to the atmosphere from the land surface provides the
lower boundary conditions for the radiation and turbulent schemes in an atmospheric
model coupled to SURFEX or forced by SURFEX output. In SURFEX, each grid box is
made up of four adjacent surfaces: one for nature, one for urban areas, one for sea or ocean
and one for lake, identified by the global ECOCLIMAP land database. The SURFEX fluxes
are the average of the fluxes computed over nature, town, sea/ocean or lake, weighted by
their respective fraction. The assimilation system at NILU is illustrated in Fig. 4 with reference to the EnKF. It
can assimilate the following data: (1) 2-m screen-level temperature (T2m) and 2-m screen-
level relative humidity (RH2m) provided, for example, by the SYNOP/CANARI 123 Surv Geophys Fig. 4.6 Data Assimilation Research Applications Table 4 shows a selection of studies using a variety of observation types to improve the
land surface state or the state in a hydraulic, vegetation or snow model coupled to it. Because of its success in highly nonlinear land surface modelling (Reichle 2008), the
EnKF has gained a lot of attention. Therefore, state estimation studies using an EnKF or
EnKS (Ensemble Kalman Smoother, where the time integration is done forwards and
backwards) are organized separately from those that use any other assimilation technique
(e.g., variational, optimal interpolation). Also shown are a few examples on parameter
estimation in land surface or forward models. While this review focuses on state estima-
tion, parameter estimation and forcing correction are of utmost importance in land surface
models. Land surface models are not chaotic and thus benefit less from state estimation
than atmospheric or oceanic applications. By contrast, parameters and forcings determine
the major part of the land surface model uncertainty, and great advances can be expected
from combining state, bias, parameter and forcing estimation (Moradkhani et al. 2005b; De
Lannoy et al. 2006; Vrugt et al. 2012). Here, we discuss a number of soil moisture and
snow-related studies done mainly for state updating, with particular attention to the con-
ceptual problems they address. Examples on evapotranspiration, surface or skin temper-
ature, LAI (leaf area index), discharge and water stage assimilation are also provided in
Table 4, but not discussed in detail. 4.5 Example of a Land Data Assimilation System 3 Exchanges between the atmosphere and land surface implemented in the SURFEX LSM. See text. Based on Le Moigne (2009) Fig. 3 Exchanges between the atmosphere and land surface implemented in the SURFEX LSM. See text. Based on Le Moigne (2009) Fig. 3 Exchanges between the atmosphere and land surface implemented in the SURFEX LSM. See text. Based on Le Moigne (2009) Fig. 4 Schematic of the NILU SURFEX-EnKF land DA system methodology. From Lahoz et al. (2010b) Fig. 4 Schematic of the NILU SURFEX-EnKF land DA system methodology. From Lahoz et al. (201 (Code d’Analyse Ne´cessaire a` Arpege pour ses Rejets et son Initialisation; Taillefer 2002)
analysis; and (2) superficial soil moisture content data from satellites (e.g., from ASCAT,
AMSR-E and SMOS). The control variables (Nichols 2010) of the NILU land DA system
are the following: •
Surface temperature; •
Mean surface temperature; •
Superficial volumetric water content; •
Mean volumetric water content of the root zone. 123 Surv Geophys 4.6 Data Assimilation Research Applications 4.6.1 Single-column Applications Because of the large impact of
parameters and forcings on soil moisture errors and biases, assimilation schemes have paid
increasing attention to including parameter estimation along with state updating, as, for
example, illustrated in Monsivais-Huerteroet et al. (2010). At present, EnKF filtering
experiments are being conducted at point-scales to further identify and address conceptual
problems with soil profile estimation, using surface observations (see, e.g., Han et al. 2012a). Another important conceptual problem with soil moisture assimilation, initially
addressed in a point-scale setting, is the direct assimilation of radiances or assimilation
using an observation operator. This is done to avoid inconsistencies between auxiliary
information that would be used in retrievals and that used in the land surface models. Entekhabi et al. (1994) estimated 1-m-deep bare soil moisture profiles using synthetic
microwave brightness temperatures. This work was extended by Galantowicz et al. (1999)
using eight days of L-band brightness temperature (Tb) data collected from a test plot in
Beltsville, USA. Pathmathevan et al. (2003) assimilated microwave observations with a
variational technique, but using a heuristic optimization, rather than an adjoint. Crosson
et al. (2002) tested Tb assimilation at the point-scale with an EKF and showed that biases
could not be overcome through assimilation. Crow (2003) successfully assimilated Tb for
soil moisture and showed improvements at the plot-scale, using either synthetic or real field
data. Crow analysed the EnKF performance in terms of the assumptions that underlie the
KF. Crow and Wood (2003) also used the EnKF at two sites within the Southern Great
Plains 1997 (SGP97) experimental domain and reported that Tb data assimilation was able
to correct for rainfall errors. Wilker et al. (2006) highlighted the difficulty in mapping
heterogeneous soil moisture into Tb using a forward operator and identified the repre-
sentativeness errors associated with these data. Similar to the above studies, Hoeben and
Troch (2000) used a KF including a forward backscatter model to explore the direct
assimilation of radar microwave signals to estimate soil moisture profiles. Snow data assimilation has conceptual problems inherent to the cumulative and temporary
nature of this variable. Slater and Clark (2006) illustrated how a square root EnKF could
improve the snow state at in situ sites in Colorado during the accumulation and melt phase. They also identified the temporal correlation in snowpacks and showed how it could limit the
efficiency of filtering if not accounted for properly. 4.6.1 Single-column Applications To explore the possibilities and limitations of assimilation schemes, numerous studies have
first explored single point-scale or grid cell-scale applications. For soil moisture assimi-
lation, conceptual problems include the propagation of information from the surface to the
entire soil profile; the optimization of assimilation techniques and update frequencies; and
the identification of an allowable level of uncertainty in surface observations to be useful in
a data assimilation scheme, mostly in view of satellite sensor design. Georgakakos and Baumer (1996) performed a sensitivity study to document the impact
of observation noise on Kalman filter (KF) results. Calvet et al. (1998) and Wingeron et al. (1999) assimilated surface soil moisture data from a soil profile in the highly instrumented
field site of the Monitoring the Usable soil Reservoir EXperiment (MUREX) in France to
update root zone soil moisture using variational approaches and investigated the impor-
tance of assimilation windows and observation frequencies. Similarly, Li and Islam (1999)
studied the effect of assimilation frequency while directly inserting gravimetric mea-
surements as surrogates for remote sensing data, and Aubert et al. (2003) suggested that a
1-week soil moisture update is sufficient. Walker et al. (2001a) showed in a synthetic
profile study that the KF was superior to direct insertion. In a subsequent study with real
data from the Nerrigundah catchment in Australia, Walker et al. (2001b) articulated the
idea that soil moisture assimilation can solve issues with errors in forcings or initial
conditions, but not errors caused by problems in the physics of the soil model. De Lannoy et al. (2007a) used an EnKF to study vertical information propagation, and
the effect of assimilation depth and frequency for an extensive set of soil profiles in an
USDA field in Beltsville, USA. This study highlighted the effect of bias propagation
through the profile and the need for bias estimation, a conceptual problem that was
addressed with a two-stage forecast and bias filter (De Lannoy et al. 2007a, b). At the same
time, Sabater et al. (2007) studied the concept of propagating surface observations to
deeper model layers using different types of filtering, using ground data from the Surface 123 Surv Geophys Monitoring of the Soil Reservoir EXperiment, SMOSREX. Camporese et al. (2009) set up
synthetic soil profile assimilation experiments studying the effect of uncertainties,
ensemble size, bias and other factors with an EnKF. 4.6.1 Single-column Applications In a synthetic study, Liston and Hiemstra
(2008) proposed a technique to update snow retroactively, which would be useful for re-
analysis applications, if observations would only be available at the end of the snow season. In situ snow data assimilation is performed operationally (see Sect. 4.7 below), usually with
simple assimilation techniques. An example where both the snow state and parameters were
estimated using an EnKF in a 1-D setting is given by Su et al. (2011). A number of point- or single-grid-scale studies have tried to relate brightness temper-
ature data to snowpack characteristics (Durand et al. 2008; Andreadis et al. 2008), in
preparation for Tb assimilation. Many of these studies highlight the large sensitivity of
snowpack estimates to model parameters (Davenport et al. 2012), which makes both
forward simulation and inversion of Tb observations for SWE estimation a difficult task. 4.6.2 Distributed Applications The most obvious advantage of remotely sensed observations is the possibility of per-
forming large-scale and spatially distributed assimilation. It should be recognized, Surv Geophys however, that despite the spatial coverage of data, for computational reasons assimilation is
often performed per column, that is, using a 1-D filter. When the vertical columns (of snow
or soil) are horizontally connected through the model physics or assimilation statistics, this
is referred to as 3-D assimilation. however, that despite the spatial coverage of data, for computational reasons assimilation is
often performed per column, that is, using a 1-D filter. When the vertical columns (of snow
or soil) are horizontally connected through the model physics or assimilation statistics, this
is referred to as 3-D assimilation. The assimilation of catchment-distributed soil moisture has often focused on the
improvement of the state or initial conditions (Pauwels et al. 2001, 2002) and parameters in
order to improve spatially integrated fluxes, such as discharge. However, it is also possible
to use soil moisture assimilation to correct rainfall estimates (Crow and Ryu 2009). At the
global scale, soil moisture assimilation will become increasingly important when coupled
to the atmosphere for climate and seasonal predictions. Spatially distributed studies initially focused on assimilation of retrievals with simple
techniques and gradually developed towards more complex schemes, with the inclusion of
forward models (observation operators) to directly assimilate, for example, microwave
observations. Initial soil moisture retrieval studies explored the performance of different
filter techniques, such as Newtonian nudging, statistical correction and statistical inter-
polation (Houser et al. 1998; Pauwels et al. 2001; Paniconi et al. 2003; Hurkmans et al. 2006), while during the last decade, variational and KF-based assimilation largely domi-
nated this research field because of the proven robustness and flexibility of these latter
techniques (Reichle et al. 2002a, b). A typical conceptual problem with spatially distributed assimilation is the use of coarse-
scale remotely sensed data to infer fine-scale information. There are many static disag-
gregation techniques that use auxiliary information to perform such a downscaling outside
the assimilation scheme. Performing dynamic disaggregation within the assimilation
scheme remains a research challenge. The latter concept consists of a 3-D filter with
inclusion of spatially correlated (fine-scale) state and (coarse-scale) observation prediction
errors and has been addressed in EnKF frameworks by Reichle et al. (2001b, 2013),
Reichle and Koster (2003), Pan et al. (2009), De Lannoy et al. (2010) and Sahoo et al. 4.6.2 Distributed Applications (2013). An important issue connected to 3-D filtering for disaggregation is the use of local
observations to update neighbouring locations, for example, to propagate from observed
swaths to unobserved locations. Often, this problem is solved with spatial interpolation or
by relying on horizontal connections in the model equations (Walker et al. 2002). Alter-
natively, such horizontal information propagation can be done within an assimilation
scheme that provides accurate error correlations between observed and non-observed
observations and forecasts (Reichle and Koster 2003; De Lannoy et al. 2012). De Lannoy
et al. (2009) used an adaptive KF to identify such spatial correlations, along with the
magnitude of the forecast error, to optimize filter performance. Han et al. (2012b) studied
the effect of spatial correlations in an OSSE (observing system simulation experiment)
with a local ensemble transform Kalman filter. Filter technical issues such as update
frequency (Walker and Houser 2004) and error estimation have also been addressed in a
spatial context. Reichle and Koster (2005) demonstrated the validity of the concept that
assimilation results should be better than either the model or observations alone. After
re-scaling satellite observations from AMSR-E and SMMR to take bias out of the system,
Reichle et al. (2007) showed that satellite observations can contribute valuable informa-
tion, even if they are not accurate. Reichle et al. (2009) further assessed the quality of
assimilation products as a function of retrieval and land surface model uncertainty in an
OSSE and showed that soil moisture retrievals can have slightly less skill than the land
surface model and still contribute to an overall higher skill in the assimilation product. This
was confirmed in a real data assimilation study by Draper et al. (2012). 123 Surv Geophys The importance of correctly specifying random errors and biases is a major conceptual
challenge in the optimization of distributed assimilation systems. Bias mitigation has
become a regular part of most soil moisture data assimilation systems (Reichle and Koster
2004; Drusch et al. 2005; Kumar et al. 2012; Sahoo et al. 2013), and random error
specifications for soil moisture data assimilation have been studied through adaptive fil-
tering (Crow and van Loon 2006; Reichle et al. 2008). Another idea with potential benefit is multi-sensor assimilation for soil moisture esti-
mation. As an example, Draper et al. (2012) showed how both active (ASCAT) and passive
(AMSR-E) microwave retrievals can contribute to a similar improvement in assimilation
results. 4.6.2 Distributed Applications Combining improved precipitation data with soil moisture retrieval assimilation
(Liu et al. 2011) and combining discharge (Pauwels and De Lannoy 2006), temperature or
LAI with soil moisture assimilation are other avenues that have been exploited for
hydrological assimilation. As already indicated for single-column applications, a major conceptual problem is the
direct assimilation of brightness temperatures (Tb) or backscatter observations from
satellite missions for soil moisture estimation. Reichle et al. (2001a, b) presented pio-
neering studies with a 3-D variational scheme to assimilate and disaggregate synthetic or
real brightness temperatures over the SGP97 study area, while Margulis et al. (2002) used
an EnKF and Dunne and Entekhabi (2006) compared an EnKF with an EnKS for the same
Tb assimilation problem. Walker et al. (2002) also assimilated Tb directly, but from SMMR
and using an EKF over Australia. Using a variational scheme, and with inclusion of both a
land surface temperature and microwave brightness temperature observation operator,
Barrett and Renzullo (2009) showed that both thermal (AVHRR) and microwave (AMSR-
E) satellite observations can provide effective observational constraints on the modelled
profile and on surface soil moisture. There are only a few studies on spatially distributed
backscatter assimilation, but in a recent OSSE using an EnKF, Flores et al. (2012) showed
the potential of the L-band radar information expected from the future SMAP mission. p
p
For snow, spatially distributed assimilation studies include snow cover area (or snow
cover fraction) and snow water equivalent (SWE) assimilation. A correct specification of
the snow-covered area is important to represent feedbacks from the land to the atmosphere,
while a good estimate of the actual amount of snow in the snowpack is of crucial
importance for flood, drought and discharge predictions (He et al. 2012). Snow cover
observations are typically fine-scale visible/near infrared observations that are only
available in cloud-free areas, while SWE measurements are typically more inaccurate
retrievals from Tb observations at a coarse scale (see Table 1). It can be expected that
multi-sensor assimilation could help to further snow estimation (De Lannoy et al. 2012). Because of its binary nature, snow cover in terms of the presence or absence of snow
cannot be assimilated with filters that rely on continuous variables. Instead, rule-based
algorithms have been proposed (Rodell and Houser 2004; Zaitchik and Rodell 2009; Roy
et al. 2010). 4.6.2 Distributed Applications However, the snow cover fraction (SCF) is a more continuous variable that has
been assimilated with KF-based algorithms (Clark et al. 2006; Su et al. 2008; De Lannoy
et al. 2012). When assimilating SCF with a Kalman filter, there is a need to relate SCF to
the actual SWE state variable through an observation operator, often defined as a snow
depletion curve. It is also possible to use visible/near infrared snow albedo observations to
update snow parameters such as grain size (Dumont et al. 2012). For snow, spatially distributed assimilation studies include snow cover area (or snow
cover fraction) and snow water equivalent (SWE) assimilation. A correct specification of
the snow-covered area is important to represent feedbacks from the land to the atmosphere,
while a good estimate of the actual amount of snow in the snowpack is of crucial
importance for flood, drought and discharge predictions (He et al. 2012). Snow cover
observations are typically fine-scale visible/near infrared observations that are only
available in cloud-free areas, while SWE measurements are typically more inaccurate
retrievals from Tb observations at a coarse scale (see Table 1). It can be expected that
multi-sensor assimilation could help to further snow estimation (De Lannoy et al. 2012). Because of its binary nature, snow cover in terms of the presence or absence of snow
cannot be assimilated with filters that rely on continuous variables. Instead, rule-based
algorithms have been proposed (Rodell and Houser 2004; Zaitchik and Rodell 2009; Roy
et al. 2010). However, the snow cover fraction (SCF) is a more continuous variable that has
been assimilated with KF-based algorithms (Clark et al. 2006; Su et al. 2008; De Lannoy
et al. 2012). When assimilating SCF with a Kalman filter, there is a need to relate SCF to
the actual SWE state variable through an observation operator, often defined as a snow
depletion curve. It is also possible to use visible/near infrared snow albedo observations to
update snow parameters such as grain size (Dumont et al. 2012). The two dominant conceptual problems with satellite-based SWE assimilation are the
coarse-scale nature and high uncertainty of the measurements. Initial attempts to assimilate
SMMR or AMSR-E SWE retrievals only yielded marginal success (Andreadis and
Lettenmaier 2006; Dong et al. 2007), because of retrieval errors due to signal saturation, 123 Surv Geophys presence of liquid water in the snowpack and multiple other factors. 4.7 Towards Operational Land Data Assimilation 4.7 Towards Operational Land Data Assimilation Land surface processes and their initialization are of crucial importance to address the
challenge of seamless prediction from weather to seasonal and climate timescales (Palmer
et al. 2008). It is well established that high skill in short- and medium-range forecasts of
temperature and humidity over land requires proper initialization of soil moisture (Beljaars
et al. 1996; Douville et al. 2000; Mahfouf et al. 2000; Drusch and Viterbo 2007; van den
Hurk et al. 2008). A similar impact from soil moisture has been established for seasonal
forecasts (Koster et al. 2004a, b, 2011; Weisheimer et al. 2011). Initialization of snow
conditions also has a significant impact on forecast accuracy at weather timescales
(Brasnett 1999; Drusch et al. 2004). Operational land data assimilation has initially focused
on ingesting precipitation observations (e.g., Saha et al. 2010; Reichle et al. 2011), but
improved snow and soil moisture state updates are now emerging, as documented, for
example, for the ECMWF Integrated Forecasting System by de Rosnay et al. (2012a). An unprecedented operational land data assimilation product will be provided by the
Global Modeling and Assimilation Office (NASA GMAO) in the form of a level 4 satellite-
based soil moisture product (Reichle et al. 2012; De Lannoy et al. 2013). The assimilation
of SMAP brightness temperatures into the Goddard Earth Observing System land surface
model will yield a global root zone soil moisture product. 4.6.2 Distributed Applications To address the coarse-
scale issue, De Lannoy et al. (2010) proposed several 3-D filter options to disaggregate
SWE data and propagate data from observed swaths to unobserved regions. These tech-
niques showed great benefit in a synthetic data study. When using real AMSR-E retrievals
(De Lannoy et al. 2012), and with bias mitigation through re-scaling added to the system,
the assimilation analyses were affected by a lack of a realistic interannual signal in the
retrievals. To address the problems with SWE retrieval accuracy, the potential of direct radiance
assimilation has been investigated (Durand and Margulis 2006; Andreadis et al. 2008;
Durand et al. 2009; DeChant and Moradkhani 2010). However, these efforts rely on a good
description of the snowpack in the land surface model, which is not always available for
large-scale applications. To address this, Forman et al. (2013) developed an artificial neural
network as a computationally attractive forward model in readiness for large-scale radiance
assimilation. In preparation for the future SMAP mission, freeze–thaw assimilation (Bateni
et al. 2013) has been investigated, because of its importance in understanding the carbon
cycle. The above studies update either snow or soil moisture separately. A major challenge for
land data assimilation is making use of total water storage (TWS) observations from
GRACE, which include soil moisture, snow and other water components at a very coarse
scale (Table 1). Total water storage can be decomposed into soil and snow components and
disaggregated to finer scales (Zaitchik et al. 2008; Su et al. 2010; Forman et al. 2012;
Li et al. 2012; Reichle et al. 2013). 5.1 Assimilated Observations •
To be useful for model development and assimilation, the dominant modes (in space
and time) of the land system must be sampled; •
To be efficient for state updating, observations need to be available at a reasonable time
interval to capture short-term dynamical variations (cf. the importance of satellite
overpass frequency; Walker and Houser 2004; Pan and Wood 2010); •
To be efficient for state updating, observations need to be available at a reasonable time
interval to capture short-term dynamical variations (cf. the importance of satellite
overpass frequency; Walker and Houser 2004; Pan and Wood 2010); •
Observations must be collected in long enough historical records to identify long-term,
climatological, statistics for bias mitigation (Reichle and Koster 2004) or trend
identification; •
Observations need to be sampled at different spatial scales to capture both local and
global processes; •
There is a need to have a reasonable signal-to-noise ratio (e.g., SMAP’s target of
brightness temperature uncertainty is 1.3 K; Entekhabi et al. 2010a), and an uncertainty
in the error description appropriate for scientific studies; •
There is a need to relate observations to key system state variables, that is, there needs
to be system observability. 5.2 Forward and Retrieval Models, with Particular Reference to Radiances
and Backscatter Processes •
To achieve appropriate retrieval accuracy, there is a need to use advanced methods to
describe physical processes in radiative transfer models (RTMs); •
To achieve appropriate retrieval accuracy, there is a need to use advanced methods to
describe physical processes in radiative transfer models (RTMs); •
When assimilating radiances at large scales (e.g., from microwave sensors), there is a
need for calibration of RTMs (De Lannoy et al. 2013; Forman et al. 2013). •
When assimilating radiances at large scales (e.g., from microwave sensors), there is a
need for calibration of RTMs (De Lannoy et al. 2013; Forman et al. 2013). 5.3 Land Surface Models •
There is a need to use advanced methods to describe physical processes (this limits
structural uncertainty) and couple land surface models with models describing more
specialized processes such as run-off routing, dynamic vegetation or snow (Pauwels
et al. 2006); •
There is a need for consistent global parameter datasets to limit predictive uncertainty
due to parameter uncertainty; •
There is a need for high-quality forcing data (this limits input uncertainty), mainly for
precipitation (Maggioni et al. 2011; Reichle et al. 2011). 5.4 Data Assimilation Challenges 5 Conceptual Problems and Key Challenges To summarize, the conceptual problems in our understanding of the hydrological cycle
over land can be grouped by observing, modelling and data assimilation systems. These are
outlined below. 123 123 Surv Geophys 5.1 Assimilated Observations Open Access
This article is distributed under the terms of the Creative Commons Attribution License
which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the
source are credited. 5.5 Validation •
Needs ground observations with substantial spatial and temporal coverage; Needs tools to address scaling and representativeness errors (Crow et al. 2012);
d
i
d ff
i
lid i
i
(
kh bi
l 2010b) •
Needs tools to address scaling and representativeness errors (Crow et al. 201 •
Needs appropriate and effective validation metrics (Entekhabi et al. 2010b). Acknowledgments
This paper arose from the International Space Science Institute (ISSI) workshop
‘‘The Earth’s Hydrological Cycle’’, held at ISSI, Bern, Switzerland, on 6–10 February 2012. A NILU
internal project supported WAL. Thanks to Alexandra Griesfeller for providing Fig. 2. 5.4 Data Assimilation Challenges 5.4 Data Assimilation Challenges •
There is a need to fill in the spatial and temporal gaps in observations (Reichle and
Koster 2003; De Lannoy et al. 2012); •
There is a need to disaggregate data in space and time and into their individual
components (Forman et al. 2012; Reichle et al. 2013); •
There is a need to ingest directly radiances or backscatter information (as opposed to
retrievals) to avoid inconsistencies between auxiliary information in retrievals and land
surface models (Crow and Wood 2003; Durand et al. 2009; Flores et al. 2012); 123 Surv Geophys •
There is a need to exploit the simultaneous use of multiple sensors (Pan et al. 2008;
Draper et al. 2012) and explore the capabilities of new sensors (Andreadis et al. 2007;
Durand et al. 2008); •
There is a need to combine state and input (forcing) information with parameter
updates (Moradkhani et al. 2005b; Liu et al. 2011; Vrugt et al. 2012); •
There is a need to explore advanced filtering techniques, for example, the use of the
particle filter to account for non-Gaussian errors (Plaza et al. 2012); •
There is a need to improve the representation of observation and forecast errors, and to
specify biases in observational and model information (De Lannoy et al. 2007b; Crow
and Reichle 2008; Reichle et al. 2008; De Lannoy et al. 2009; Crow and van den Berg
2010); •
There is a need to preserve water balance in the land system (Pan and Wood 2006;
Yilmaz et al. 2011) and draw lessons from the information in the assimilation
increments; •
There is a need to have access to adequate computational resources. 5.5 Validation 6 Conclusions To understand the hydrological cycle over land, we need to make observations and develop
models that encapsulate our understanding. These models have a basis on the information
gathered from observations, as well as on previous experience, and are used to project our
understanding into the future by making predictions. A crucial element in this procedure is
confronting models with observations. Data assimilation, which combines observational
and model information, provides an objective method to confront models against obser-
vations and add value to both the model and the observations. Data assimilation adds value
to observations by filling the gaps between them and adds value to models by constraining
them with observations. In this paper, we touch on the main conceptual problems that limit
a full integration of land surface models and observations by reviewing progress in land
surface data assimilation research over the last decade. Collectively, the advent of new satellite missions, the increasing attention to forecast
uncertainty due to errors in the land surface model structure, parameters and input, and the
development of advanced assimilation techniques will eventually close the largest gaps in
our understanding of the hydrological cycle over land. Acknowledgments
This paper arose from the International Space Science Institute (ISSI) workshop
‘‘The Earth’s Hydrological Cycle’’, held at ISSI, Bern, Switzerland, on 6–10 February 2012. A NILU
internal project supported WAL. Thanks to Alexandra Griesfeller for providing Fig. 2. 123 123 Surv Geophys Appendix: Sensor acronyms Appendix: Sensor acronyms AACE
Australian Airborne Cal/Val Experiments (for SMOS)
AMSR
Advanced Microwave Sounding Radiometer on EOS Aqua
ASCAT
Advanced SCATterometer
ASTER
Advanced Spaceborne Thermal Emission and reflection Radiometer
AVHRR
Advanced Very High Resolution Radiometer
CONAE
COmisio´n Nacional de Actividades Espaciales (National Space
Activities Commission)—Argentina Space Agency
CoReH2O
COld REgions Hydrology high-resolution Observatory
ERS
European Research Satellite
ESA
European Space Agency
GOES
Geostationary Operational Environmental Satellite
GPM
Global Precipitation Measurement
GRACE
Gravity Recovery And Climate Experiment
JERS
Japanese Earth Resources Satellite
MODIS
MODerate resolution Imaging Spectroradiometer
MUREX
Monitoring of the Usable Reservoir EXperiment
NASA
National Aeronautics and Space Administration
SCLP
Snow and Cold Land Process
SMAP
Soil Moisture Active and Passive
SMMR
Scanning Multichannel Microwave Radiometer
SMOS
Soil Moisture and Ocean Salinity
SMOSMANIA
Soil Moisture Observing System-Meteorological Automatic Network
Integrated Application
SMOSREX
Surface MOnitoring of the Soil Reservoir EXperiment
SSM/I
Special Sensor Microwave Imager
SWOT
Surface Water Ocean Topography
TRMM
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1. INTRODUCTION –
A RATIONALE FOR THE STUDY At the heart of the Christian faith is a particular understanding of God,
and the task of theology is to give an account of that vision under specific
historical conditions. That is the challenge, burden, and privilege of
theology. This research focuses on how that assignment is undertaken in
contemporary Systematic Theology and proposes, in a modest manner, a
specific direction for the development of a doctrine of God. An academic discipline mediates the task of giving an account. What
Christians believe can obviously be expressed in myriad ways such as
doxologically and liturgically – that is first-order embodiment. People
convey their deepest convictions in prayers, hymns, and sermons. But
there is another rational, analytic, and coherent way to do this – that is the
domain of intellectual disciplines – that is second-order activity. From the
earliest attempts to present the truths of the Christian faith in a synthesis,
starting with Origen’s On first principles, theologians have described their
understanding of God. It is possible to write a history of this disciplinary
reflection, and how the intellectual constructions have changed over
time. One may even label this endeavour as “theo-epistemes”, to
borrow a notion from the philosopher Foucault.1 These descriptions or
constructions display deeper dynamics; thinking about God took place
according to the available patterns of knowledge that were current at the
time. The elements selected for discussion and the order of presentation
are all significant. This study focuses specifically on these elements: What
should be addressed in a Christian doctrine of God? The title of this short book – Considering the doctrine of God: Fragments
on Trinity, discourses, and time – conveys a number of important
emphases. This is no comprehensive theology of God; this is meta-
reflection, an investigation into the practice of writing a doctrine about
God. This is preliminary work, exhibiting thinking that is still in progress. It ponders construction; it is not the edifice itself. This research conveys
one firm conviction: the Christian approach to God should be consistently
trinitarian. In a Christian doctrine of God, the confession of God as triune
is central. This is the ultimate symbol in the Christian faith, the one element
that assigns meaning to everything else. A sense of time is critical; thinking
takes place in a historical moment against a background of work done
in the past, with a sense of the future. 2022 2022 Acta Theologica Supplementum 34 1
For Foucault (1970:xxii), an “episteme” refers to implicit rules that govern what is constituted as
forms of legitimate knowledge for a particular period. Venter the influences of the present context. It is reflection in conversation with
a wider community of thinkers; hence, the emphasis on discourse. Much
of the work is mere listening and communicating to the interested reader
what others have said. Systematic Theology happens in a network of fellow
scholars; there is contestation, diverse opinions, and mutual enrichment.2 the influences of the present context. It is reflection in conversation with
a wider community of thinkers; hence, the emphasis on discourse. Much
of the work is mere listening and communicating to the interested reader
what others have said. Systematic Theology happens in a network of fellow
scholars; there is contestation, diverse opinions, and mutual enrichment.2 A work like the present one is obviously the fruit of personal interest. But there may also be a more academic warrant. The attentive student of
Systematic Theology and of the evolving currents and trends, specifically
concerning the doctrine of God, may perceive two subtle developments: it
seems as if the enthusiasm for trinitarian adventures experienced since the
1970s is subsiding, and one no longer comes across major original works
on God. Few contemporary work can be compared to the comprehensive
and penetrating studies by Moltmann, Pannenberg, Jenson, Kasper, and
Jüngel, to mention a few. One could have given the present investigation
a different and more succinct title: Speaking God after the trinitarian
renaissance. The critical word is obviously “after”. Does it signify an end
and a new beginning, or the continuation after new insights have been
internalised? Maybe the historical moment is too ambiguous; there is still
too much contestation to suggest an answer. In light of the discussion
and of newer sensibilities, what should be the path forward? The work is
interested in a modest contribution to this question. It may be productive to examine briefly the doctrines of God presented
in three excellent monographs on “Dogmatics” and register some of the
trends observed therein. A theo-epistemic approach is interested in design,
rubrics, foci, as well as in dialogues or voices. The outstanding feature
of the work by Migliore (2014) is the consistent focus on the Trinity. The
biblical, historical, and constructive sections are preceded by a description
of the “problem of God in modern theology”, in which challenges such as
justice and feminism are mentioned. 1. INTRODUCTION –
A RATIONALE FOR THE STUDY Speaking and writing about the
Christian God is no sanitised practice, immunised from the pressures and 1 1 DOI: http://dx.doi.org/10.18820/23099089/actat.Sup34.1 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 2
See the massive study of Collins (1998) on the sociology of philosophy and the central role of
intellectual communities in the history of the development of ideas. Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 the structural design, one comes across the focus on the Trinity, which
is the “toegangspoort” to the doctrine of God (2012:83). The treatment
is informed by a thorough knowledge of contemporary discourses. Two
features are conspicuous: an interaction with the monotheism of Islam
and treatment of the so-called practical significance of the Trinity. This
is basically interpreted as a regulative function for doing theology, by
considering the various configurations of the mutual relations of the three
Persons (2012:109), and not as expected in social ethical terms. The
second major structural mark of the dogmatics by Van den Brink and Van
der Kooi is the space given in a separate chapter to the names of God
and the attributes. In this instance, one observes, again, the consistent
employment of trinitarian thought: it functions as a “ordeningsprincipe” for
the doctrine of the attributes (2012:137). The authors, however, still think
in terms of an old convention of distinguishing categories for attributes, in
this case those of “transcendentie en toewyding” (2012:137). According to
them, divine attributes do give a glimpse of the being of God. The dogmatics by Kärkkäinen (2019), a condensed version of his five-
volume study A constructive Christian theology for the pluralistic world,
is an impressive study. Kärkkäinen, a Finnish evangelical theologian
in the USA, has an acute sense of the changing global world and the
role of world religions. His doctrine of God is also from a trinitarian
perspective. Outstanding features of this approach are a discussion of the
contemporary secular world and the atheistic critique of faith in God; a
novel section attending to the panentheistic relation of God to the world; a
new identification of hospitality as attribute of God; an extensive attention
to world religions, and a trinitarian orientation of the problematic. The three studies by Migliore, Van den Brink and Van den Kooi, and
Kärkkäinen, although different in many respects, do evidence how
doctrines of God should be restated in light of changing scholarship
and social conditions. The fundamental optic of the Trinity, and how that
functions in speaking the divine, is arguably the dominant impression one
gets of the approaches. There are clearly novel issues requiring attention,
especially those in the work of Kärkkäinen. The proposal of this short study centres on four elements that will also
be the rationale for its structure. Venter Migliore’s (2014:69) central question
is: “Is it possible to retrieve and re-present the Christian doctrine of the
Trinity in a contemporary idiom and in all its revolutionary significance?”
He treats the divine attributes in a separate section, after the trinitarian
explanations, and follows to a great extent Barth with a dialectical pairing,
for example the love of God is vulnerable and yet unconquerable (2014:88). As a Calvinist, Migliore concludes his doctrine of God with the section on
divine election, but he views it in strict trinitarian terms. The Systematic Theology by Van den Brink and Van der Kooi (2012)
is a comprehensive study with a dominant Eurocentric orientation. In 2 2022 3
References indicated as “recommendations” will not be listed in the Bibliography again. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Acta Theologica Supplementum 34 A contemporary doctrine of God should
address, minimally: • A genealogy of how the Christian understanding of God has developed
over time and an indication of the current profile. • A genealogy of how the Christian understanding of God has developed
over time and an indication of the current profile. • A grammar of fundamental elements constituting an understanding of
God, accounting for the who, what and where questions. • A grammar of fundamental elements constituting an understanding of
God, accounting for the who, what and where questions. 3 2.1
Big history and multiple theo-trajectories Usually, a sense of historical development of notions of the divine is
conspicuously absent in treatments of the doctrine of God. The pattern
is basically generic: there is a departure from a stable notion of God and
then a discussion about trinitarianism. This has been the typical traditional
approach. One can easily substantiate this claim with an overview of
systematic doctrines of God, for example, of the last few decades. For
one embodiment of this approach, see the comprehensive work by Horton
(2011). More recent approaches realise that the split between one God and
the Trinity is untenable, and that one should start right away with the triune
God. This was clearly seen, for example, in the three examples referred
to in the Introduction by Migliore, Van den Brink and Van der Kooi , and
Kärkkäinen. But still, even in these works, there is no acknowledgement
that behind trinitarianism is a vast history of evolution of notions of the
divine. A theo-episteme, sensitive to the turn to history, especially since
the nineteenth century, should go in another direction. The radical shifts
in conceptualising the divine should be attended to. Only then could
one focus on the Trinity, but even that is under construction. What is
expressed, in this instance, is a sense of “big story”, of an understanding
of human civilisation as old and of drastic evolutionary changes that have
taken place over centuries. Excellent sources are also available in this
regard.4 The well-known book by Armstrong, A history of God (1993), is
an expression of what is at stake in this instance. There could obviously
be different approaches as to how to capture and categorise the historical
shifts. At fundamental level, one could speak of at least six trajectories:
(i) the origin of religion and the “birth of the gods”; (ii) tribal religion and
the emergence of Yahwism; (iii) the articulation of monotheism; (iv) the
formulation of metaphysical substantial trinitarianism; (v) the embrace of
relational trinitarianism, as well as the turn away from classical theism
and (vi) the development of anatheism. This proposal has its limitations; it
focuses on the genealogy of the Hebraic-Christian God: its predecessors
and possible future(s). A full history of the divine would have to include the
gods and notions of transcendence and ultimacy in other world religions. Venter • An approach as to how intellectual understanding or doctrinal
exposition can be integrated with a way of life. • An approach as to how intellectual understanding or doctrinal
exposition can be integrated with a way of life. • The current concern about contextuality, the imperative to engage with
present challenges and which would include the positionality of the
author him or herself. • The current concern about contextuality, the imperative to engage with
present challenges and which would include the positionality of the
author him or herself. This proposal will be argued with extensive referencing to resources in
recent scholarship. The contribution of this approach lies not only in the emphasis on the
continued importance and centrality of the Trinity, but also in a number of
novel suggestions: that a genealogy is indispensable for understanding the
current trajectory of God reflection; that a focus of divine action (the where
question) should be placed in the doctrine of God, and not in inferential
doctrines such as providence; that spirituality and doctrine should be tied
together, and that theo-construction deserves theological examination. It is hoped that this study will elicit further interaction and that it may
stimulate conversation about the way ahead of thinking God responsibly
and imaginatively in our time. The overall form and presentation has been intentionally designed: the
work comprises twenty-five short thematic reflections and discussions,
each one with recommended reading. The pithy and controlled lay-out
reinforces the preliminary and fragmentary character. These are initial
explorations towards a larger work. The literature recommendations
are crucial; they convey the sense of being part of larger discourses.3
The selected form may serve students and seasoned theologians by
introducing them to the scholarly questions and by motivating alternative
interpretations of the doctrine of God. Generating knowledge about God is understandably self-implicating. It betrays not only personal concerns and interests, but also personal
experiences and a personal fondness for specific thinkers and texts. 4 4 4 2022 Acta Theologica Supplementum 34 Emergence of monotheism The articulation of monotheism – the existence of one single divine
reality – is widely considered a major occurrence in the history of religion. Generally, this is distinguished from “monolatry” – the worship of one God
amidst the existence of others – and is associated with the Babylonian
exile. The impact of the events is obvious in Deutero-Isaiah (Isa. 40-55). The crisis led to intense theological reflection. What one encounters in
Deutero-Isaiah is a claim – “there is no other” – and an emphasis on the
uniqueness of YHWH. The connection between monotheism and exile is
fairly obvious and this major shift in God conceptualisation was based
on a fundamental conviction about the power of YHWH in history. The
impact on monotheism is a highly complex field of enquiry and discussion
is still continuing. Venter conditions, intellectual contestations, and ultimately corresponding
notions of the human self. Detailed studies would disclose how each
historical period perceives the divine and what attributes are ascribed
to God. It would also reveal the immense struggle to name the divine in
available thought patterns, for example, from Hebraic to Hellenistic to
German Idealistic to postmodern ones. Apart from historical integrity, a
genealogical approach would be heuristic; it would avoid anachronistic
mix-ups and it would assist the task for contemporary construals. In this
section, trajectories three to six – from the articulation of monotheism to
anatheism – will be briefly mapped. The remainder of the study will explore,
in consecutive chapters, some aspects of these four trajectories in greater
detail. The first two trajectories – origin of the gods and of Yahwism - will
not be discussed in this work, as they require sophisticated knowledge the
present author is somewhat reticent to engage with at this stage. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 2.1
Big history and multiple theo-trajectories Such an approach would situate Systematic Theology in a larger
intellectual context and express a stronger sense of historical
respectability. An approach acknowledging “trajectories” would reveal
a sense of the immense complex interplay between dominant patterns
of conceptualisation, specific thought categories, formative material 4
See, for example, Harari (2011), Christian (2018), and Fernández-Armesto (2019). 5 Considering the doctrine of God Emergence of substantial trinitarianism The next trajectory moves to the fourth century AD, with the formulation
of the trinitarian confession of the divine identity, employing metaphysical
categories of Greek thought available at that time. The importance of this
century for the establishment of the specific Christian understanding of
God can hardly be emphasised enough. At stake was the articulation that
God is one in substance/nature/essence, and simultaneously differentiated
as three Persons – Father, Son, and Spirit. It took the church a long time to
reach a satisfactory understanding of the unity and diversity of the divine. Two specific councils – Nicaea 325 and Constantinople 381 – are crucial
for the final terminology such as homoousios and the acceptance of the
so-called “Cappadocian settlement”. For the church, Sabellianism, the
appearances of one God in various forms, and subordinationism, Jesus as 6 Acta Theologica Supplementum 34 2022 unique but not of the same nature as the Father, were not acceptable. Two
intellectual achievements should be specifically identified: the use of the
term homoousios at Nicaea to name the co-substantiality of Jesus Christ
with the Father, and the Cappadocian insight that being is never “bare”,
but intrinsically personal and relational. Emergence of relational trinitarianism The next trajectory brings one to the twentieth century, with the articulation
of the so-called social Trinity, which forms part of the wider movement,
the “Trinitarian Renaissance”. The names of Barth and Rahner form the
background to the new interest. Theologians such as Moltmann and
Boff argue that God has a life of God’s own, that God’s very nature is
relational, and that God is a divine community. The starting point for
trinitarian reflection is the oikonomia, the work of God in history; the three
Persons should be taken seriously, and God is affected by history. The
relationship between immanent and economic Trinity and the reality of
history are central in the discourse. Several interpreters find the impetus
for the new interest already with Hegel and his turn to history and the quest
for new categories for dynamic thinking, specifically relational ones, and
the occupation with concrete history. The resistance to this re-imagining
has been and continues to be intense. The effects of the Trinitarian
Renaissance are fascinating: it revitalised a doctrine that many considered
arcane and without any practical relevance. The social ethical implications
became fairly obvious. Venter Christian tradition and with trinitarianism. To name God, in a postmodern
sense, as “The Impossible”, he suggests understanding God as the
Incomprehensible, and the Hidden. In this trajectory, one clearly finds new
categories of speaking, of a re-location of the presence of the divine, but
also an explicit framing in terms of the ethical. Christian tradition and with trinitarianism. To name God, in a postmodern
sense, as “The Impossible”, he suggests understanding God as the
Incomprehensible, and the Hidden. In this trajectory, one clearly finds new
categories of speaking, of a re-location of the presence of the divine, but
also an explicit framing in terms of the ethical. After this introductory and orientating section four of these trajectories
will be explored chronologically in greater detail and aspects of these will
be described in the ensuing sections. A full and systematic treatment of a
“genealogy” of God remains a future assignment. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Emergence of anatheism The final trajectory must be placed primarily within the discipline of
Continental Philosophy of Religion of the second half of the twentieth
century. A constellation of ideas converged in this instance: Heidegger’s
rejection of onto-theology and the ethical turn to “the face of the other” in
the philosophy of Levinas. One encounters a farewell to the metaphysical
God of classical theism, the so-called “omni-monster”. The (violent)
historical conditions of the twentieth century and the philosophical
critiques of Western metaphysics converged in postmodern philosophers
of religion such as Marion, Caputo, and Kearney. The power and action of
the divine has become immensely problematic; “God” should happen in
the ethical encounter with the Other. Substantial or relational categories of
thinking have been replaced by a dynamic one of “event”. Kearney’s notion
of anatheism aptly denotes this trajectory: it entails faith after atheism. The
work of the Christian systematic theologian Tracy should be mentioned in
this regard. He agrees with most of the emphases of these philosophers
of religion; yet he has a greater appreciation for some continuity with the 7 5
For a recent overview of the history of scholarship on Ancient Israelite religion, see Lewis
(2020:17-47).
6
Miller (2000a:393) views this in terms of continuity and discontinuities – common ground with the
wider religious world and differentiation from it. Recommended further reading The recommendations cover the spectrum of ideas raised in this section:
work on “big history”: an overview of human civilisation that also
attends to shifting patters of thought; one major proposal on the origin
of theistic ideas; a new and voluminous study on the development of
Israelite religion; a comprehensive volume on contemporary trinitarian
scholarship, and a work that maps post-theistic paths in the contemporary
religious landscape. Emery, G. & Levering, M. (Eds)
2011. The Oxford handbook to the Trinity. Oxford: Oxford University Press. https://doi.org/10.1093/oxfordhb/9780199557813.001.0001 Emery, G. & Levering, M. (Eds)
2011. The Oxford handbook to the Trinity. Oxford: Oxford University Press. https://doi.org/10.1093/oxfordhb/9780199557813.001.0001 FernÁndez-Armesto, F. 2019. Out of our minds: What we think and how we came to think it. London:
OneWorld. https://doi.org/10.1525/9780520974364 FernÁndez-Armesto, F. 2019. Out of our minds: What we think and how we came to think it. London:
OneWorld. https://doi.org/10.1525/9780520974364 Lewis, T.J. 2020. The origin and character of God: Ancient Israelite religion through the
lens of divinity. Oxford: Oxford University Press. https://doi.org/10.1093/
oso/9780190072544.001.0001 Smedes, T.A. 2016. God, iets of niets? De postseculiere maatschappij tussen geloof en
ongeloof. Amsterdam: Amsterdam University Press. Smedes, T.A. 2016. God, iets of niets? De postseculiere maatschappij tussen geloof en
ongeloof. Amsterdam: Amsterdam University Press. Torrey, E.F. 2017. Evolving brains, emerging Gods: Early humans and the origins of religion. New York, NY: Columbia University Press. https://doi.org/10.7312/torr18336 Torrey, E.F. 2017. Evolving brains, emerging Gods: Early humans and the origins of religion. New York, NY: Columbia University Press. https://doi.org/10.7312/torr18336 Torrey, E.F. 2017. Evolving brains, emerging Gods: Early humans and the origins of religion. New York, NY: Columbia University Press. https://doi.org/10.7312/torr18336 8 Acta Theologica Supplementum 34 2022 2.2
Monotheism and trauma How the Old Testament functions in Systematic Theology could be
a research theme of immense proportions. It is an open question as to
whether the developing state of scholarship is carefully addressed in
an interdisciplinary manner. There are definitely abundant references to
text verses, but, and this is my hunch, hardly any attention is paid to the
discourses on the Old Testament as a disciplinary field. There is a definite
lack of engagement with the field of History of the Religion of Israel. As
a rule, there is a propensity to focus on and appreciate the theologies
of the Old Testament. A historically sensitive doctrine of God cannot
do without an acquaintance with Israel’s faith as part of a religion with
many dimensions. Typical questions about the origin and development
of the conceptions of the divine belong to the discipline of History of
Religion, which is undergoing its own constant disciplinary shifts and
developments.5 This involves a theological datum (see Miller 2000a:393).6 I
suggest that the theological implication is twofold: How novel trajectories
emerge through human intellectual re-imagination, and what the shape of
a mutated image of God entails. This historical knowledge is mediated
through the discipline of History of Israelite Religion. These insights are
eventually helpful when speaking about re-construction in our time. The literature on the History of Israelite Religion includes a wide array
of focal interests such as, for example, different forms of religion, the
origin of Yahwism, the prevalence of aniconism, the issue about the
feminine, specifically Asherah, and iconography. Obviously, I cannot
attend to these in this purview. One aspect of the field, the relation to
the wider religious environment, should be briefly referred to, because
it may signal novel developments. In a most helpful and balanced
overview, Miller (2000b:23-29) categorises this in three ways: “Yahweh
out of the Gods”, “The Gods in Yahweh”, and “Yahweh against the
Gods”. The following issues are at stake in this instance: Israel was part
of a polytheistic world, and a close reading of the Old Testament texts
conveys that Yahweh split off from the El figure. Miller (2000b:25) points
to clan religion, Amorite religion, and Canaanite religion as the “matrix”
out of which Israel’s God developed. At the same time, elements of other
god figures such as, for example, elements of Baal were incorporated
and arguably have to do with an assumption of power. For a recent overview of the history of scholarship on Ancient Israelite religion, see Lewi
(2020:17-47). Miller (2000a:393) views this in terms of continuity and discontinuities – common ground with the
wider religious world and differentiation from it. Venter definitive identity formation, by placing YHWH in opposition to the other
gods, for example, the goddess figures. The focus and interest for Christian theology would obviously be in
YHWH as the God of Israel.7 Excellent studies are available, and one should
refer to these at least. The single best volume is arguably Römer’s The
invention of God (2015). In this comprehensive study, Römer discusses
the name itself, the geographical origin, the role of the Midianites, and
how YHWH became the God of Israel and Judah. He covers the historical
trajectories up to the articulation of monotheism. The work by Smith
(2001; 2002) on the early history of YHWH and on monotheism is solid and
deserves attention. A Christian doctrine of God should engage with these
and tease out the implications for a historically informed view. My interest
in this short section is specifically on the radical shift from monolatry to
monotheism, the shift from an exclusive concentration on YHWH to the
belief that there is ultimately only one deity. The crystallisation of monotheism – the existence of one single divine
reality – is widely considered a revolutionary occurrence in the history of
religion. Generally, this is distinguished from monolatry – the worship of
one God amidst a recognition of the existence of others – and the critical
insight in scholarship is the conviction that the emergence should be
linked to the Babylonian exile of 597/587 BCE. Albertz’s (1994a; 1994b)
two-volume History of Israelite religion is the main source consulted in this
instance. Albertz is also an established scholar on the exilic period.8 As to
the question of the age and origin of monotheism, Albertz opines that its
development was not pre-programmed from the beginning, but that it had
to be fought out in many social conflicts. He is convinced that something in
the Yahwistic religion led to the formulation of the prohibition of alien gods. Albertz (1994a:62) locates this tendency in the exclusiveness intrinsic to the
religion in the extraordinary combination of social and religious conditions
of the Exodus group from which it emerged. Under the experiences of
liberation and the extreme conditions of life in the wilderness, a personal
relationship, which had a certain intrinsic exclusiveness, developed with
Yahweh (1994a:175). As long as the fight was only for survival, a single
religious symbol sufficed; Yahweh had only one task – to secure their survival
(1994a:63). 7
This seemingly innocent title has intense layers of association. In a recent thorough study, Stahl
(2020) argues that this title has been neglected in academic scholarship, and that Judah took the
title over from Israel after their fall as ascription for YHWH to strengthen royal support after the
influx of immigrants to Jerusalem. This conveys the impression of how crucial titles were and what
historical and political ideologies were connected thereto.
8
See Albertz’s extensive study on the exilic period (2003). 2.2
Monotheism and trauma There is also the 9 Venter Venter Considering the doctrine of God Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 to monolatry or monotheism. Changed social circumstances increased
the possibilities for syncretism and polytheism. The fight for exclusiveness
was simultaneously a fight against social and political developments. It
was no coincidence that the monotheistic tendency was only realised fully
during the exile. On the way towards that, Albertz points out a number
of historical developments. In the Jehu revolution, the Yahweh religion
showed its intolerance, for the first time, and the exclusiveness took
on fanatical features. Hosea’s fundamental criticism of the monarchy
and his denouncement of the Yahweh cult occurred on the basis of an
appeal to the relationship with Yahweh in the early period, which acquired
a certain critical function. The Deuteronomic movement, with its reform
slogan “hear, Israel, Yahweh, our God, Yahweh is one” (Deut. 6:4), sought
to establish monolatry and mono-Yahwism in an attempt to safeguard
the identity of Israel and national unity (1994a:206). With the Deutero-
Isaiah group during the exile, one finds for the first time a formulation of
a consistent monotheism (1994b:417). Their concern was not to ensure
the sole worship of Yahweh like earlier theologians, but rather to make it
possible for the people in exile to have faith in the universal historical action
of their god Yahweh and to dispel anxiety (1994b:418). Albertz (1994b:418)
emphasises that this was a development within Israel, without external
stimuli, grounded in Yahweh’s power. The argument in the judgement
discourses pointed to the distinctive action of Yahweh in history. The crisis
led to intense theological reflection; what one encounters in Deutero-
Isaiah is a counter-narrative, and the claim is repeatedly made “there is
no other” (Isa. 45: 5, 6, 14, 18, 21, 22, etc.), and the uniqueness of YHWH
is emphasised (for example, Isa. 42:8, 43:10-11, 44:6, 8). The connection
between monotheism and exile is fairly obvious. There remain speculative
views about the intellectual dynamics behind Deutero-Isaiah and the
eventual impact of Judean self-construction. Albertz’s (1994b:414‑426)
view is exceedingly important for Deutero-Isaiah. It was produced by a
group of theologians gathered around a master who came from circles of
temple singers and cult prophets with nationalistic sentiments (1994b:415). The message of the text was fairly innovative, but also offensive, and was
based on a fundamental conviction about the power of YHWH in history
(1994b:416-418). Their re-interpretation of God tended to universalism,
and to a critique of domination. Venter This disposition towards one god does not automatically lead 10 2022 Blenkinsopp, J.
2008. The Midianite-Kenite hypothesis revisited and the origins of Judah.
Journal for the Study of the Old Testament 33(2):131-153. https://doi.
org/10.1177/0309089208099253 Venter situation of absolute political helplessness” (1994b:425). Smith (2001:193;
see entire section 179-194) refers to a “new stage of rhetoric” found in
Israelite religion. In this instance, something novel, a historical novum with
extensive ramifications emerged. The impact of the events, described in 2 Kings 24-25, on the conception
and transformation of Yahwism is found in Deuteronomy 4 as post-exilic
literature and then in Deutero-Isaiah (Isa. 40-55). Increasingly, one finds
studies that describe the political, social, and psychological devastation
of these events for Judah – deportations, violence against women,
children and the elderly, and plundering of the temple and the city. A most
fruitful shift in scholarship has been the explicit exploration of the exile in
terms of trauma studies; the name of Carr (see, specifically, 2014:67-90)
deserves special mention. In a recent article, Markl (2020) consolidated
research and extensively linked exile, monotheism, and trauma; he refers
to the “birth trauma of monotheism” (2020:2). Markl’s (2020:19) comment
is pertinent: “[T]he monotheistic claims in Deutero-Isaiah are a powerful
instrument of resilience against the background of Exile as cultural
trauma.” New trajectories in God thinking emerge where old ones no longer
capture the imagination. New ways to conceptualise the divine crystallise
in desperation. The impact on monotheism is a complex field of enquiry. The work
by the Egyptologist Assmann should be acknowledged in this context. Monotheism amounted to a transformation of the world; it shaped the
Western image of man, but it came at a “price” – exclusion; a certain
kind of truth emerged: absolute, metaphysical and fideistic (Assmann
2010:15). One could only speculate about the monotheistic self: the
human being who is more secure, more integrated, but also dangerously
prone to certainty. Lynch, M.J.
2014. Mapping monotheism: Modes of monotheistic rhetoric in the Hebrew Bible.
Vetus Testamentum 64:47-68. https://doi.org/10.1163/15685330-12341141 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Considering the doctrine of God Acta Theologica Supplementum 34 The fusion of the divine and political power
was dissolved (1994b:420, 424). In this instance, Albertz (1994b:421) notes
“a clear transformation of traditional notions”, which was also an “ongoing
process of reflection” (1994b:423). The monotheism, in this instance, had
two characteristics: one tendency towards universalism and another to
be critical of domination (1994b:420). It is particularly significant “to
point to the fact that Israel made the breakthrough to monotheism in a 11 Recommended further reading The following sources discuss the significance of the Midianite
provenance for the origin of Yahwism, various proposals in contemporary
scholarship on the origin of Israel’s God, the problematic nature of the
term “monotheism” and its occurrence in the Old Testament text, as well
as its potential impact. 12 Acta Theologica Supplementum 34 2022 Schwartz, R.M. 1997. The curse of Cain: The violent legacy of monotheism. Chicago, ILL:
University of Chicago Press. Sommer, B.D. 2016. Monotheism. In: J. Barton (ed.), The Hebrew Bible: A critical companion
(Princeton, NJ: Princeton University Press), pp. 239-270. https://doi.org/10. 1515/9781400880584-012 Van Oorschot, J. & Witte, M. (Eds)
2019. The origins of Yahwism. Berlin: W. de Gruyter. Van Oorschot, J. & Witte, M. (Eds)
2019. The origins of Yahwism. Berlin: W. de Gruyter. 9
See Huijgen’s excellent study (2017). Venter In this section, I will address the work of the Old Testament scholar W. Brueggemann. He is truly one of the great “God thinkers” of our time, and in
his prolific output he does not shy away from difficult textual traditions and
from going against the grain, sometimes fairly provocatively. Trinitarian
theology that values the Old Testament should consider his work and
come to terms with it in some way. In a discussion of “Brueggemann’s
God”, Fretheim (1998:25) opines that one finds in his theology a Reformed
restatement of the sovereignty of God with his emphasis on an unsettled
and unsettling God. According to Fretheim (1998:33), Brueggemann’s
remarkable range of descriptors for God such as “savage, odd, abusive,
mean-spirited, wild, self-indulgent, unreliable, unstable, capricious,
irascible, irrational, sulky” points to this sovereignty which admits of
no qualification. I now turn to Brueggemann’s major work, Theology of the Old
Testament (1997).10 The reader must be explicitly aware of certain
principles that are operative throughout his theology. First, he is forthright
about his anti-essentialist stance. In an important footnote (1997:65, n. 11), he also mentions the problematic nature of the speech/reality relation. According to him, speech leads reality in the Old Testament, and speech
constitutes reality. God is endlessly in the process of being rhetorically
reconstructed. Secondly, Western modes of thinking, characterised by a
refusal of ambiguity and a propensity to give universalising closure, do
not convey the testimony of the Old Testament. Brueggemann’s work
epitomises an attempt to retrieve and embody the typical Jewish mode
of thinking: discourse is polyvalent, and no particular attempt is made to
resolve contradictions. Rhetorical strategies of ambiguity, incongruity,
irony, and metaphor abound, and God is characteristically “in the fray”
(1997:83, 111). Israel’s life as a theological enterprise consisted in coming to terms
with YHWH as an elusive but dominating Subject. Their primary rhetorical
responsibility was to try and bring YHWH, who refused exhaustible
domestication, to adequate speech. Brueggemann’s proposal to employ
the metaphor of testimony as organisation principle for an entire theology
of the Old Testament is his specific contribution to this field of study. A
core testimony deals with verbal sentences, adjectival markings, and
nouns to characterise YHWH. These utterances disclose a profoundly
disjunctive rendering of YHWH, which forms the crux of Brueggemann’s
view. In the Old Testament, there is also evidence of a counter-testimony
that struggles with YHWH’s hiddenness, ambiguity, and negativity. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 10
All references will be to this work, unless indicated otherwise. 2.3
Plurality and complex characterisation An appeal to the Bible is obviously of central concern in the construction
of a doctrine of God. When focusing on the Trinity, it becomes a complex
undertaking, especially with reference to the Old Testament. It has
become untenable in critical scholarship to “find the Trinity” in the Old
Testament. Argumentation to establish some form of continuity between
the Old Testament and a full-blown classical trinitarian confession has
become more sophisticated.9 A second trend, and this applies to a more
general interest in God, is to be sceptical about levelling attempts, in
other words, approaching the canonical text as if there were stability and
univalence to the presentation of the divine. Noll’s (2001:16) words, “the
changeability in divine personality is the central motif in twentieth-century
biblical scholarship”, convey a wide assumption nowadays. At stake, in
this instance, is the recognition of intra-canonical plurality which is a major
challenge to biblical scholarship, to theology in general and, specifically,
to speaking God. The Bible attests to an immense variety of traditions, text
types, theologies, and interests. There are various rhetorical strategies
such as insensitivity, dismissal, or ingenious integration to come to
terms with it. My approach is to activate an antenna for this variety and
explore its implications. It is a more authentic approach to the historical
conditions of origination of the biblical texts, and it also corresponds to the
nature of the subject matter under consideration, namely the divine. One
of the specific tasks of a theology of the Trinity is to attend to the many
portrayals of YHWH in the Old Testament and to relate these specifically to
the fourth-century settled trinitarian doctrine of God. The identification and
formulation of this specific task differs from the typical discussion of “the
Trinity and the Old Testament”. Conventionally, the Trinity is associated
with love, the God who is for us. Recognition of Old Testament plurality
complicates this. 9
See Huijgen’s excellent study (2017). 13 Considering the doctrine of God Acta Theologica Supplementum 34 2022 his nature, being, or attributes. Brueggemann discusses the following
in depth: the God who creates (most mature testimony), who makes
promises (oddest testimony), who delivers (most revolutionary testimony),
who commands (most pervasive testimony), and who leads (most intimate
testimony). A second group of the core testimony pertains to the uses of
adjectives that are characteristic of Israel’s speech about YHWH, and they
bespeak relationship and classical vocabulary (omnipotent, omniscient,
omnipresent) as conspicuously absent; the primary propensity is to focus
on YHWH’s fidelity, and not on power. By using nouns to characterise YHWH, Israel assigns constancy and
substance to YHWH. Two categories of metaphors can be distinguished
in the Old Testament, namely governance and sustenance. The first group
of metaphors, which identifies YHWH as judge, king, warrior, and father,
pertains to the use of power (1997:233). Each of these nouns testifies to
a contradiction in the very character of YHWH, a positive and negative
inclination. Severity could be rational and relative to the maintenance of
order, but sovereignty runs, occasionally, beyond reason at a destructive
rate (1997:249). Brueggemann (1997:249) refers to “an ominous dimension
to Yahweh”. Israel does not begin its speech about YHWH with some generic
notion of God; YHWH provided the peculiar norms whereby “goodness”
was understood (1997:144). The largest thematisation concerning God is
that “YHWH is at the same time sovereign and faithful” (1997:283). The
“most crucial” utterance about YHWH, the “substance” of what could be
said about YHWH centres on the capacity for sovereignty and solidarity
(1997:268, 271). To Brueggemann’s mind, this insight confronted Israel
with “a massive Holy Problem”: at the core of YHWH’s life is a “profound
disjunction”, a “profound, unresolved ambiguity” (1997:227, 268, 311). When power or sovereignty and solidarity or fidelity are in relative balance,
the righteousness of YHWH – the most comprehensive category for
Old Testament theology (1997:303) – can be said to resolve the tension
(1997:283). A convergence of sovereignty and compassion renders a
coherent picture of YHWH’s constancy and reliability (1997:306). This
balance is, however, not everywhere present. Even though fidelity is
emphatically affirmed, sovereignty does not always converge with fidelity. The lack of self-restraint and an unfettered and recurring show of self-
assertion constitute the problematic. Especially the noun metaphors
disclose “a dimension of fierceness that tilts toward potential violence”
(1997:275). Venter Israel
is characteristically concerned with the action of YHWH, and not with 10
All references will be to this work, unless indicated otherwise. 14 Acta Theologica Supplementum 34 Venter During exile and especially by the imaginative efforts of prophets such
as Hosea, Jeremiah, and the late Isaiah, one finds a new development in
YHWH’s own life: metaphors of relationship between husband and wife,
and between parent and child transpose the theme of the covenant into
a practice of pathos (1997:298f). This establishes a major rearticulation
of YHWH; an upheaval in YHWH’s life and character (1997:302). Despite
these efforts to define YHWH by pathos, Brueggemann (1997:303) believes
that “the Old Testament witnesses to a persistent tension that does not
admit of resolution”. A process of cross-examination of the core testimony can be detected
in the Old Testament. The discrepancy between lived reality and the
core testimony occasioned questions about YHWH’s reliability, fidelity,
and sovereign power. Especially experiences of unbearable injustice
such as the exile led to complaints. Questions such as How long? Why? Where? establish the counter-testimony of Israel’s speech about YHWH. Similarly, the presence of disjunction, incongruity, and dissonance in the
textual articulation of YHWH is a theological datum and necessitates a
revised narrative about YHWH. Brueggemann deals with the evidence
in three categories: hiddenness, ambiguity, and negativity. The tension
between the core testimony and the counter-testimony belongs to the
very substance and character of the Old Testament faith (1997:400). This
dialectical and disputatious quality defines this faith. These textual voices
move Brueggemann to conclude that YHWH could at times be abusive,
contradictory, and unreliable. He even concludes that “YHWH is, on
occasion, an unprincipled bully, who will coerce, manipulate, and exploit to
have YHWH’s own way” (1997:362), and the “counter-testimony bespeaks
something profoundly unreliable about YHWH” (1997:372). What is the impact of this faith? Brueggemann (1997:296) mentions that
“One never knows whether YHWH will turn out to be a loose cannon”. He is
forced to admit: “Yes, the faith of Israel is not without anxiety” (1997:282),
“making a relationship with Yahweh endlessly demanding and restless”
(1997:227). In a seminal article, Brueggemann (1995:459) explores his
vision of the presentation of God for human self-construction: pluralism
invites a rethinking of the self; there could not be a one-dimensional self:
“‘Many selves of the self’ is in partnership with the ‘many-selved’ God”
(1995:460). As the divine self is under negotiation, so is the human self. Brueggemann’s creative and courageous view is exceedingly important
for a Christian and trinitarian approach to God. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 Old Testament theology must reckon with this ominous aspect:
“YHWH has a potential for extraordinary destructiveness” (1997:249,
275). Finally, YHWH “cares most about YHWH’s own self” (1997:290). 15 Considering the doctrine of God ,
1996. God: A biography. New York: Simon & Schuster. Patrick, D. 1981. The rendering of God in the Old Testament. Philadelphia, PN: Fortress. Patrick, D. 1981. The rendering of God in the Old Testament. Philadelphia, PN: Fortress Seibert, E.A. 2009. Disturbing divine behaviour: Troubling Old Testament images of God. Minneapolis, MN: Fortress. Seibert, E.A. 2009. Disturbing divine behaviour: Troubling Old Testament images of God. Minneapolis, MN: Fortress. Venter His distinction of a core
testimony from a counter-testimony, and his insistence that the God is not
a “patron of conventional truth” (2005:26), but One who remains elusive
and irascible. Interestingly, he (1995:467, n 9) remarks that his view is not 16 Acta Theologica Supplementum 34 2022 inimical to the Christian tradition, suggesting that “[t]he doctrine of the
Trinity allows for great elasticity in the discernment and articulation of
God”. What the pluralising in the Old Testament and God’s own pluralising
character imply for a rich multifaceted trinitarian conception should be
imagined. The frank acknowledgement of a variety of traditions, of a “dark
side”, and of God as character in a drama are advances in scholarship that
should be accounted for in a doctrine of God. Recommended further reading These few recommendations convey an impression of the plurality of
methodological approaches, of the potential of narrativity to understanding
God as “character”, and of problematic textual testimonies. Gordon, R.P. (Ed.)
2007. The God of Israel. Cambridge: Cambridge University Press. (
)
2007. The God of Israel. Cambridge: Cambridge University Press. Joyce, P.M. & Rom-Shiloni, D. (Ed.)
2015. The God Ezekiel creates. London: Bloomsbury. 2015. The God Ezekiel creates. London: Bloomsbury. Miles, J. 1996. God: A biography. New York: Simon & Schuster. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter of the resurrection experience and its causal link to the formulation of
Christologies; the utterances of Christologies themselves, their background
and significance;11 the practice of worshipping Jesus; the construction of
God in the New Testament texts, and the explanation for the exceptional
development of Early Christianity.12 These issues cannot be dealt with in
detail within the purview of this text and how it was delimited. Attention
will be paid to two New Testament scholars, namely Schnelle and Hurtado,
who wrote extensively and authoritatively on these matters. Their basic
interpretation of this period is indicative of contemporary scholarship and
is fruitful for the unfolding of the argument. After an extensive Theology of the New Testament (2009), that was
structured according to four major transformations among the followers
of Jesus after his death, Schnelle developed a major history of the New
Movement of Christ Believers, entitled The first one hundred years of
Christianity (2020). He identifies the emergence of Christology as the first
major transformation.13 He is interested in the emergence of Christianity as a
new independent religious movement, as distinct from Judaism. The Easter 11
Capes’ (2019) chapter and Smith’s (2019) article capture the contemporary state of scholarship
particularly well. The figures of Bousset and Hengel loom large in the background and the
question is whether the devotion to Jesus and a high Christology were initially due to a Hellenistic
background or whether they were already present in the earliest Jewish communities. Intrinsically,
it was disputed whether Judaism and Hellenism could be neatly compartmentalised. There is
clearly a much greater appreciation of Jewish antecedents nowadays (Capes 2019:165-168). It also involves the question as to whether the Christological development happened slowly
and incrementally, or momentously. There is a third fundamental issue in recent discourse: Was
monotheism at that time strict or flexible? (Smith 2019:196). A more flexible approach allows for
a special (even divine) status to Jesus, without assigning a position to him equivalent to YHWH. From the mid-1990s, one perceives the formation of an “Early High Christology Club” (Capes
2019:173) that shares common convictions: the focus should be on devotional practices; the
worshipping of Jesus took place post-resurrection within Jewish circles. Memory of the life
of Jesus, the resurrection, and exegesis stimulated these occurrences. The debates among
Hurtado, Bauckham, and Dunn and the new interest in the life of the earthly Jesus are particularly
noteworthy. 11
Capes’ (2019) chapter and Smith’s (2019) article capture the contemporary state of scholarship
particularly well. The figures of Bousset and Hengel loom large in the background and the
question is whether the devotion to Jesus and a high Christology were initially due to a Hellenistic
background or whether they were already present in the earliest Jewish communities. Intrinsically,
it was disputed whether Judaism and Hellenism could be neatly compartmentalised. There is
clearly a much greater appreciation of Jewish antecedents nowadays (Capes 2019:165-168).
It also involves the question as to whether the Christological development happened slowly
and incrementally, or momentously. There is a third fundamental issue in recent discourse: Was
monotheism at that time strict or flexible? (Smith 2019:196). A more flexible approach allows for
a special (even divine) status to Jesus, without assigning a position to him equivalent to YHWH.
From the mid-1990s, one perceives the formation of an “Early High Christology Club” (Capes
2019:173) that shares common convictions: the focus should be on devotional practices; the
worshipping of Jesus took place post-resurrection within Jewish circles. Memory of the life
of Jesus, the resurrection, and exegesis stimulated these occurrences. The debates among
Hurtado, Bauckham, and Dunn and the new interest in the life of the earthly Jesus are particularly
noteworthy. 12
See, for example, Ehrman (2018), who, interestingly, connects the “worship of the one God” to
the success of Early Christianity (2018:111-116). One would have expected that an exclusionary
insistence would be off-putting; it simply had the opposite effect: “It was this claim that led to the
triumph of Christianity” (2018:116). 2.4
Resurrection, worship and Christologies The argument about a genealogy now moves to a new trajectory – the
articulation of a trinitarian notion of God. As the path to an exclusivist
monotheistic view was long and winding, the one towards the fourth-
century settlement is equally demanding to navigate. A journey from the
historical person of Jesus of Nazareth to Constantinople in 381 should
be traversed. One may justifiably divide this in two movements: from
the ministry and crucifixion of Jesus to the emergence of Christologies
honouring his special status, such as the title logos, and from the post-
canonical period in the second century to the fourth century. This
section focuses on the historical establishing of Jesus as a figure worthy
of worshipping. As could be expected, the scholarship on this period, especially if one
is interested in the notion of God, is fairly voluminous. A number of specific
research foci can be identified that receive active attention. For example,
the understanding of, and the approach to monotheism; the interpretation 17 Considering the doctrine of God 13
For a full discussion, see Schnelle (2009:163-192). The other transformations he distinguishes
include the mission to the Gentiles without the precondition of circumcision, as embodied by Paul;
the composition of the Gospels as response to crises, and the production of literature such as the
Deutero-Pauline letters, the catholic epistles, and the Johannine literature as engagement with the
wider environment. The content of this rich monograph on New Testament theology cannot be
captured in a short space. One is struck by the attention to the inherent canonical plurality, as well Venter The notion of a crucified man as messiah was a new way of thinking –
blasphemous to Jews (Deut. 21:22-23) and ridiculous to Greeks (1 Cor. 1:23). Any possibility of cultural plausibility was turned upside down. Furthermore, Jesus was elevated to a unique proximity to God (Schnelle
2020:91): the name of God was given to him (Phil. 2:9-10); he was the
image of God (2 Cor. 4:4). It is noteworthy that, at the beginning of the
new movement, “stood a thoroughly creative process” (Schnelle 2020:92). Schnelle (2009:192) concludes his Christological discussion with the view
that, in this instance, there is “an exclusive monotheism in binitarian form”;
a “God who is defined Christologically”. What makes Schnelle particularly
relevant for my own line of argumentation is his attention to the role of
a re-interpreted image of God. The view of God is at the centre of the
movement and its new narrative (Schnelle 2020:467). In a world marked by
growing dissatisfaction with the Graeco-Roman view, the early Christian
view of God gained some attraction. There was something fascinatingly
personal to it. This God was defined by concrete action in the person of
Jesus, who was in an exceptionally intimate relation with the Father (as
evidenced prominently in the Gospel of John). Secondly, the nature of this
God of Jesus Christ was understood in terms of life and love (see Rom. 4:17 [“who gives life to the dead and calls into being the things that do not
exist”] and 1 John 4:8b, 16b [“God is life”]). Schnelle (2020:473) makes a
remarkable statement: “Here nothing less than a totally new image of God
is introduced in intellectual history.” This redefined notion of monotheism –
God understood in terms of the person of Jesus, in terms of the cross, and
in terms of the resurrection and of love – contributed to the attractiveness
of Early Christianity and its success (Schnelle 2020:560). events as well as the appearances and experiences of the Resurrected One,
in particular, form the foundation, “the initial force” (2020:82), of the new
movement. It is important to note that the Easter event “set off a creative
interpretative process” (2020:85). Venter As a means of making sense of Jesus
as the Messiah of Israel, Christology was based on three realities: the pre-
Easter claims of Jesus (for example, forgiveness of sins like God does);
the content of his teaching (for example, God’s unlimited love), and the
Easter event complex of cross, resurrection, and appearances. Schnelle
(2020:87) also points out, as part of this dynamics, the experiences of the
Spirit and the role of a Christological re-interpretation of Scripture. Psalm
110:1 is a key passage signifying Jesus’ dignity. The introduction of Jesus
as the “founder of a new discourse” and a “new religious world” transpired
in this process, and this is central to Schnelle’s reconstruction (2020:89). The notion of a crucified man as messiah was a new way of thinking –
blasphemous to Jews (Deut. 21:22-23) and ridiculous to Greeks (1 Cor. 1:23). Any possibility of cultural plausibility was turned upside down. Furthermore, Jesus was elevated to a unique proximity to God (Schnelle
2020:91): the name of God was given to him (Phil. 2:9-10); he was the
image of God (2 Cor. 4:4). It is noteworthy that, at the beginning of the
new movement, “stood a thoroughly creative process” (Schnelle 2020:92). Schnelle (2009:192) concludes his Christological discussion with the view
that, in this instance, there is “an exclusive monotheism in binitarian form”;
a “God who is defined Christologically”. What makes Schnelle particularly
relevant for my own line of argumentation is his attention to the role of
a re-interpreted image of God. The view of God is at the centre of the
movement and its new narrative (Schnelle 2020:467). In a world marked by
growing dissatisfaction with the Graeco-Roman view, the early Christian
view of God gained some attraction. There was something fascinatingly
personal to it. This God was defined by concrete action in the person of
Jesus, who was in an exceptionally intimate relation with the Father (as
evidenced prominently in the Gospel of John). Secondly, the nature of this
God of Jesus Christ was understood in terms of life and love (see Rom. 4:17 [“who gives life to the dead and calls into being the things that do not
exist”] and 1 John 4:8b, 16b [“God is life”]). as the space accorded to various theologies proper, that is, presentations of God (for example,
Schnelle 2009:205, 660, 752). Venter 12 12
See, for example, Ehrman (2018), who, interestingly, connects the “worship of the one God” to
the success of Early Christianity (2018:111-116). One would have expected that an exclusionary
insistence would be off-putting; it simply had the opposite effect: “It was this claim that led to the
triumph of Christianity” (2018:116). 13
For a full discussion, see Schnelle (2009:163-192). The other transformations he distinguishes
include the mission to the Gentiles without the precondition of circumcision, as embodied by Paul;
the composition of the Gospels as response to crises, and the production of literature such as the
Deutero-Pauline letters, the catholic epistles, and the Johannine literature as engagement with the
wider environment. The content of this rich monograph on New Testament theology cannot be
captured in a short space. One is struck by the attention to the inherent canonical plurality, as well 13
For a full discussion, see Schnelle (2009:163-192). The other transformations he distinguishes
include the mission to the Gentiles without the precondition of circumcision, as embodied by Paul;
the composition of the Gospels as response to crises, and the production of literature such as the
Deutero-Pauline letters, the catholic epistles, and the Johannine literature as engagement with the
wider environment. The content of this rich monograph on New Testament theology cannot be
captured in a short space. One is struck by the attention to the inherent canonical plurality, as well 18 Acta Theologica Supplementum 34 2022 events as well as the appearances and experiences of the Resurrected One,
in particular, form the foundation, “the initial force” (2020:82), of the new
movement. It is important to note that the Easter event “set off a creative
interpretative process” (2020:85). As a means of making sense of Jesus
as the Messiah of Israel, Christology was based on three realities: the pre-
Easter claims of Jesus (for example, forgiveness of sins like God does);
the content of his teaching (for example, God’s unlimited love), and the
Easter event complex of cross, resurrection, and appearances. Schnelle
(2020:87) also points out, as part of this dynamics, the experiences of the
Spirit and the role of a Christological re-interpretation of Scripture. Psalm
110:1 is a key passage signifying Jesus’ dignity. The introduction of Jesus
as the “founder of a new discourse” and a “new religious world” transpired
in this process, and this is central to Schnelle’s reconstruction (2020:89). Venter Hurtado’s scholarship on early Christian devotional practices and
Christology exemplifies truly ground-breaking research and deserves
careful attention in a genealogy of a Christian God. Already with his
book One God, one Lord: Early Christian devotion and ancient Jewish
monotheism (1988), he chartered a new direction. This culminated in his
seminal tome Lord Jesus Christ: Devotion to Jesus in Earliest Christianity
(2003). Hurtado’s book, strikingly entitled How on earth did Jesus become
a God? (2005), summarises crucial aspects of his work in a fairly condensed
space: questions about monotheism, the centrality of experience, as well
as the social and political consequences of the devotion to Jesus. Basic
to Hurtado’s contribution is a prioritising of devotional practices, and not
ideas; for example, phenomena such as singing of hymns, prayers to Jesus,
use of Jesus’ name at rituals (for example, at baptism), and the common
meal as the Lord’s Supper (2010b:172). His assumption is that Jewish
monotheism could accommodate special figures without diminishing the
place of God. A typical expression of his position is found in Revelation
5:13 (“To him who sits on the throne and to the Lamb be praise and honour
and glory and power, for ever and ever!”). Hurtado’s view of this is of
utmost importance: it is a “unique re-shaping of monotheism”, a “highly
innovation in the monotheistic tradition of the time”, a “‘mutation’ within
second-temple Jewish tradition” (2010b:172, 173, 176). This involves not
only some continuity with the Old Testament, but also a development. Hurtado’s (2010a) later work on God in the New Testament is also
particularly relevant and important, as it redresses the general neglect
of this theme in New Testament scholarship. Theologising about God
in the New Testament involves making inferences based on God’s acts
(2010a:35). The identity of God is understood in connection with Jesus,
and this implies “a profoundly amended portrayal of ‘God’” (2011a:38). Hurtado gives prominence to the notion of “binitarian” worship. In this later
work (2010a), he assigns a larger space to the Spirit, by mentioning the
“triadic shape of the God-discourse in the NT” (2010a:99). Interestingly, he
emphasises that the “triadic shape” also reflects the “triangular” form of
early Christian experience, which reflects fairly stable relationships among
Father, Jesus, and Spirit (2010a:101). This major “reconfiguring of [the]
God-discourse” had a much wider impact. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Schnelle (2020:473) makes a
remarkable statement: “Here nothing less than a totally new image of God
is introduced in intellectual history.” This redefined notion of monotheism –
God understood in terms of the person of Jesus, in terms of the cross, and
in terms of the resurrection and of love – contributed to the attractiveness
of Early Christianity and its success (Schnelle 2020:560). 19 Considering the doctrine of God Venter In one of his last books on the
distinctiveness of Early Christianity, Hurtado (2016:62-66), like Ehrman and
Schnelle, points to the unique Christian view of God as a factor in the
growth of the new movement. In this instance, perspectives have not been given the same weight
previously: the recognition of the impact of the resurrection on worship
practices, and a sense that the reconfigured conception of God should
receive due attention and be central in the narrative of Early Christianity 20 Acta Theologica Supplementum 34 2022 and in New Testament discourse. The famous lament by Dahl (1991:154)
that “God [is a] neglected factor in New Testament theology” has been
heeded. There are not only investigations of this theme nowadays, but also
a sense of the significant novel profile that the divine assumed in light of
the Jesus events. Recommended further reading The recommendations are discussions of the complex phenomenon
of monotheism in the New Testament era, a seminal treatment of the
historicity of the resurrection and its link to Christology, proposals on how
Jesus’ divinity emerged, and various interpretations of God in the New
Testament texts. 2008. Jesus and the God of Israel. Grand Rapids, MI: Wm B. Eerdmans. Das, A.A. & Matera, F.J. (Eds)
2002. The forgotten God: Perspectives in biblical theology. Louisville, KY:
Westminster John Knox Press. s,
&
,
J (
s)
2002. The forgotten God: Perspectives in biblical theology. Louisville, KY:
Westminster John Knox Press. Ehrman, B.D. 2014. How Jesus became God: The exaltation of a Jewish preacher from
Galilee. New York: HarperOne. Stuckenbruck, L.T. & North, W. (Eds)
2004. Early Jewish and Christian monotheism. London: T. & T. Clark. Wright, N.T. Stuckenbruck, L.T. & North, W. (Eds)
2004. Early Jewish and Christian monotheism. London: T. & T. Clark. Wright, N.T. 2003. The resurrection of the Son of God. Minneapolis, MN: Fortress. Venter Considering the doctrine of God Hence, in this section, I will focus mainly on the fourth century and
these two councils. There is excellent scholarship on Patristics, or Early Christian Studies, in
general. For example, the Cambridge history of Christianity Vol 1 Origins to
Constantine (Mitchell & Young 2006), the Oxford handbook of Early Christian
studies (Harvey & Hunter 2008), the Routledge companion to Early Christian
thought (Bingham 2010), and the Wiley Blackwell companion to patristics
(Parry 2015) furnish fascinating treatments of the horizon of the emergence
of a historically new conception of God. Theology is placed within material
contexts of concrete life in its geographical and social dimensions. People
of that time emerge in their diversity with multiple religious practices. The
fourth century has been studied intensively and outstanding works are
available, such as the older Hanson (2005, org. 1988) and, more recently,
Behr (2004), Ayres (2004), and Anatolios (2011). Despite the advances
made, critical questions remain about the various reconstructions, as
ably pointed out by Coakley (2007); one issue that remains disputed is the
relation between the East and the West (2007:128, 131). According to Ayres
and Radde-Gallwitz (2008:873), a fundamental division between Latin
and Greek trinitarian theology can no longer be claimed. Von Harnack’s
thesis about the triumph of Hellenisation in Early Christianity continues to
be relevant, although this also has been increasingly discredited (Ayres
& Radde-Gallwitz 2008:865). The interaction between biblical Christianity
and its cultural and philosophical embeddedness remains a dynamic that
deserves careful attention. Some understanding of the chronology is inevitable for background
information. McGuckin (2011:49), with a Greek orientation, refers to “five
great acts of a play” after the New Testament era, and proceeds to identify
the second-century theologians, the third-century Apologists, the genius
of Origen, the fourth-century Nicene and post-Nicene reactions, and the
long quieting-down aftermath. Smith (2011:110-117) divides the fourth-
century narrative into five parts: Arius and Alexander, Council of Nicaea
and its aftermath, the Anti-Macellan phase, ascendancy of the Homoians,
and the endgame of Eunomians, Cappadocians, and the Council
of Constantinople. As the trinitarian developments, after the closing of the New Testament
canon, are rather complex and intricate, conventionally one encounters
distillation efforts to simplify discourses and controversies in neat rubrics. Boff (1988:43-99) submits a rather good illustration of this. 2.5
The fourth century and metaphysical
trinitarianism The Patristic scholar Anatolios makes two statements that are exceedingly
relevant to my understanding of the genealogy of God:
The Trinitarian controversies of the fourth century constitute the
most crucially formative period in the development of the Christian
doctrine of God (2007:432)
and
My premise is that if we wish to understand trinitarian doctrine, we
must observe how it came to be formulated in the councils of Nicaea
(325) and Constantinople (381) (2011:1). 21 Considering the doctrine of God Considering the doctrine of God Venter He describes
the erroneous ways (Modalism, Subordinationism, and Tritheism), the
theologians who created the trinitarian language (Irenaeus, Origen,
Tertullian, the Cappadocians, Augustine, and Thomas Aquinas), the official 22 Acta Theologica Supplementum 34 2022 credal statements (Nicaea, Constantinople), and the key terms (substance,
hypostasis, processions, begetting, spiration). This is helpful for a first
orientation, but it does not convey the historical struggle, contestation,
and slow emergence of a particular vision of God. Before paying attention to the primary councils, I stipulate a number
of disparate comments that might be pertinent to grasping something of
the developments: • During the second century, the notion of a logos theology appeared in
cosmological debates. This incidentally produced the first conceptual
model with explanatory power. The logos was considered the
immanent presence of the immutable divine in the material and created
order. Theologically, this could allow for combining monotheism with a
confession of Jesus as the power of God. • A note on Latin trinitarian theology should be raised as the narrative
is so predominantly Greek. Tertullian from Carthage in North Africa is
usually regarded as the font of Latin trinitarian theology and associated
with coining the term “Trinitas” and for speaking of divine one-ness
and differentiation as “one substance and three persons”. The patristic
scholar Barnes (2011:70-75) points out some deeper currents in Latin
trinitarian theology: its internal disposition was anti-monarchical, that
is, anti-modalist. “Sight” plays an important role in this tradition, and the
critical question is: How is the Father seen in the Son and the Son seen
in the Father? A biblical passage such as John 14:9 (in relation to 14:10)
is key in this instance. The typical Latin understanding is that “works
indicate power”. Divine substance is noted in the works that could be
performed; the basis for unity is this: common power conveys common
substance. Barnes’ (2011:74) view that the language of substance was
not so prominent in the fourth century is noteworthy: “The common
language for describing trinitarian unity is ‘one power’”. The divinity of
Jesus was argued for in this way. The form of Latin theology consisted
of the following: one-ness of God is indicated by unity of works and
power; distinction by causal relations; the three are always irreducible
and that is indicated by the word “person”. • A note on Latin trinitarian theology should be raised as the narrative
is so predominantly Greek. 14
For brief, but clear treatments, see McGuckin (2011:60-62) and Young (2006:463-466). Venter “hypostasis” (understood as single concrete being) “made” by an
eternal generation from the Father. With this, Origen accomplished two
things: overcoming modalism, and simultaneously opening a space
for a subordinationist approach to Jesus Christ. He also declined to
use the ousia language of God, because of its potential materialist
implications. His successors accepted the ontic distinction between
Father and Word and the use of “hypostasis”, but this fundamental
insight was tarred by hierarchical associations. His views were taken
up in two directions (McGuckin 2011:62): by Arius, who advocated the
special creaturely status of Jesus, on the one hand, and by the Nicene
theologians, who developed the notion that Jesus was “eternally
begotten” and one with God, on the other. Some distinction and nuance
are required in this instance. The issue was not whether the Son was
divine , but whether he was true God, equal with the Father (see Smith
2011:118). In a world structured hierarchically, there were also levels
of divinity.15 “hypostasis” (understood as single concrete being) “made” by an
eternal generation from the Father. With this, Origen accomplished two
things: overcoming modalism, and simultaneously opening a space
for a subordinationist approach to Jesus Christ. He also declined to
use the ousia language of God, because of its potential materialist
implications. His successors accepted the ontic distinction between
Father and Word and the use of “hypostasis”, but this fundamental
insight was tarred by hierarchical associations. His views were taken
up in two directions (McGuckin 2011:62): by Arius, who advocated the
special creaturely status of Jesus, on the one hand, and by the Nicene
theologians, who developed the notion that Jesus was “eternally
begotten” and one with God, on the other. Some distinction and nuance
are required in this instance. The issue was not whether the Son was
divine , but whether he was true God, equal with the Father (see Smith
2011:118). In a world structured hierarchically, there were also levels
of divinity.15 • The role of hermeneutics and exegesis cannot be stressed enough. Bates (2015:12, 175) appropriately speaks about “reading as birth” and
“reading God right”. The controversies were basically a disagreement
about interpreting Scriptures. Ayres (2019:439) underlines this: “The
trinitarian controversies of the fourth century were exegetical.” In a
fuller treatment, which cannot be undertaken in this article, this aspect
warrants attention. • The role of councils is increasingly studied. 6
The scholarship on Arius is vast. The magisterial study by R. Williams (2001) should be mentioned Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter 15
See also the important discussion on “Christ in a world of hierarchies” by Young (2013:374-378).
16
The scholarship on Arius is vast. The magisterial study by R. Williams (2001) should be mentioned. 5
See also the important discussion on “Christ in a world of hierarchies” by Young (2013:374-378 Venter Tertullian from Carthage in North Africa is
usually regarded as the font of Latin trinitarian theology and associated
with coining the term “Trinitas” and for speaking of divine one-ness
and differentiation as “one substance and three persons”. The patristic
scholar Barnes (2011:70-75) points out some deeper currents in Latin
trinitarian theology: its internal disposition was anti-monarchical, that
is, anti-modalist. “Sight” plays an important role in this tradition, and the
critical question is: How is the Father seen in the Son and the Son seen
in the Father? A biblical passage such as John 14:9 (in relation to 14:10)
is key in this instance. The typical Latin understanding is that “works
indicate power”. Divine substance is noted in the works that could be
performed; the basis for unity is this: common power conveys common
substance. Barnes’ (2011:74) view that the language of substance was
not so prominent in the fourth century is noteworthy: “The common
language for describing trinitarian unity is ‘one power’”. The divinity of
Jesus was argued for in this way. The form of Latin theology consisted
of the following: one-ness of God is indicated by unity of works and
power; distinction by causal relations; the three are always irreducible
and that is indicated by the word “person”. • The thought of Origen of Alexandria is widely considered as decisive for
the history of trinitarian discourse.14 He continued the logos trajectory
and developed a complex system influenced by Middle Platonism. Jesus as Word or Wisdom of God is the ontologically mediating link
between the Transcendent God and creaturely beings. He is a distinct • The thought of Origen of Alexandria is widely considered as decisive for
the history of trinitarian discourse.14 He continued the logos trajectory
and developed a complex system influenced by Middle Platonism. Jesus as Word or Wisdom of God is the ontologically mediating link
between the Transcendent God and creaturely beings. He is a distinct 14
For brief, but clear treatments, see McGuckin (2011:60-62) and Young (2006:463-466). 23 Considering the doctrine of God 17
In his substantial work on the fourth century, Anatolios (2011:30) employs will vs. being as
structural principles to group thinkers and positions on the Trinity.
18
A recently published excellent volume of essays addresses a wide range of fascinating issues; see
Kim (2021) in the recommended further reading.
19
Note the crucial part: “We believe … and in one Lord Jesus Christ, the Son of God, begotten of
the Father, the only-begotten; that is, of the essence/substance of the Father, God of God, Light
of Light, very God of very God, begotten, not made, consubstantial with the Father.”
20
See Prestige (1952:233): “The Cappadocian Settlement finally fixed the statement of Trinitarian
orthodoxy in the formula of one ousia and three hypostases”.
21
This will be discussed in the following chapter on Zizioulas’ interpretation of the Cappadocians. Venter The striking title of
MacMullen’s work, Voting about God in Early Church councils (2006)
underlines what was at stake: God’s identity was determined in a
context of debate and power relations. The operative rationality of this
period and of the main players should be mapped. Important studies
in this regard have been published on literacy, the intellect in patristic
theology, the rise of Christian intellectuals, Christian discourse, and
Christian paideia. The intellectual quality of the generative process is
obvious and should be expressly studied. The Christian view of God
was born in argumentation, in intellectual activity. With the dawn of the important fourth century, a trinitarian sensibility
– divine unity and triadic distinction – pervaded in the worship of the Early
Church, although conceptual ambiguities remained (Anatolios 2007:431). The Egyptian elder Arius16 provoked the controversy with his strong
insistence on God’s utter transcendence, and the subordinate position of 24 2022 Acta Theologica Supplementum 34 Venter of relationship of origin: The Father is self-existent, the Son begotten,
and the Spirit spirated. Hypostasis as person conveys real distinction of
God godself (Smith 2011:116). The one selfsame “ousia” is differentiated
in three hypostases. “Agennêtos” discloses the order of relations in
God, and not the divine essence (see Anatolios 2007:445). The Council
of Constantinople, summoned by Emperor Theodosius in 381, brought an
end to the disputes. The formula one nature, three persons (mia ousia,
treis hypostases) would become the main way of speaking the Christian
identity of God. A historical note is pertinent at this point. It was only at the
Council of Chalcedon (451) that the archdeacon of the city read out a creed,
assumed to be the confession of Constantinople. It is generally assumed
that there was no Constantinopolitan Creed, as the Council understood
itself as a continuation or restatement of Nicaea (see Anatolios 2011:26). A long process of intellectual struggle and contestation has come to an
end.22 In an in-depth investigation of the terminology involved, Lienhard
(1999:121) concludes that the settlement was “a gain – in clarity – but it
was also a loss”. Fixed terms narrow the range of speaking about God. of relationship of origin: The Father is self-existent, the Son begotten,
and the Spirit spirated. Hypostasis as person conveys real distinction of
God godself (Smith 2011:116). The one selfsame “ousia” is differentiated
in three hypostases. “Agennêtos” discloses the order of relations in
God, and not the divine essence (see Anatolios 2007:445). The Council
of Constantinople, summoned by Emperor Theodosius in 381, brought an
end to the disputes. The formula one nature, three persons (mia ousia,
treis hypostases) would become the main way of speaking the Christian
identity of God. A historical note is pertinent at this point. It was only at the
Council of Chalcedon (451) that the archdeacon of the city read out a creed,
assumed to be the confession of Constantinople. It is generally assumed
that there was no Constantinopolitan Creed, as the Council understood
itself as a continuation or restatement of Nicaea (see Anatolios 2011:26). A long process of intellectual struggle and contestation has come to an
end.22 In an in-depth investigation of the terminology involved, Lienhard
(1999:121) concludes that the settlement was “a gain – in clarity – but it
was also a loss”. Fixed terms narrow the range of speaking about God. 22
For a fascinating study of the Council, see Freeman (2008), who acknowledges the far-reaching
consequences of the Council, but interprets it as the suppression of plurality and diversity.
23
For a discussion of these three implications, see Dünzl (2007:133-139). Considering the doctrine of God Considering the doctrine of God Venter Acta Theologica Supplementum 34 the Word. As Jesus was generated/begotten by the Father, he could never
be equal or co-eternal to God; at most, He could be the highest instance
of creaturehood. Hence, the slogan associated with Arius: “There was
once when the Son was not”. In this “framework of a graded hierarchy of
transcendence”, unity between the Father and the Son was one of will, not
of substance.17 “Arianism” became a label discrediting all subordinationist
interpretations of Jesus’ divine status. The doctrinal disagreement and
the subsequent division in the empire prompted Emperor Constantine
to summon a Council at Nicaea (325).18 This combination of empire,
church, and doctrinal formulation could be problematic; however, the
precise motivation of the emperor should be clarified: it was less a matter
of interest in the subject matter than a concern about the division in his
empire. His interest was in the unity of religion “to ensure for himself the
protection of the supreme deity” (Dünzl 2007:50). The Council’s decision
and credal formulation were decisive in the history of the church and for
her understanding of the divine, especially the use of the term “homo-
ousios” (consubstantial, of one being with) to convey Jesus’ status.19 The
Council did not generate the anticipated peace and stability; its reception
was fairly conflictual. The term “homo-ousios” was ambiguous; it had
materialist connotations, might allow even Sabellian interpretations, and
the New Testament distinction between the Father and the Son was not
adequately reflected in the creed. After Nicaea, Athanasius, who attended
the Council as young deacon, increasingly played a most polemical and
consequential role in defending the decision for thirty years. The “Cappadocian Settlement”20 would prove to be seminal.21 The
Cappadocians made creative conceptual moves to steer the church to
greater clarity and eventual unity. Basil insisted on the distinction between
the terms “ousia” and “hypostasis”, which were previously often used as
synonyms; this allowed for a supplement to the central Nicene term to
enable speaking of unity and differentiation (Anatolios 2007:440). Gregory
of Nazianzus, in particular, clarified the term “hypostasis” in this context
(McGuckin 2011:65). It refers to the distinction of the Godhead in terms 25 Considering the doctrine of God 3
For a discussion of these three implications, see Dünzl (2007:133-139). 24
In a larger purview, one could (should!) distinguish between St Basil the Great, St Gregory of
Nazianzus, and St Gregory of Nyssa, and their distinctive views. In this section, the collective
would be employed. Venter The significance of what has taken place and accomplished in this
period, following the closing of the New Testament canon up to the end
of the fourth century, can hardly be gauged. The conventional maxim –
God revealed godself as Father, Son, and Spirit – should obviously be
understood as a historically layered process with complex dynamics. The
very notion of revelation requires reconstruction. Theological motivations,
philosophical influences, hermeneutical practices, geographical influences,
as well as social and cultural realities have driven the entire process from
the crucifixion to the creedal statement of Constantinople. Revelation
was redefined as being historically mediated by intellectual discourse;
God is disclosed in deliberation. A revolutionary conception of the divine
crystallised. Deep structures of Christian soteriology have been identified
– salvation is the work of the triune God and salvation means being drawn
into the life of God self.23 Finally, a certain way of thinking matured and
has defined Christian theology ever since – it became a “discourse of
precision’’ (Young 2006:468); the church developed a unique paideia in the
Graeco-Roman world. 26 Acta Theologica Supplementum 34 2022 Recommended further reading The recommendations focus on an authoritative reading of the fourth-
century, Patristic biblical exegesis, a feminist perspective on the period, a
study of how the Christian emphasis on language facilitated the formation
of a totalising discourse, and a detailed investigation of various aspects of
the Council of Nicaea. Barnes, M.R. 1998. The fourth century as trinitarian canon. In: L. Ayres & G. Jones (eds),
Christian origins: Theology, rhetoric and community (London: Routledge),
pp. 47-67. Barnes, M.R. 1998. The fourth century as trinitarian canon. In: L. Ayres & G. Jones (eds),
Christian origins: Theology, rhetoric and community (London: Routledge),
pp. 47-67. Blowers, P.M. & Martens, P.W. (Eds)
2019. The Oxford handbook of early Christian biblical interpretation. Oxford:
Oxford University Press. https://doi.org/10.1093/oxfordhb/9780198718390. 001.0001 Burrus, V. 2000. “Begotten, not made”: Conceiving manhood in Late Antiquity. Stanford,
CA: Stanford University Press. https://doi.org/10.1515/9781503618312 Cameron, A. 1991. Christianity and the rhetoric of empire: The development of Christian
discourse. Berkeley, CA: University of California Press. https://doi.org/10. 1525/9780520915503 Kim, Y.R. (Ed.)
2021. The Cambridge companion to the Council of Nicaea. Cambridge:
Cambridge University Press. https://doi.org/10.1017/9781108613200 2.6
Being as personal - an interpretation of
the Cappadocians Conventionally, the role of the Cappadocians24 in the trinitarian
controversies of the fourth century is acknowledged. The question is: What
is the extent of their contribution? The Greek Orthodox theologian Zizioulas
(1931-) may be a noteworthy interpreter, as he has an acute sense of the
idea-historical significance that took place, but also of the doctrine of the
Trinity’s implication for a wider philosophical horizon. Zizioulas (1991:29)
makes an astounding claim: 27 27 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God The doctrine of the Trinity has a great deal to contribute to
Man’s quest for personhood, freedom and community and the
world’s survival. To grasp why he could make such a statement, one should attend to his
understanding of the insights generated by the Cappadocians. Acta Theologica Supplementum 34 2022 The difference from Augustine can be referred to clarify the view of the
Cappadocian: God is understood primarily as substance, and the persons
as relations within the substance. Origin and unity lie with substance; with
the Cappadocians, it is different: it is associated with personhood and the
Father is the origin and source of unity (see Zizioulas 1991:31, notes 22-
24). Zizioulas is fairly critical of the Western trinitarian tradition, especially
its refusal to make the Father the originating cause. This failure resulted in
an inability to deal with freedom and otherness (Zizioulas 2006:36). One may enquire about the way in which Zizioulas interprets ousia. Zizioulas (1995:49) is quite reticent about this: one cannot speak “about the
substance of God nothing can be said at all”. Properties (idiomata) belong
to the Persons; about the substance; apart from saying it is one, simple
and uncompounded, one is rendered silent by unknowability. He (2008:53)
clearly prioritises the plurality of persons as basis for his ontology. To say
that God is “love” is to refer to the three Persons and their relationships. It is interesting to note that Zizioulas is clear as to how one should deal
with attributes; not as part of a general discussion of God, but expressly
of the Trinity, otherwise one “demote[s] the question of which God [one is]
referring to” (Zizioulas 2008:67). Zizioulas is emphatically clear as to the implications of his interpretation
of the Cappadocians; he is specifically interested in anthropology. Human
beings have been made in the image of this trinitarian God; hence, a
view of man should reflect divine personhood, and not divine nature. The focus is on person, communion, relationality, love, freedom, and
particularity. Identity can be understood only in terms of relationality, as
was also pointed out by Gregory of Nazianzus. He also raises the notion
of otherness, which is prominent in contemporary thought. Otherness is
not a mere ethical matter, but one of ontology (2006:11). The problem with
Arianism was its intolerance of multiplicity in the divine being (2006:33). Zizioulas (2006:33) even refers to the “ontological ultimacy of otherness”. The perennial problem in trinitarian thought was the denial of otherness or
the secondary placing after substance. Zizioulas’ theology and interpretation received mixed reaction. His
attitude to ousia is problematic; it is simply too negative, and he may
be overstating the role of the Father. To grasp why he could make such a statement, one should attend to his
understanding of the insights generated by the Cappadocians. One should first consider the background to their theology. They
responded mid-fourth century to the contentious trinitarian views of
Sabellianism and Eunomianism. The notion of prosopon, with its theatre
association of mark, could create the impression of roles assumed by One
God. The Cappadocians (especially Basil) countered this, by replacing
prosopon with hypostasis and dissociated it from ousia (Zizioulas 1995:47). In this instance, one already finds a “historic revolution” (Zizioulas 1995:47),
as hypostasis had previously been identified with substance. Now to the
second challenge: Eunomius, a sophisticated Arian thinker, linked the
substance of God to the idea of “unbegotten” (agennetos) and concluded
that Jesus as being “begotten” cannot be homo-ousios with God. The
Cappadocian ingenious counter-interpretation was that “unbegottenness”
belongs to personhood, and not to substance (Zizioulas 1995:49). The
ramifications of this move were far-reaching. The notion of hypostasis (person) emerged as a distinct category
in ontology. Properties are connected with personhood, thus also
associations
of
uniqueness
and
irreplaceability. Simultaneously,
relationality receives attention, as a person cannot be understood without
that. When the implications are considered, the significance starts to
crystallise. If unbegottenness is linked to the person of the Father, the
cause of divine existence is not ousia/substance – the Father is the arche. The Cappadocians replaced substance with personhood, as causing
principle in ontology. According to Zizioulas’ (1995:54) interpretation:
“What causes God to be the Person of the Father?”. At the same time,
freedom as part of causation emerges at this point. This runs counter to
typical Greek philosophy that posited nature or substance prior to person,
oneness to the many, and necessity to freedom. On account of these
insights, Zizioulas (1995:52) views the Cappadocians as “revolutionary
thinkers in the history of philosophy”. Zizioulas’ interpretation should be teased out slightly. There is no bare
essence; “personal communion lies at the heart of divine being” (Zizioulas
2008:53). The “what” of God should be approached together with the
“how” question. “The ‘how’ question is as ontologically fundamental as
the ‘what’ question” (Zizioulas 2008:57). One cannot talk about the being
of God, without speaking simultaneously about the divine persons. Being
or essence and person are co-fundamental. 28 Recommended further reading The following focus on the theology of Zizioulas, in general, specific
readings of the Cappadocians, a dismissive critique of his work, and
exceptional treatment of communion and otherness by Zizioulas himself. Knight, D.H. (Ed.)
2007. The theology of John Zizioulas. London: Routledge. Knight, D.H. (Ed.)
2007. The theology of John Zizioulas. London: Routledge. Meesters, A.C. 2012. The Cappadocians and their trinitarian conceptions of God. Neue
Zeitschrift für Systematische Theologie und Religionsphilosophie 54(4):396-413. DOI: 10.1515/nzsth-2012-0017. Plantinga, C. Jr. 1986. Gregory of Nyssa and the social analogy of the Trinity. The Thomist
50:325-352. https://doi.org/10.1353/tho.1986.0017 Turcescu, L. 2002. “Person” versus “individual”, and other modern misreadings of
Gregory of Nyssa. Modern Theology 18(4):527-539. https://doi.org/10.1111/
1468-0025.00202 Zizioulas, J. 1994. Communion and otherness. St Vladimir’s Theological Quarterly 38:347-361. Venter (2006:171); such a God would be “an anthropomorphic monstrosity”. At
stake in Zizioulas’ interpretation may be the “surplus of meaning” in a text. His prioritising of hypostasis, relationality, freedom, and alterity associated
with the Trinity may have enduring validity. (2006:171); such a God would be “an anthropomorphic monstrosity”. At
stake in Zizioulas’ interpretation may be the “surplus of meaning” in a text. His prioritising of hypostasis, relationality, freedom, and alterity associated
with the Trinity may have enduring validity. Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 Wilks (1995) gives a helpful and
sensitive evaluation of Zizioulas’ interpretation, questioning whether
Zizioulas represents the Cappadocians fairly and whether his accents are
justified, but also acknowledges that Zizioulas allows the Trinity to speak
to contemporary concerns. He flatly rejects some of the critique levelled
against him, for example that he succumbs to contemporary existential
thought and transforms the divine persons into modern individuals 29 29 Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 to point to the so-called “turn to relationality” (see Shults 2005:5-9). At
stake, in this instance, is the notion of “category” in philosophy that has
been prominent in metaphysics since Aristotle and the dominance of
“substance” over against “relation”. This has been questioned by early
modernist thinkers. A philosopher such as Hegel followed Kant’s privileging
of relation as category. A host of scholars such as Husserl, Heidegger,
and Whitehead have started to assign primacy to relationality. This turn
was also reinforced by developments in natural and social sciences. Shults (2005:9) makes an interesting comment: what is noted in theology
is fundamentally a “re-turn to relationality”, as it has always been in some
way inherent in Christian thought. to point to the so-called “turn to relationality” (see Shults 2005:5-9). At
stake, in this instance, is the notion of “category” in philosophy that has
been prominent in metaphysics since Aristotle and the dominance of
“substance” over against “relation”. This has been questioned by early
modernist thinkers. A philosopher such as Hegel followed Kant’s privileging
of relation as category. A host of scholars such as Husserl, Heidegger,
and Whitehead have started to assign primacy to relationality. This turn
was also reinforced by developments in natural and social sciences. Shults (2005:9) makes an interesting comment: what is noted in theology
is fundamentally a “re-turn to relationality”, as it has always been in some
way inherent in Christian thought. Social trinitarianism basically embodies a shift to personalistic,
dynamic, and relational modes of thinking of God. The fundamental issue
is how personhood is interpreted (see Plantinga, Thompson & Lundberg
2010:131),25 as its meaning could never be detached from prevailing
conceptions in society. This was also noted in the previous section on
Zizioulas’ interpretation of the Cappadocians. The typical Western and
pervasive position, represented by Augustine and Thomas, has recourse to
the notion of “subsistent relations” to come to terms with the personhood
of the Father, Son, and Spirit. Persons are basically relations (paternitas,
filiatio, and spiratio). The basic assumption of this classical view is divine
simplicity, and the primary analogy is a one-person model (Plantinga,
Thompson & Lundberg 2010:132-135). An accent on simplicity tends to
level trinitarian distinctions among persons; this is the reason why one
also finds an Augustinian preference for psychological analogies. 25
Thompson completed a PhD on the social Trinity at Princeton (Imitation Trinitatis: The Trinity as
social model in the theologies of Jürgen Moltmann and Leonardo Boff [1996]). His 1997 article is
arguably one of the best available to understand this approach to the Trinity.
26
This issue distinguishes a social analogy from other trinitarian constructs (Thompson 1997:27,
and especially 29). 2.7
Twentieth-century renaissance and
social trinitarianism During the twentieth century, there was a major shift in dominant God
thinking, with the same innovative profile as the previous ones, the
emergence of exclusive monotheism and substantial trinitarianism. One
witnessed an astonishing interest in the Trinity and a prolific output of
reflection on the various related dimensions of trinitarian theology. Central
to this so-called trinitarian renaissance is the advocacy of a social model of
the Trinity and, correspondingly, an enthusiastic application of trinitarian
motifs to social ethical questions. Like the previous theo-trajectories, one
also finds, in this instance, a long preparation with multiple precursors,
but eventually also proponents of this approach with clear views of which
one can take note. The long road to theologians such as Moltmann, Boff,
Lacugna, Volf, and Gunton, for example, cannot be understood without
prior philosophical developments. One way to come to terms with this is 30 2022 Venter this argumentation. Proponents of this view are convinced that it better
clarifies the Christian vision of God, gives more adequate expression to
what personhood entails, and provides a more fruitful model for Christian
community and society than the typical Western view (see Plantinga,
Thompson & Lundberg 2010:143). Thompson (1997:40) is explicit on this
insight: “A social analogy better clarifies for us our fundamental vision
of God.” What transpires in this instance is the emphatic preference for
a specific point of departure: the biblical, salvific history of the Father,
Son, and Spirit; thus the oikonomia. This steers us towards a social
understanding. Concomitantly, its obvious ethical thrust may also be the
chief impulse to the renewal and enthusiasm of the Trinity. Moltmann’s work became particularly associated with the label “social
Trinity”. History is a dominant motif in his theology overall, but at the same
time one finds a strong eschatological orientation in his thought from his
early epoch-making work Theology of hope (1967). This eschatological
history is essentially the history of God. Already in his theology of the
cross (The crucified God [1974]), one sees that the death of the Son is
an intra-trinitarian event, and that the triune God is not immutable. God’s
action in the world has a retroactive effect on God’s relationship to
Godself; “the cross puts its impress on the inner life of the triune God”
(Moltmann 1981:161). In his mature trinitarian theology (specifically The
Trinity and the kingdom of God [1981]), his starting point is the history
of Jesus the Son and the fellowship of the three divine Persons. This
is different from the dominant Western tradition that begins with the
unity. The subsequent theological task to be completed is to argue for
the unity of God. Moltmann (1981:177; 2000b) does this with an appeal
to the notion of perichoresis. He finds the typical traditional attempts to
argue substantially or monarchically unacceptable. One should note that
Moltmann’s understanding is different from Zizioulas’, as he balances
nature and person: “The trinitarian Persons subsist in the common divine
nature; they exist in their relations to one another” (1981:173; the entire
section 171-178 is crucial). Because of their mutual love, the Persons exist
totally in the other. This dynamic moves beyond personhood, relationality
– something “takes place in the Trinity itself”; hence, the reference to the
“history of God” (1981:174). 27
An astute interpreter of the major trinitarian thinkers, Grenz (2004:82) considers this “the most
significant aspect” of Moltmann’s trinitarian theology. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Acta Theologica Supplementum 34 Social
trinitarianism, as part of a process of finding an appropriate connotation
of divine personhood, conceptualises the three persons as “three discrete
centers of thought, will and consciousness” (Plantinga, Thompson &
Lundberg 2010:137).26 Analogies such as a community or society emerge
in this instance. The major logical challenge for this approach is obviously
the question of divine unity. Instead of positing this in the divine ousia/
substance, or monarchically in the person of the Father, the proposal
is to appeal to the ancient notion of perichoresis. By mutual indwelling,
fellowship, and love, the Father, Son, and Spirit are the one God. This
is a corrective to the autonomous understanding of personhood by a
“person-in-relation” approach. John 17 looms large in the background in 31 Considering the doctrine of God Venter Interestingly, the heading to the entire section
is also called “The life of the Trinity”. A prominent feature of his view is to
emphasise the “openness” of the Trinity27 – that is, God invites people into
the divine fellowship. Subsequently, space plays a prominent place in his 32 Acta Theologica Supplementum 34 2022 thought (see, for example, Moltmann 2000:318). The particular quality of
the social life of God has political implications – thought (see, for example, Moltmann 2000:318). The particular quality of
the social life of God has political implications – the Trinity corresponds to a community in which people are defined
through their relations with one another and in their significance
for one another (Moltmann 1981:198; see the entire critical section
191-200); hence, the human community must “mirror” the triune God (Moltmann
1983:53). Moltmann has an intricate position on the political significance
of the Trinity. He places this in the broad frame of “freedom” – “The
doctrine of the Trinity must be developed as the true theological doctrine
of freedom” (1981:192). For this to be achieved, one must overcome
unqualified monotheism and monarchism by perichoresis – the sociality
of the divine Persons. The combination of the dismissal of substantial
categories, the strong emphasis on divine persons and divine sociality, the
focus on perichoresis, and the insistence on political implications render
this Trinitarian approach an exceptional social interpretation. The social model of the Trinity addresses two fundamental realities
that should be acknowledged as belonging inherently to constructing a
doctrine of God: an awareness of a changing intellectual landscape and
a need to relate theological construals to social dilemmas. It was born
from this double sense of responsibility. The recourse to a social approach
came surprisingly naturally; the fellowship of three distinct realities, so it
seems, requires a social interpretation. Furthermore, because one deals
with an ultimate horizon, the application to social exigencies is also
easy and obvious. The theological problem is that classical Western
trinitarianism did not exhibit the potential for these twofold reflexes. Boff’s (1988) monograph is arguably the best example that embodies this
clearly. He (1988:111-122, 123-154) discusses explicitly the Trinity “in a
changed cultural situation”, and the Trinity “as basis for social and integral
liberation”. In the next chapter, I will describe the resistance to a social
model. Two arguments in support of such an approach may be noted. Venter and postmodernity as dialectic; the trinitarian rhetoric issues a powerful
critique: it “sets forth a narrative of peaceful, superabundant donation”
(Cunningham 2003:197). To the notion of relationality, the discourse on the
Trinity brought gifts of association unheard of previously.28 and postmodernity as dialectic; the trinitarian rhetoric issues a powerful
critique: it “sets forth a narrative of peaceful, superabundant donation”
(Cunningham 2003:197). To the notion of relationality, the discourse on the
Trinity brought gifts of association unheard of previously.28 29
The list of theologians issuing critical voices is much longer. One can easily add names. One
esteemed Patristic scholar, Ayres, should be mentioned for one contribution by him (2010). Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 28
This will be described in later chapters. Venter The
notion of person, despite its complex history of interpretation, continues
to denote particularity and uniqueness that is ecstatic and relational in
a way that no other term can do (see Godzieba 2011:187-190). It has
displayed a surplus of meaning that has been fruitfully explored, especially
in light of the turn to relationality. Secondly, relationality has been part
of the grammar of trinitarian language from the beginning, but it has
acquired a certain pregnant relevance in the twentieth century that was
unprecedented. An interpreter such as Cunningham (2003:191) links this
directly with postmodernity and the proclivity for difference that was built
into trinitarian theology. He, however, views the relationship between Trinity 33 Recommended further reading The recommendations include an article by Moltmann, in which he
summarises his own view. Two articles discuss specifically Moltmann’s
trinitarian view, and one investigates the widespread use of perichoresis. The last recommendation is an exchange on two major approaches to the
Trinity – the classical vs the relational. Mcdougall, J.A. 2003. The return of trinitarian praxis? Moltmann on the Trinity and the Christian
life. The Journal of Religion 83(2):177-203. https://doi.org/10.1086/491276 Mcdougall, J.A. 2003. The return of trinitarian praxis? Moltmann on the Trinity and the Christian
life. The Journal of Religion 83(2):177-203. https://doi.org/10.1086/491276 Moltmann, J. 2000. Perichoresis: An old magic word for a new trinitarian theology. In: M.D. Meeks (ed.), Trinity, community, and power (Nashville, TN: Kingswood Books),
pp. 111-125. Otto, R.E. 2001. The use and abuse of perichoresis in recent theology. Scottish Journal of
Theology 54(3):366-384. https://doi.org/10.1017/S0036930600051656 Rossi-Keen, D.E. 2008. Jürgen Moltmann’s doctrine of God: The Trinity beyond metaphysics. Studies in Religion/Sciences Religieuses 37(3-4):447-463. https://doi.org/10. 1177/000842980803700304 Sexton, J.S. (Eds)
2014. Two views on the doctrine of the Trinity. Grand Rapids, MI: Zondervan. Acta Theologica Supplementum 34 2022 In an often-quoted article, Kilby (2000) points out that the chief strategy
by theologians who try to revive the doctrine of the Trinity is to advocate
a social analogy. In this process, three movements take place: the term
“person” is reinterpreted along communitarian lines; preference is given to
the Cappadocian Fathers, and perichoresis is invoked to account for divine
unity. The main thrust of her unease is summarised with the accusation
of projection: [M]uch of the detail is derived from either the individual author’s or
the larger society’s latest ideals of how human beings should live in
community (2000:441). What is projected unto God is immediately reflected back onto the
world. She believes that thinking about the Trinity was derailed, once
the question of its relevance was raised. The moment Kilby starts to
delineate her alternative to the project of social trinitarianism, one senses
how problematic her own position is. She (2000:443) relegates trinitarian
propositions to second-order reflection and continues: “[O]ne should
renounce the very idea that the point of the doctrine is to give insight
into God.” She thus distances herself from the insight that the confession
of the Trinity is the fundamental Christian identification of God. In later
contributions, she (2010; 2014) emphasises an apophatic approach and
tries to explain what the political implication might imply. Holmes (2009:81), a scholar well acquainted with contemporary
trinitarian discourse, acknowledges the usefulness of the doctrine, stating
that it has been “extraordinarily generative for ecclesial, social and political
practice”, but continues to critique the contemporary trend to explore the
practical significance of the doctrine. He advances three arguments. First,
the ethical usefulness is overstated, according to him, and the political
utility can only be achieved by some “massaging” of the doctrine (2009:84). Secondly, contemporary social trinitarianism deviates from the original
Cappadocian Fathers. It is important to note, in this instance, that Holmes
accepts only one form of distinction between the divine hypostases, that is,
relations of origin. He dismisses any further qualification, for example, that
these relationships could be relations of mutual love (2009:85). Thirdly, he
questions the Biblical basis of social trinitarianism. Again, his own position
is noteworthy: the heart of the biblical witness is monotheism, and then he
adds astoundingly that this emphasis is “supplemented by a brief coda
or appendix suggesting that this One God is in fact triune” (2009:87). 2.8
Critical responses. End of the trinitarian
renaissance? These theological developments provoked an interesting reception. The
new appreciation of the Trinity as unique Christian understanding of God
has been widely registered and viewed as constructive. Some dissenting
voices should, however, not be ignored. Scholars such as Kilby, Holmes,
and Tanner articulated explicit critiques of social trinitarianism and its
concomitant political applications.29 As they are respected theologians,
their views cannot be dismissed. 34 Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 30
For a thorough discussion of “participation”, see Cunningham (1998a:7-25), who has a
sophisticated view and cannot easily be enlisted by the minimalists. His notion strictly aims at
countering “any attempt to understand them (i.e., the three divine persons) as independent
existences” (1998a:10). He wants to overcome any trace of Western hyper individualism. He
continues, and this makes his view unique: “[W]e are called to live lives of mutual participation”
(1998a:13). Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Acta Theologica Supplementum 34 His
alternative is Christology: What social trinitarianism tries to address with
the doctrine of the Trinity, the Father did with reference to Christology. In a recent monograph, Holmes (2012b) argues that twentieth-century 35 Considering the doctrine of God Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 to argue that Jesus’ own life towards other people is the example to
be followed. Despite the persuasive power of her argument, at least
two comments could be made. The manner in which Tanner deals with
divine relationality borders on sheer reification; speaking about the Trinity
presupposes a strong sense of the metaphoric quality of all God talk. Mimesis need not require the direct correspondence. She does not argue
properly why participation is necessarily political. A clear shift of category
takes place. She emphasises soteriology, whereas social trinitarians argue
for a social ethic.31 to argue that Jesus’ own life towards other people is the example to
be followed. Despite the persuasive power of her argument, at least
two comments could be made. The manner in which Tanner deals with
divine relationality borders on sheer reification; speaking about the Trinity
presupposes a strong sense of the metaphoric quality of all God talk. Mimesis need not require the direct correspondence. She does not argue
properly why participation is necessarily political. A clear shift of category
takes place. She emphasises soteriology, whereas social trinitarians argue
for a social ethic.31 Van den Brink (2014) responded comprehensively to the charges of the
critics of social trinitarianism and persuasively rebutted the main objections. His reading of the resurgence is also noteworthy: he connects the renewed
interest in the Trinity primarily to the social approach (2014:332). Four
outstanding insights distinguish this interpretation from previous ones
(2014:336): the focus on a three-personal God, a relational ontology, a
historical orientation, and an emphasis on the practical relevance. Van
den Brink’s critical treatment considers the problems identified by Holmes
and Kilby which he then dismisses with strong counter-arguments. The
accusation of lack of unanimity and of projection is quixotic, as it could
be levelled against any theological position. The alleged misreading of
the Fathers does not undo the social trinitarians’ commitment to basic
Nicene Christianity (three hypostases and one ousia) (2014:341). The
prominence of the distinctive Persons in the oikonomia and the flexible
notion of second-temple monotheism lend social trinitarianism a strong
biblical basis. Statements about the immanent Trinity, which the naysayers
consider to be mere second-order perspectives, could be argued in light
of the Rahner axiom (identity between economic and immanent Trinity). 31
In a recent contribution to the debate, Irvin (2011:402) gives the impression that he
unproblematically combines both motifs and approaches; see, for example, his statement: “[T]he
more closely one enters into the Trinitarian life of communion with God through Christ in the Spirit,
the more one’s life can be expected to reflect these divine attributes.” Venter theology deviates substantially from Patristic, Medieval and Reformational
accounts. To dismiss some of the major theologians such as Barth, Rahner,
Zizioulas, Pannenberg, Moltmann, Jenson, Boff, Volf, and Plantinga in
this manner is indeed a brave intellectual feat. The doctrinal culprit was
Hegel, who located the fundamental distinction in reality in the Father-
Son relation, and not in the God-world relation. Modern theologians have
consistently followed Hegel in this regard (Holmes 2012b:186). It is not
possible to engage fully with Holmes’ position. In his theology, divine
simplicity functions so strongly that the distinctions among the three divine
persons, manifest in the history of the divine economy, are eclipsed when
one ventures to state something of the divine immanent identity. Finally,
the question concerning the political and the divine disappears totally in
his polemical approach. The quality of Tanner’s argument, resisting social trinitarianism,
requires careful attention. In several works (2004; 2012), she articulated her
rejection of this trend in theology. She points out that the preference for a
social analogy for the Trinity is a particular modern development, starting
in the late nineteenth century. Her dismissal is based on two perspectives. Tanner believes that the progressive political potential of the Trinity has
been overestimated. The Trinity could be interpreted differently; especially
the traditional order of the persons (taxes), and the language of Father/Son
could support hierarchical relations and sexism. Trinitarianism could be as
dangerous as monotheism. She accuses proponents of social trinitarianism
of modifying the problematic features inherent in the doctrine. The second
major problem she addresses is the fundamental difference between God
and human beings. The nature of divine relationality is different from that
of creatures: “Divine persons … seem much more relational than human
beings.”; human character is not in the same intimate way bound up with
relations (2012:379). The movement from God to humanity is the crucial
issue to address. Her own strategy and alternative is to give preference
to participation30 and Christology. According to Tanner (2004:328), the
economic Trinity closes the gap by incorporating human beings within it. Human beings are “swept up” into the Trinity; they should not “become
like” (2012:383) the triune God. Hope for the radical renewal of the human
condition lies with this incorporation and not with imitation. She continues 36 2022 Venter Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Van den Brink (2014:332) is genuinely concerned that the critique and
dismissal of a social understanding will lead us back to the situation in which the doctrine of the Trinity
as just an inutile piece of speculative thinking, safely stored in a far-
away barn of the theological tradition. It is unavoidable to raise the question as to whether the trinitarian
enthusiasm has exhausted itself, and whether the renaissance is morphing
into something different. Some years ago, Peterson (2016:12) asked
whether “the current fecundity could be symptomatic of the doctrine of
the Trinity’s ill-health”. Tonstad (2016:1) put it bluntly in the same year:
“Trinitarian theology has lost its way.” A few years later, Harvey (2018:6)
warned that the backlash has gained so much momentum that the “entire
project is in danger of becoming a footnote, bracketed in time and no
longer worthy of investment”. Barrett (2021) recently advocated an
“unmanipulated” approach. Historical conditions are changing, and a new
and younger generation of scholars are emerging with novel antennas and
interests. There have been maximalists and minimalists, and this divide will
likely continue. Acta Theologica Supplementum 34 In response to the conventional allegation of tritheism, it is interesting
to note that Van den Brink shows that social trinitarians employ the
notion of “person” to critique individualist understandings; a trinitarian
theology generates its own conception of personhood (2014:347). Having
addressed the typical problems raised against social trinitarianism, Van
den Brink argues for a continued appreciation thereof, in light of its biblical
foundation, the truth of revelation (what God discloses in the economy is
true about Godself), and the identity of the economic and immanent Trinity
(2014:349). The very understanding of salvation corresponds fittingly to a
relational approach, as believers participate in the life of the triune God. 37 Mosser, C.
2009. Fully social trinitarianism. In: T. McCall & M.C. Rae (eds), Philosophical and
theological essays on the Trinity (Oxford: Oxford University Press), pp. 131‑150.
https://doi.org/10.1093/acprof:oso/9780199216215.003.0008 Recommended further reading The recommendations focus on various positions on social trinitarianism, a
discussion of one specific question – that of simplicity – and an evaluation of
possible contributions made by theologians of the trinitarian renaissance. Dolezal, J.E. 2014. Trinity, simplicity, and the status of God’s personal relations. International Journal of Systematic Theology 16(1):79-98. https://doi. org/10.1111/ijst.12016 Dolezal, J.E. 2014. Trinity, simplicity, and the status of God’s personal relations. International Journal of Systematic Theology 16(1):79-98. https://doi. org/10.1111/ijst.12016 Hasker, W. 2010. Objections to social trinitarianism. Religious Studies 46:421-439. https://
doi.org/10.1017/S0034412510000107 Hasker, W. 2010. Objections to social trinitarianism. Religious Studies 46:421-439. https://
doi.org/10.1017/S0034412510000107 Mosser, C. 2009. Fully social trinitarianism. In: T. McCall & M.C. Rae (eds), Philosophical and
theological essays on the Trinity (Oxford: Oxford University Press), pp. 131‑150. https://doi.org/10.1093/acprof:oso/9780199216215.003.0008 Mosser, C. 2009. Fully social trinitarianism. In: T. McCall & M.C. Rae (eds), Philosophical and
theological essays on the Trinity (Oxford: Oxford University Press), pp. 131‑150. https://doi.org/10.1093/acprof:oso/9780199216215.003.0008 38 Acta Theologica Supplementum 34 32
Bowen & Simmons (2022:2) align themselves with this pluralist position and consider scholars
such as Marion, Westphal, and Hart to also belong here. Acta Theologica Supplementum 34 2022 Plantinga, C. Jr
1989. Social Trinity and tritheism. In: R.J. Feenstra & C. Plantinga jr., Trinity,
incarnation, and atonement (Notre Dame, IN: University of Notre Dame Press),
pp. 21-47. Venter, R. 2019. Taking stock of the trinitarian renaissance: What have we learnt? HTS
Teologiese Studies/Theological Studies 75(1), a5407. https://doi.org/10.4102/
hts.v75i1.5407. 33
For a clear and succinct discussion of these developments, see Jonkers (2005:3-7).
34
For an excellent treatment, see Peacocke (1998), who describes Heidegger’s complex use of
terms such as “theology”, “philosophy”, and thinking. He also accounts for how Heidegger leaves
the question open for the possibility of God (1998:187-192).
35
One of the best introductory overviews is arguably the work by Gschwandtner (2013), who gives
an informative description of the major thinkers in the movement. Considering the doctrine of God Considering the doctrine of God 2.9 A final and current trajectory in a proposed theo-genealogy concerns the
relationship between Theology and Philosophy of Religion. A vigorous
discourse since the second half of the previous century in continental
circles among philosophers holds particularly challenging resources for
thinking about the Christian God. This interaction between Theology
and Philosophy of Religion, which Gunton (1996:306) aptly labels
“indispensable opponent and conversation partner”, does not consistently
receive its due attention. This section briefly maps the horizon, and the
next two sections deal with one major philosopher of religion, R. Kearney,
and with a prominent theologian, D. Tracy, respectively. Tracy has taken
this discourse with utter seriousness. A productive place to start may be the influential article by Janicaud
(2001) on the “theological turn” in French phenomenological philosophy. He accuses well-known thinkers such as Levinas, Henry, and Marion of
allowing theology to compromise phenomenology, by leaving room for
the god question. He resisted this imperialism, as theology’s focus on
God and phenomenology’s focus on intentional consciousness make
for two separate disciplines. The reception of Janicaud’s claim has
continued and, after thirty years, the controversy remains unabated. In a
recent evaluation, Bowen and Simmons (2022:2) identify three attitudes:
those who think that Janicaud was right, those who reject his position,
and those who consider him partially right – the pluralists who deem the
two disciplines to be distinct but advocate a discursive interaction.32 It
is critical to identify the background to this discourse: the influence of
Heidegger to overcome metaphysics as onto-theology. He rejects the
structure of thinking, as positing God as the absolute ground of being by
way of analogical thinking. The path of Hegel, presenting the apogee of
metaphysical thought, via Nietzsche and Husserl with their destruction
of metaphysics and the introduction of phenomenology, stopped with 39 Considering the doctrine of God Considering the doctrine of God 36
For a recent and informed discussion of a phenomenology of kenosis, see Cassidy-Deketelaere
(2022), who identifies various senses of kenosis, and finally argues for a kenotic turn of
contemporary phenomenology itself, which results in a radical de-theologising of the enterprise.
37
For a fascinating treatment, see Kearney (2021:573, 583). Venter Heidegger.33 “Onto-theology”, the neologism coined by Heidegger,
describes the nature of the metaphysical enterprise. For Western
metaphysics, since the Greeks, ontology and theology went hand in hand. The deity enters to function as causa prima, the ultimate Ratio, unifying
as generative ground the wholeness of the whole. Heidegger exposes the
West’s fanatical quest for the final ground of being. Worth noting, in this
instance, is exactly whát Heidegger rejects: the metaphysical God, the
traditional God of reason. The death of God does not necessarily suggest
atheism and nihilism. A different kind of thinking, the meditative, may
allow the holy to unveil itself.34 The quest in Continental Philosophy of Religion has been for thinking
Transcendence beyond onto-theology subsequently. Thinkers such
as Levinas, Ricoeur, Derrida, Marion, Caputo, and Kearney, as well as
debates on the nature of phenomenology, on weak thought, the event,
alterity, kenosis, and apophasis have all come to constitute a vibrant and
multifaceted discourse.35 Despite big differences, Simmons (2008:926)
identifies five commonalities in the movement: there is an explicit conviction
that what these scholars are doing is philosophy and not theology;
there is a connection between God and the Other; kenosis is central for
understanding God; the focus is on the immediacy of religious experience,
expression is difficult, and a post-foundationalist epistemology of trust
is present. The central concern is to think and speak God beyond onto-theology. The challenge is how to do this in a more authentic way, to refer to the
divine not contaminated by being, or otherwise than being. This involves
two moves (see Jonkers 2005:11): to think à Dieu, towards the totally other,
but also adieu, that is, attending to radical Alterity ánd saying farewell to
onto-theology. The quest is for a divine God. The critique of ontotheology
is inherently a critique of idols; it tries to master transcendence to the
extent that one could say that “every concept of God does violence to
God” (Schrijvers 2006:303, 313). New categories of thinking have emerged,
which try to avoid the typical casual pattern, asserting God as the
prima causa. 40 7
For a fascinating treatment, see Kearney (2021:573, 583). Recommended further reading The following works represent an excellent overview of the religious views
of major philosophers from Kant to the present; two classic texts by Marion
and Caputo; a treatment of the idiosyncratic views of Heidegger, and one
well-informed description of current French phenomenology. Caputo, J.D. 2006. The weakness of God: A theology of the event. Bloomington, IN: Indiana
University Press. Goodchild, P. & Phelps, H. 2017. Religion and European philosophy: Key thinkers from Kant to Žižek. London: Routledge. https://doi.org/10.4324/9781315642253 Gschwandtner, C.M. 2019. What is phenomenology of religion? (Part II): The phenomenology of
religious experience. Philosophy Compass 14:e12567. https://doi.org/10.1111/
phc3.12567. Marion, J-L. 1991. God without being. Chicago, ILL: University of Chicago Press. Vedder, B. 2007. Heidegger’s philosophy of religion: From God to the Gods. Pittsburgh,
PN: Duquesne University Press. Acta Theologica Supplementum 34 2022 Basically, in all the projects from Levinas to Caputo, one finds the
emphasis on excess (see Gschwandtner 2013:287). Marion is of particular
relevance and importance, in this instance. The central premise of his
phenomenology is the primacy of givenness. This is a radicalisation of
Husserl’s phenomenology, as it liberates it from metaphysics. Givenness
is unconditional and irreducible (see James 2012:21-25) and includes
phenomena of saturation. This refers, for Marion, to givenness free from
any conditioning. These appear outside of the Kantian categories of
understanding (quality, quantity, relation, or analogy) (James 2012:32),
beyond any dependence on a finite horizon. The work by Caputo is also worth mentioning. In his work, one comes
across notions that could be considered in a constructive Systematic
Theology of God. In his radical hermeneutics, he emphasises “event”, the
weakness of God, a theology of “perhaps”. The critical question, however,
is: To what extent has he taken deconstruction to its final consequences? What remains of “God”? For him, “God” is a name for a call that “insists”,
rather than exists. It is an event that translates into a form of life. Kenosis,36
in his philosophy of religion, could result in a total eclipse of God. Ultimately,
he speaks about an “impossible God”.37 Otherness or alterity plays a crucial role in this discourse. Otherness
allows for some form of transcending. The critique of ontotheology is,
indeed, this quest for a space of transgression, or for an openness for
Otherness (Henriksen 2010:163). This will become clear in the philosophy
of Kearney in the next section. Verhoef (2017:180, 181) summarises the impact of the discourse
in Continental Philosophy of Religion well. The space for theology has
not been eliminated; it has been transformed. A radical metaphysical
understanding of God is no longer tenable. Continued reinterpretation
is required. 41 Considering the doctrine of God Venter Venter Recommended further reading Acta Theologica Supplementum 34 2022 Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 it is a dark bottomless abyss, a void of empty space. It is a-theological
and a-donational. According to Kearney, deconstruction has a preferential
option for khora, and is more “archi-ultimate than God” (2003:203, 205). What he finds problematic, for example in Caputo’s thought, is that khora
is an alternative to theology; it is either khora or God. He himself considers
it a “third way of posse”, a space where the divine may dwell, where there
could be a “play between khora and hyperousia” (2003:193, 208). In this
regard, Kearney (2003:207) makes a significant remark: “not every notion
of the Trinitarian God … is a fetish of presence or hyperessence”. Kearney established himself as a major post-metaphysical thinker
with his work The God who may be (2001a). Central in this study is an
interpretation of Exodus 3:14. Equating YHWH with being - God is
Being itself - inaugurated ontotheology. Kearney (2001b:155) submits
an alternative interpretation: “I am who may be”, which he labels “onto-
eschatological”, or a “poetics of the possible”. God’s esse is revealed as
posse: “God neither is nor is not, but may be” (Kearney 2001a:1). Kearney
steers a course, in this instance, between atheism and ontotheological
theism. His positioning within the wider postmodern naming of God is
critical, in this instance. If God has no being (like Marion’s view), alterity is so
other, so unrecognisable that it becomes impossible to distinguish it from
monstrosity (Kearney 2001b:167, 180 n45). Kearney’s proposal suggests
a new interpretation of God and being, a “rediscovery of posse in esse”
(2006:15). God keeps godself open for a future. Within the broader context
in the narrative, there is a displacement from an ancestral deity to a salvific
God. And then – and this is crucial in Kearney’s thought – a commitment to
a shared history of becoming (2001b:161): a relationship is established “of
mutual answerability and co-creation” (2001b:162); “God can be God only
if we enable this to happen” (2001a:2); and “Divinity capacitates humanity
and, in return, humanity reactivates divinity” (2006:16). Kearney is right:
“So much depends, then, on what we mean by the possible” (Kearney
2007:58). Kearney’s (2006:5) train of thought moves obviously into the terrain
of the eschatological. He describes his own contribution as a “fourth
reduction”, an eschatological one. 2.10 Anatheism - The God who may be Richard Kearney, a prominent participant in the contemporary Continental
Philosophy of Religion movement to think the divine in a post-
ontotheological fashion, has made unique contributions. What makes
this renaissance man such an attractive thinker is not only his impressive
acquaintance with philosophical traditions, but also his openness
to religious thought in general, his aesthetic antenna, and his acute
political awareness. The prominent motifs in his philosophy such as the
“possible”, eschatology of everyday life, alterity, bodiliness, and touch are
exceptionally promising for an engagement by Christian theology. Terms
such as “anatheism”, carnal and diacritical hermeneutics have become
associated with his name. His resolute commitment to move beyond the
omni-God, the Alpha God of traditional theism, must be borne in mind. A reader is constantly confronted by novel interpretations in his “theo-
poetics”. His difference from Caputo is of importance and should be
carefully noted. This is seen in a pronounced manner in his impressive
discussion of khora (Kearney 2003:193-211). This enigmatic notion, first
mentioned by Plato, has intrigued thinkers about its precise referent. For
thinkers such as Derrida and Caputo, this has no meaning, no identity; 42 Venter Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God in the color of their eyes, in the lines of their hands and fingers, in
the cracked tone of voices, in all the tiny epiphanies of flesh and
blood (2006:7). in the color of their eyes, in the lines of their hands and fingers, in
the cracked tone of voices, in all the tiny epiphanies of flesh and
blood (2006:7). This micro-eschatological “possibilizes” the other three reductions and
signals a return to poetics (2006:12). Kearney coined the term “anatheism” to refer to his position of a God
after God, of returning to God after having left God. This represents for him
a historical-cultural phenomenon; it coincides with the cultural situation
that comes after the death of God. He is fairly sensitive about what we
mean when speaking of God – “so much depends … on what we mean by
God” (Kearney 2010:xiv). Anatheism opens a space for questioning God, a
space for endless interpretation for the Absolute and requires pluralism and
not absolutism. This differs from atheism and theism, as it resists absolute
positions against, as well as for the divine (2010:16). It should be noted
that Kearney’s position is fairly nuanced. Anatheism is not a dialectical
third term that supersedes theism and atheism; it contains a moment of
both (Kearney 2017:35). It entails a retrieval of liberated faith. Within this
context, one can clarify what Kearney (2009:167) wants to leave behind: the
God of metaphysical causality and theodicy; this is his adieu. God is not a
thing out there that can be described in an essentialist manner: “God is a
call, cry, summons” (Kearney 2016:141). He thinks of God in terms of the
“more” the “surplus”, and the “surprise”, the “enabling God” (2009:182). His
eschatological thrust surfaces again, in this instance, as the eschatology
of the possible. The space of anatheistic thinking does not represent a
fait accompli, but a “wager” to be made again and again (2009:1983). This
is a central motif in his philosophy and is not about calculation and blind
leaps, but about imagination and hospitality (2010:xvii). It is about re-
imagining the Sacred in “the least of these” (2017:35); Kearney’s (2007:53)
God is “a God of ‘small things’”, a “kenotic God” (2016:258). One should
also refer in this context to his preference for theo-poetics, as anatheism
requires it (2017:37). Acta Theologica Supplementum 34 The well-known context, in this
instance, is the radicalisations of phenomenology, that is, the reorientation
of consciousness – transcendentally (essence), ontologically (being),
and donologically (gift) by Husserl, Heidegger and Marion successively. Kearney’s (2006:6) redirection is to the everyday, the manifestation of
the divine 43 Venter 38
The influence of Etty Hillesum should be acknowledged, in this instance. Venter It is not about abstract systems of thought, but about
imagination. Kearney thinks, in this instance, and it is critical to see this,
in terms of “mutual recreation between the human and the divine … God
co-depends on us”; “divine poiesis relates to human praxis” (2017:32, 34):
“It is we who must help God to be God” (2007:55).38 Theopoesis brings us to Kearney’s appreciation for artistic expression
and also for the Rublev’s icon of the Trinity. He is convinced that artwork
goes deeper and wider than theoretical theology (2017:41). In the icon,
Kearney highlights several aspects: the perichoretic dance by the three 38
The influence of Etty Hillesum should be acknowledged, in this instance. 44 Acta Theologica Supplementum 34 2022 persons, but then especially also the empty chalice. This is a “drama of lived
hospitality” (2017:30). The chalice represents a “fourth person”; it is the
chora of “endless possibility” (2017:39). Kearney is especially interested in
this space – “a symbol of the gap in our horizons of time and space where
the radically Other may arrive” (2010:25). This fourth dimension signifies
the empty space of the ordinary (2006:10), where the eschatological will be
realised in the presence of the alien in our midst (2010:29). Relationships
and spaces go together: for Kearney, this triune God is the “interstitial
deity” (2016:252); “in the beginning was the Relationship” (2017:41). There is a certain attractiveness in Kearney’s project to imagine the
divine anew after ontotheology. At the same time, the systematic theologian
cannot suppress some “anxiety” (see Venter 2018:247-250). Benjamins
(2015:120) rightly points out that Kearney does not consider the possibility
of a God who reveals or identifies godself. This kenotic God requires some
form of sacramental mediation, where an interplay between the self and the
stranger can take place. Tracy (2016:222-228) credits Kearney for having
succeeded in uniting a philosophical vision with a way of life – anatheism is
finally a way of life (2016:222). But he also has critical questions, especially
about the understanding of power, kenosis, and messianism in Kearney’s
work. For me, the critical question is whether the successive reductions in
the phenomenology have truncated the divine that we end up with a notion
of the divine who is so dependent on us, that not even anatheism could
save God, as “God is up to us, in the end” (Kearney 2016:250). Venter Might this
be the final theo-trajectory before an aggressive atheism triumphs? Kearney, R. & Zimmermann, J. (Eds)
2016. Reimagining the sacred: Richard Kearney debates God. New York: Columbia
University Press. https://doi.org/10.7312/columbia/9780231161039.001.0001 Venter Manoussakis, J.P. 2006. After God: Richard Kearney and the religious turn in continental philosophy. New York: Fordham University Press. Veldsman, D.P. & Steenkamp, Y. (Eds)
2018. Debating otherness with Richard Kearney: Perspectives from South Africa. Durbanville: AOSIS. https://doi.org/10.4102/aosis.2018.BK94 Veldsman, D.P. & Steenkamp, Y. (Eds)
2018. Debating otherness with Richard Kearney: Perspectives from South Afr
Durbanville: AOSIS. https://doi.org/10.4102/aosis.2018.BK94 Recommended further reading The following recommendations include an interesting autobiographical
sketch by Kearney himself, a series of conversations he held with prominent
contemporary philosophers on God, a critical description of his project
by Caputo, a long interlocuter, and two volumes of essays engaging with
aspects of Kearney’s oeuvre. Caputo, J.D. 2021. Where is Richard Kearney coming from? Hospitality, anatheism, and
ana-deconstruction. Philosophy and Social Criticism 47(5):551-569. https://doi. org/10.1177/01914537211021929 Kearney, R. 2018. Where I speak from: A short intellectual autobiography. In: D.P. Veldsman
& Y. Steenkamp (eds), pp. 31-62. https://doi.org/10.4102/aosis.2018.BK94.02 Kearney, R. & Zimmermann, J. (Eds)
2016. Reimagining the sacred: Richard Kearney debates God. New York: Columbia
University Press. https://doi.org/10.7312/columbia/9780231161039.001.0001 45 Venter Considering the doctrine of God 39
In the last phase of this theological development, Tracy increasingly employs the idea of “Absolute
Infinity”. His promised big book is allegedly an exploration of this in a trinitarian fashion (see
interview with Woodward, 2019).
40
The idea of the “tragic” in Tracy’s thought warrants a separate study. He regards natural
catastrophes as “tragic necessities” (Tracy 2016:28). At the same time, he wants to free the
human from the Augustinian heritage that blames humanity for all suffering and evil (Tracy
2005a:106). 2.11 The impossible God - Incomprehensible
and hidden Together with the Old Testament scholar Brueggemann, the philosopher
of religion Kearney, and the systematic theologians Barth and Moltmann,
David Tracy should be considered a truly major god thinker of the past
one hundred years. This Catholic theologian from Chicago is “God-
obsessed” (Gibson 2010:14), a sustained effort to name God for the
contemporary era. He has become exceptionally influential with his
approach to theology as hermeneutical and public, with key terminology
such as “publics of theology” and the “fragment”, and with notions such
as “plurality and ambiguity” and “mystical-prophetic”. Since his Gifford
lectures (1999/2000), he has indicated that he is working on a “big book”
on God that has not yet been published. In several articles and interviews,
he has, however, delineated the main contours of his thinking on God. What makes him particularly relevant for my line of argumentation,
especially in this section on a theo-genealogy, is his intentional effort
to move beyond the relational paradigm and to articulate an explicit
postmodern vision of God. As background to Tracy’s thought, one should bear in mind his acute
antenna to “name the present”, that is, to engage with postmodernity and
to critique modernity, and to be sensitive to the many forms of suffering. As
a postmodern thinker, he wants to break with totality systems and attend
to the “other” (Malcolm 2002:27). Consequently, he resists the reduction
of everything to more of the same, and the exclusion of what is different
(Tracy 2000:68). He views “radical suffering” as the principal philosophical
and theological issue of our time (Tracy 2016:27). The meaning of suffering
and the reality of the other destroy confident versions of history and allow
for the “return of God” (Tracy 1994:43). The notion of “form” is central to Tracy’s approach to name God anew. One cannot understand the Western intellectual if the question of form
is not taken prominently into account (Tracy 2005b:49). He situates the
recent interest in form in the context of the resistance to the embrace of
modernity. Theologically, the Word has always taken form in Christ and
the Trinity (Tracy 2005b:50). It is crucial for Tracy and for his subsequent 46 Acta Theologica Supplementum 34 2022 construal of a vision of God to identify two basic religious forms in the
Bible: the prophetic and the sapiential or meditative (Tracy 2005b:54). He combines these two in the category of the prophetic-mystical. Venter appears as more than relationality; it is a transgressive, excessive, sheer
gift (Tracy 2011:127). For Tracy, biblical books such as Job, Lamentation
and Song of Songs become important traditions conveying these accents. Tracy (2011:124) keeps on emphasising that this impossible, hidden,
incomprehensible, and excessively loving God is radically disruptive; there
is no onto-theological deity in this instance. One final remark about Tracy and the doctrine of the Trinity may
be pertinent. Part of his project is to overcome the fatal separations of
modernity, one of which is the division between theology and spirituality. In this context, writing about the trinitarian “form of the divine reality”,
he defends the “form of speculation”. In addition to realistic forms of
narrative, doctrine, and liturgy to give expression to the economic Trinity,
Tracy (1999:287) allows for speculation about the immanent Trinity. He
(2002:29) considers this advocacy a shift in his thinking: I have been driven to think that some such speculation is needed if
we are to speak of the immanent Trinity. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 2.11 The impossible God - Incomprehensible
and hidden The
prophetic is associated and radicalised with the apocalyptic and gives
expression to the ethical (Tracy 2005b:55). The sapiential or meditative
form is, in turn, linked to love, as well as to dark realities such as lament
or tragedy (Tracy 2000:75). By linking the task of naming God with “form”
and substantiating it in this manner opens vast possibilities for speaking
the divine . Tracy has an exceptional historical command of the theological tradition
and wants to move beyond the Patristic and Medieval focus on God as
intelligence and love, and the modernist one on relationality. He credits
modernity, under the influence of Hegel, with the outstanding achievement
of naming God in terms of relationality (Tracy 2011:123). He, however, has
his reservations about its overconfidence and non-apophatic character
(Tracy 2011:123); hence, his project to move beyond it. Theo-logy – the
speaking of the logos under conditions of intelligibility (Tracy 1994:37)
– implies for him as postmodern to employ the idea of the “Impossible”
(Tracy 2011:124), a term which he finds already with Kierkegaard (Tracy
2000:71) and later with Derrida (Tracy 2006:343) and which he uses to fight
systems of totalisation.39 He then develops his understanding of God as “Impossible”, in line with
his interpretation of forms, from two specific perspectives: hiddenness and
incomprehensibility. This is his move beyond the modernist paradigm of
relationality. For Tracy, the major historical exponent of hiddenness is to be
found with Luther. His own interpretation is fairly unique. He connects it to
history, and with the suffering of the non-person, those who are oppressed
and marginalised, on the one hand, and with the “abyss, chasm, chaos,
horror”, on the other (Tracy 2000:82). This understanding of hiddenness
is novel; it allows for a historical-political reading (Tracy 1996:9), and it
retrieves motifs such as lament and tragedy40 that were hitherto neglected. For Tracy, Dionysius the Areopagite exemplifies thinking about God and
incomprehensibility. The limits of language come into focus with this
emphasis, as well as a new estimation of the apophatic tradition. Love 47 47 Woodward, K.L.
2019. In praise of fragments: An interview with David Tracy. Commonweal
Magazine, 25 September: 11 pages. 2020b. Metaphysics, theology, and the mystical. In: E. Howells & M.A. McIntosh
(eds), The Oxford handbook of mystical theology (Oxford: Oxford University
Press), pp. 425-449. https://doi.org/10.1093/oxfordhb/9780198722380.013.22 41
The exact function of placing the doctrine at the end is interpreted in various ways. An astute
scholar such as Schüssler Fiorenza (2005:175) does not regard this as a “marginalization”, but
rather as the “conclusion” by Schleiermacher of his emphasis that God is love and not power.
42
The pronoun as used by Barth is reflected, in this instance. Recommended further reading The sources recommended include a full treatment of Tracy’s oeuvre and
his theology, a recent interview with him, and three of his outputs, namely
a collection of essays, and articles on the infinite and on spirituality. The sources recommended include a full treatment of Tracy’s oeuvre and
his theology, a recent interview with him, and three of his outputs, namely
a collection of essays, and articles on the infinite and on spirituality. Okey, S. 2018. A theology of conversation: An introduction to David Tracy. Collegeville,
MN: Liturgical Press. Tracy, D. 2018. God as infinite: Ethical implications. In: M. Renaud & J. Daniel (eds),
God and the moral life (London: Routledge), pp. 135-156. https://doi. org/10.4324/9781315142975-9 Tracy, D. 2018. God as infinite: Ethical implications. In: M. Renaud & J. Daniel (eds),
God and the moral life (London: Routledge), pp. 135-156. https://doi. org/10.4324/9781315142975-9
2020a. Fragments: The existential situation of our time. Chicago, ILL: University
of Chicago Press. https://doi.org/10.7208/chicago/9780226584508.001.0001
2020b. Metaphysics, theology, and the mystical. In: E. Howells & M.A. McIntosh
(eds), The Oxford handbook of mystical theology (Oxford: Oxford University
Press), pp. 425-449. https://doi.org/10.1093/oxfordhb/9780198722380.013.22
Woodward, K.L. 2019. In praise of fragments: An interview with David Tracy. Commonweal
Magazine, 25 September: 11 pages. Tracy, D. 2018. God as infinite: Ethical implications. In: M. Renaud & J. Daniel (eds),
God and the moral life (London: Routledge), pp. 135-156. https://doi. org/10.4324/9781315142975-9
2020a. Fragments: The existential situation of our time. Chicago, ILL: University
of Chicago Press. https://doi.org/10.7208/chicago/9780226584508.001.0001
2020b. Metaphysics, theology, and the mystical. In: E. Howells & M.A. McIntosh
(eds), The Oxford handbook of mystical theology (Oxford: Oxford University
Press), pp. 425-449. https://doi.org/10.1093/oxfordhb/9780198722380.013.22
Woodward, K.L. 2019. In praise of fragments: An interview with David Tracy. Commonweal
Magazine, 25 September: 11 pages. 2020a. Fragments: The existential situation of our time. Chicago, ILL: University
of Chicago Press. https://doi.org/10.7208/chicago/9780226584508.001.0001 2020b. Metaphysics, theology, and the mystical. In: E. Howells & M.A. McIntosh
(eds), The Oxford handbook of mystical theology (Oxford: Oxford University
Press), pp. 425-449. https://doi.org/10.1093/oxfordhb/9780198722380.013.22 Woodward, K.L. 2019. In praise of fragments: An interview with David Tracy. Commonweal
Magazine, 25 September: 11 pages. 48 Acta Theologica Supplementum 34 2022 3. ELEMENTS OF A TRINITARIAN GRAMMAR 3.1
Who is God? Trinitarian fundamentals and
enduring contributions Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter 3.1.1 God as event – Barth’s contribution Barth’s accomplishments are vast, and there are many good studies
of his doctrine of God. In a recent general overview of his doctrine of
God, Sonderegger (2019:213, 225) rightly labels it “startling new” and
“revolutionary”. One cannot think about the Christian naming of God as
triune and not attend to the grammar that transpires in Barth’s treatment. In this short description, five major perspectives are merely identified. First, in Barth’s (1975:303) multi-volume Church dogmatics, the
location of the Trinity in Volume 1/1 is decisive. By placing it there,
Barth intentionally wants to emphasise that “its content [is] decisive
and controlling for the whole of dogmatics”. No student of the trinitarian
doctrine can miss the contrast with Schleiermacher’s positioning thereof
at the end of The Christian faith.41 Decisions about doctrinal structuring
ultimately have a fundamental impact. The Trinity, however, is more than
the conclusion of an argument; it allows for a basic optic, a way of viewing
the Christian interpretation of reality. Secondly, Barth (1975:311) firmly connects the Trinity to the doctrine
of revelation as the self-interpretation of God. The Trinity answers the
question: Who is God? This is the “business of the doctrine of the Trinity”
(Barth 1975:301). The Trinity is the “basic presupposition of the doctrine
of God” (Barth 1975:312). Connecting the Trinity so intimately to revelation
emphasises three aspects: God reveals himself through himself, and he
reveals himself.42 The subject, the act and the effect of revelation are one,
or the Revealer, the Revelation and the Revealedness (Barth 1975:296). Revelation is the self-interpretation of this God. Thirdly, Barth (1975:479) closely ties statements of the immanent Trinity
to God’s work in the economy. What one encounters in revelation “is His
reality in all depths of eternity”. God is “precisely the One He is in showing
and giving Himself” (Barth 1975:382; see also 479). Grenz (2004:48)
comments, in this instance, that the economic Trinity is the noetic starting
point for Barth, but the immanent Trinity retains the ontic priority. Barth 49 Considering the doctrine of God Venter (1957:317) does not shy away from speaking about “otherness in God” and
of “relationality”: “He is in Himself … relationship, the basis and prototype
of all relationship” (Barth 1957:641). It may be worth noting, in this instance,
that, when Barth (1975:432) discusses Jesus as Eternal Son of God, he
considers the “begetting” as “the divine mystery”. Already when God
begets the Son, God negates in himself self-isolation: “from all eternity …
God is orientated to the Other” (Barth 1975:483). Torrance (2000:85) refers,
in this context, to the coherence of Barth’s theological enterprise; he
remarkably holds together the articulation of the interrelatedness of God’s
triune being and the nature of God’s being in relation to human beings. Barth makes striking statements with regard to the essence of God, the
divine as such: the fellowship in Godself is extended to humanity – what He seeks and creates between himself and us is in fact nothing
else but what He wills and completes and therefore is in Himself
(Barth 1957:275). Fellowship, relationality, and love go together. Fellowship, relationality, and love go together. Fellowship, relationality, and love go together. Fourthly, in Church dogmatics 2/1, there are far-reaching perspectives
on God, to which one can only briefly refer. Barth (1957:260, 263)
innovatively refers to “God’s life”, resisting an absorption of the doctrine of
God in a doctrine of being: “God’s Godhead consists in the fact that it is an
event”. God is who He is in his works (Barth 1957:260). There is something
radically dynamic to his approach to God; God should be understood as
event, as act, as life (Barth 1957:264). To say that God is, is to say that God
loves. Being and act are the same; they go together. Fifthly, “what makes God God” is the fact that God loves in freedom
(Barth 1957:273). For Barth, the answer to the questions about who God
is, or what nature God is, is the same: The One who loves in freedom
(Barth 1957:331). Finally, the long section on the divine “perfections” is
fairly impressive. It follows the discussion of the Trinity, and the attributes
are treated in dialectical pairs such as, for example, patience and wisdom,
constancy and omnipotence. The order of treatment – only after a
discussion of the Trinity – is a far-reaching insight. One cannot discuss
this in full in this purview. Two notes can be offered. Barth’s originally
can clearly be noted when he thematises God’s spatiality in a discussion
about omnipresence – “God possesses space” (see Barth 1957:468, and
the entire section 461-490). When concluding with the glory of God, Barth
(1975:640-677) interprets it in aesthetic terms – “God is also beautiful”; “He
is the basis and standard of everything that is beautiful and of all ideas of
the beautiful” (Barth 1957:656). Without a trinitarian perspective on God, 50 Acta Theologica Supplementum 34 2022 one could hardly have ascribed these qualities to the divine .43 Surprising
avenues open when one resists a prior metaphysical determination of what
deity entails, but allows God to determine Godself in revelation. The richness of Barth’s theology cannot be captured in this summary,
but the direction of his thinking is clear. The positioning of the doctrine in
an overarching scheme, the emphasis on divine agency, the intimate link
between immanent and economic Trinity, the prioritising of eventfulness,
relationality, love, and freedom, as well as a trinitarian determination of
divine perfections are grammatical achievements that have until now not
been overtaken in contemporary discourse. Fellowship, relationality, and love go together. Ultimately, Barth (1975:3)
accomplished what he set out to do in terms of the discipline of Dogmatics,
a distinct field of study tasked with examining the content of the church’s
distinctive talk about God. He did so in such a manner that it should be
heeded in future God talk. 44
The first volume of Jenson’s Systematic Theology – The triune God (1997) will be used as primary
source for this summary of his views.
45
According to Grenz (2004:110), this is Jenson’s central contribution to trinitarian theology.
46
In a recent contribution, Jenson (2018:14) returns to the crucial decision a theologian should
make about the ontological weight which the overall narrative character of the Bible conveys and
the plot the story tells. He stresses the continued task of revising Greek religious metaphysics by
biblical gospel narrative. Jenson identifies himself thus with a strong interpretation of the Rahner
rule (see Grenz 2004:112). Recommended further reading eco
e ded u
e
ead
g
Major and standard interpretations of Barth’s doctrine of God and of his
trinitarian views are recommended, as well as more recent explorations of
aspects of his thought in this regard by well-known Barth scholars. Major and standard interpretations of Barth’s doctrine of God and of his
trinitarian views are recommended, as well as more recent explorations of
aspects of his thought in this regard by well-known Barth scholars. Habets, M. & Tolliday, P. (Eds)
2011. Trinitarian theology after Barth. Eugene, OR: Pickwick. https://doi. org/10.2307/j.ctt1cgf2j9 Johnson, W.S. 1997. The mystery of God: Karl Barth and the postmodern foundations of
theology. Louisville, KY: Westminster John Knox. JÜngel, E. 2001. God’s being is in becoming: The trinitarian being of God in the theology
of Karl Barth. A paraphrase. Translated by J Webster. Grand Rapids, MI:
Eerdmans. https://doi.org/10.5040/9780567690906 McCormack, B.L. 2019. Trinity. In: P.D. Jones & P.T. Nimmo (eds), The Oxford handbook of Karl
Barth (Oxford: Oxford University Press), pp. 227-245. https://doi.org/10.1093/
oxfordhb/9780199689781.013.15 Price, R.B. 2011. Letters of the divine word: The perfections of God in Karl Barth’s Church
Dogmatics. London: T. & T. Clark. 43
See Barth’s express comments on space (1957:468), and on beauty (1957:659). 51 Considering the doctrine of God Venter 3.1.2 God and temporal events –
Jenson’s contribution Robert Jenson (1930-2017) is one of the prominent theologians of the so-
called trinitarian renaissance of the last half of the twentieth century. To
appreciate his contribution, one should place him in the context of the
reception of Barth. His work God after God: The God of the past and the
God of the future, seen in the work of Karl Barth (1969/2010) is also a
major study of this theologian’s trinitarian theology. Some of the central
motifs (identity of God, eternity and time, as well as beauty) of Jenson’s
project cannot be understood without considering the ground-breaking
achievement of Barth. Jenson is, for this reflection, relevant because of his
appreciation of the importance of the God issue for systematic theology,
and for several critical insights into construing a Christian doctrine of God. The notion of “identity” is central in Jenson’s view of the Trinity. He
considers Barth’s question, “Who is God”, as part of the doctrine of the
Trinity, as one of his greatest achievements. Jenson (1997:60)44 follows
Barth in this regard, stating that “[t]he primal systematic function of
trinitarian teaching is to identify the theos in ‘theology’”. It is interesting to
note that Jenson (1997:12, 42) finds this identification of God in the biblical
narrative of the resurrection. For him, the temporality of events such as
the exodus and the resurrection is outstanding: temporal events belong
to God (1997:49).45 God is identified by and with these events, which
Jenson (1997:71) labels as dramatic narratives, “in the life of God”.46 The
biblical God is not immune to time. Having stated this, Jenson (1997:55)
moves subsequently to the issue of eternity as the chief diagnostic
question of a religion. With this as background, Jenson (1997:75) proceeds
argumentatively to the dramatis dei personae – the “characters of the
drama of God”, and the deepest mystery of God’s identity is that the God
of Israel appears as one of the personae dramatis, thus necessitating the
doctrine of the Trinity (Jenson 1997:89). Jenson’s reading of the Patristic
dilemma concerning time/eternity is fairly impressive, referring to attempts
to escape from captivity in time by asserting the deity’s capacity for degrees
of divine ness or time-denying predicates such as impassibility (Jenson 52 Acta Theologica Supplementum 34 2022 1997:94). The church’s most decisive achievement was to differentiate the
God of the Gospel from the God of that culture (Jenson 1997:103). 47
Jenson’s (1997:108) critique of the Patristic period should be noted: the indecision about
impassibility, the loss of an interpretation of Logos as God’s speech, and restriction of relations to
origin and the ignoring of God’s eschatological character. Venter (2004:415) speaks about the “eternal political life of God”. Totus Christus
implies that the second person of the Trinity includes the church within
this moral space. (2004:415) speaks about the “eternal political life of God”. Totus Christus
implies that the second person of the Trinity includes the church within
this moral space. Like his great predecessor Barth, Jenson appreciates beauty. For him,
God is also an event, because something happens between Jesus and his
Father in their Spirit, and this harmonious perichoresis sings beautifully. Ultimately, Jenson (1997:236) can claim: “God is a great fugue.” There is something deeply attractive in Jenson’s theology; he allows
an arcane doctrine to become intellectually intriguing. His connection of
the trinitarian doctrine with notions of identity, with narrative, and with
temporality are enduring feats. The re-valuation of pneumatology, the
consistent relation of immanent and economic Trinity, the invitation of
“roominess”, and the acknowledgement of beauty are evidence of his
stature as major trinitarian thinker. Considering the doctrine of God Venter Venter Considering the doctrine of God Considering the doctrine of God 3.1.2 God and temporal events –
Jenson’s contribution In this
instance, one encounters a decision about the “eventful differentiation in
God himself” (Jenson 1997:102).47 Jenson then makes his decisive move:
the action of the triune God is limitless; God takes time, and his eternity
is “temporal infinity” (Jenson 1997:217). He interlinks love, infinity, time,
and eternity. The importance Jenson assigns to pneumatology should be
highlighted. Jenson (1997:154) credits Barth for appreciating the Trinity
as the framework within which theological puzzles are to be addressed,
but he is critical whether Barth does not remain trapped in the typical
Western conception of the Spirit as the bond of love between Father and
Son, which results in a mere binity. Jenson (1997:157) moves way beyond
this: “The Spirit is the Liveliness of the divine life because he is the Power
of the divine future”; that is, “the fulfilment not only of created life but of
the divine life”. Jenson (1997:157) tries to redress a long-standing tradition
to prioritise the protological character of the divine to the detriment of
the eschatological. In this instance, Jenson (1997:160) appeals to the
resurrection – the “great occurrence of dramatic causality in God”. Three
elements transpire in Jenson’s trinitarian identification of the Christian
God: narrativity, temporality, and eschatology (see Grenz 2004:113). Jenson raises the issue about “the point of trinitarian theology”, a
cypher one often finds in a bewilderingly diverse state of scholarship
which is helpful to guide thinking. Jenson’s (1995b:37) answer is helpful. The function of trinitarian theology is to stress that the biblical story of God
and human beings is true for God himself. Here again, he returns to the
question about temporality and eternity. God’s eternity is not simply the
absence of time. As God’s life is “constituted in a structure of relations,
whose content is narrative”, time is the “accommodation God makes in his
living and moving eternity, for others than himself” (Jenson 1995b:40). The
triune life is open to others; it is “roomy” (Jenson 1997:226). And that room
that God makes is our creaturely time. Jenson’s (2004:414) originality is noticeable in his view that one could
talk about a “polity in God”. Entry into the kingdom is also entry into the
triune life and the polity that this represents. One finds in the triune God
a plurality of social personae, each with his/her own role and in total
self-giving to the others. Without hesitation, and on this basis, Jenson 53 3.1.3 The Trinity as practical doctrine –
LaCugna’s contribution The story of LaCugna’s trinitarian work, her concentrated intellectual
endeavour, and her premature death at the age of forty-four years, is part
of the history of the trinitarian renaissance and needs to be told. Catherine
Mowry LaCugna (1952-1997), former Professor of Theology at the
University of Notre Dame, produced a remarkable corpus of work on the
Trinity in her short academic career. Her award-winning book God for us:
The Trinity and the Christian life (1991a) and an array of substantial articles
and contributions to volumes are evidence of her intense and exclusive
focus on the doctrine of the Trinity. Apart from honouring her contribution
to this field, I believe that her work comprises some of the most significant
challenges to the trinitarian discourse. Not only will her book God for
us: The Trinity and the Christian life remain one of the seminal reference
sources, but her research highlights some of the most prominent issues
in trinitarian discussion. The ambitious scope of her project should not
escape notice. Ultimately, she wants to restore the doctrine of the Trinity
to the centre of theology and Christian life. Basic to her theology and, arguably, one of her most decisive
contributions to the trinitarian discourse is her rephrasing of the central
problem to be addressed. The conundrum of oneness and threeness and
how to establish reconciliation is not the issue as one finds so prominently
in Latin trinitarian reflection; it is rather the question of how the trinitarian pattern of salvation history is to be
correlated with the eternal being of God (LaCugna 1991a:4). The central theme of the doctrine of the Trinity, the “subject matter” is the
relationship between the oikonomia and the theologia (LaCugna 1991a:22). The central theme of the doctrine of the Trinity, the “subject matter” is the
relationship between the oikonomia and the theologia (LaCugna 1991a:22). LaCugna (1991a:7) wants to address the “virtually total irrelevance
of the doctrine of the Trinity”. She describes the eclipse and neglect of
the doctrine theologically, spiritually, and politically after its emergence
as the defeat of the Trinity. Central to her work is identifying the cause
for a rupture between theologia and oikonomia, starting with the Nicene
decision that solidified in the theologies of Thomas Aquinas and Gregory
Palamas. The fatal historical impetus came from Arius and the ensuing
polemic which resulted in the homoousios decision of Nicaea. Recommended further reading Various discussions of Jenson’s theology, in general, and of his trinitarian
views are recommended. Various discussions of Jenson’s theology, in general, and of his trinitarian
views are recommended. East, B. 2017. What is the doctrine of the Trinity for? Practicality and projection in Robert
Jenson’s theology. Modern Theology 33(3):414-433. https://doi.org/10.1111/
moth.12334 Grenz, S.J. 2003. The divine fugue: Robert Jenson’s renewed trinitarianism. Perspectives in
Religious Studies 30(2):211-216. Gunton, C. (Ed.)
2011 (2000 original). Trinity, time and church: A response to the theology of
Robert W. Jenson. Eugene, OR: Wipf & Stock. Verhoef, A. 2011. Timelessness, Trinity and temporality. Acta Academica 43(2):82-112. Wright, S.J. & Green, C.E.W. (Eds)
2017. The promise of Robert W. Jenson’s theology. Minneapolis, MN: Fortress. https://doi.org/10.2307/j.ctt1pwt849 54 54 2022 Acta Theologica Supplementum 34 48
The seminal text is Rahner’s The Trinity, originally published in 1967. Interestingly, LaCugna
wrote an extensive introduction to the 1997 English edition. For an excellent discussion of various
interpretations, see Sanders (2001). Considering the doctrine of God Considering the doctrine of God Venter 3.1.3 The Trinity as practical doctrine –
LaCugna’s contribution Arius
diverted Christian theology from the oikonomia to an ontology of theologia. Increasing attention was directed towards the intradivine relationality, to
God in se, with a subsequent disconnection of the reflection on God from
soteriology or God for us (LaCugna 1991a:12). This disjunction is of cardinal 55 Venter The interrelationships of divine persons 56 Acta Theologica Supplementum 34 2022 are not located in an intradivine sphere, but in the mystery of the economy,
“which is where God exists anyway” (LaCugna 1991a:369, n. 1). are not located in an intradivine sphere, but in the mystery of the economy,
“which is where God exists anyway” (LaCugna 1991a:369, n. 1). Having established the essential unity of the oikonomia and the
theologia, LaCugna infers two principles for the practice of doing trinitarian
theology: all thinking about God must start with the economy of God,
and all reflection must be related to soteriology. According to her, “[t]he
economy of salvation is the basis, the content and the final criterion for
every statement about God” (LaCugna 1991a:22). Soteriology becomes
decisive for thinking about the Christian God. Although she does not
emphasise it strongly and explicitly, but the motif that can be gleaned from
the whole tenor of the argument is her conviction that trinitarian theology
could be done according to pre-Nicene principles. Prior to Nicaea, the
emphasis was on the economy and not on the intradivine self-relationality
of God (LaCugna 1991a:101, 223). Unlike some prominent trinitarian thinkers, LaCugna (1992:681) retains
the category person and redefines it. The shift in her project to a more
Eastern-oriented and -informed trinitarianism, from substance ontology to
relational ontology, is also most conspicuous at this point. By following
the Eastern line, LaCugna opts for an ontology with personhood and
relationships as critical and definitional categories. She continues using the
category “person” because beneath it lies the category of relation (LaCugna
1985:648). It is noteworthy that, for her, desire as yearning for the other out
of love becomes one of the features of personhood (LaCugna 1991a:351). However, throughout the work, certain categories for understanding
the nature of the Christian God as Trinity crystallise and she obviously
views them as not only revolutionising the doctrine of God, but also as
incapacitating all endeavours of approaching the God issue generically. The Christian God is to be understood in terms of self-communicating
love, relationality, differentiated personhood, communion, fecundity, and
ecstasy (see her detailed exposition, LaCugna 1991a:288-292). The mystery and incomprehensibility of God is one of the themes
usually treated alongside the doctrine of the Trinity. LaCugna’s project
shows no exception to this standard practice; how she understands it
and what function it plays deserves careful attention. Venter importance for LaCugna: it provides the hermeneutical key to interpret the
history of the doctrine and to articulate a vision for its rehabilitation. She accepts Rahner’s well-known axiom – the economic Trinity is the
immanent Trinity, and vice versa48 – as a good starting point to rethink the
Christian doctrine of God (see the extensive treatment in LaCugna 1985). Rahner’s proposal functions as an epistemic principle for her: the only
starting point could be the economy of salvation. LaCugna (1991a:221)
expressly parts ways with Rahner on one crucial issue: she cannot accept
that economic distinctions are grounded in distinctions “in” God. This
leads to the crux of LaCugna’s project. The relation between economic
and immanent Trinity may simultaneously be the central and the most
controversial aspect of her work. LaCugna is unambiguous about what is
unacceptable; she expressly rejects the possibility of “a deus absconditus
who lurks behind deus revelatus” (LaCugna 1991a:211, 322); “a hidden
realm of intradivine relations” (:222); “an intradivine realm” (:223); “an
intradivine Trinity of persons” (:224); a “God in se” (:225); “a transeconomic
perspective” (:227); “the notion of God’s inner life” (:229), and “two sets
of communion” (:274). She is adamant that “there is nothing ‘in’ God
… whether it be attributes or relations or a Trinity of persons” (:225). In
addition, she abandons the terms “economic Trinity” and “immanent
Trinity” (:223). She (1991a:383) puts her case in strong language: Indeed, the ultimate theological error, the ultimate non-orthodoxy
or heresy or untruth about God, would be to think of God as living
in an altogether separate household, living entirely for Godself, by
Godself, within Godself. After this account of the negative dimension of her vision, one can
delineate her proposal. According to her, the distinction between economic
Trinity and immanent Trinity is strictly conceptual, and not ontological. She
abandons the terms “immanent Trinity” and “economic Trinity”, and only
uses the categories oikonomia and theologia. According to her, oikonomia
is not the Trinity ad extra, but “the comprehensive plan of God reaching from
creation to consummation”; theologia, on the other hand, is not the Trinity
in se, but simply the “mystery of God” (LaCugna 1991a:223). LaCugna’s
(1991a:221) revision of the Rahner axiom is as follows: “theologia is fully
revealed and bestowed in oikonomia, and oikonomia truly expresses the
ineffable mystery of theologia”. Venter cataphatic theology, or between silence and predication is crucial in
trinitarian theology (LaCugna 1991a:333, 361). Apophatism protects the
mystery of God; kataphasis permits statements to be made about the
revelation of God in Christ and the Spirit. cataphatic theology, or between silence and predication is crucial in
trinitarian theology (LaCugna 1991a:333, 361). Apophatism protects the
mystery of God; kataphasis permits statements to be made about the
revelation of God in Christ and the Spirit. The form of theological language that is commensurable with God’s
economy is doxological. A distinguishing feature and, arguably, one of
the major contributions of LaCugna’s trinitarian project emerges in this
instance: her insistence on the importance of liturgy for the doctrine. She asserts that the overarching purpose of trinitarian theology is the
praise of God (LaCugna 1991a:360). Trinitarian theology, as liturgical
and doxological theology, shifts the attention away from the speculative
conundrum about the oneness and threeness of God. The primary function
of the liturgy for trinitarian reflection is thus to restrain speculation and to
enable it to balance the incomprehensibility of God with our knowledge of
God in Christ (LaCugna 1989b:6, 8). One’s impression is that, despite her thorough historical knowledge
and impressive constructive theological ability, her deepest sentiments
are liturgical, ecclesiastical, and practical. The axiom of her project is the
following: “The doctrine of the Trinity is ultimately a practical doctrine with
radical consequences for Christian life” (LaCugna 1991a:10). Basic to her
argument is that the Christian life should be a form of life appropriate to
God’s economy. This includes the individual spiritual journey, ecclesial
arrangements and socio-political practices. The trinitarian doctrine
communicates an existential message of participation in the eternal
relationality of God (LaCugna 1991b:178). In this regard, baptism plays a
significant role in her understanding of the individual life before God. She
understands baptism as “an incorporation … into the history and story
of God, into the life and heart and identity of God” (LaCugna 1989a:248). As such, it also establishes a radical ontological change. To live in the
name of the triune God means appropriating the identity of God and living
God’s life (LaCugna 1991a:382, 400, 404). As baptism is also entry into a
new social order carrying the ethical imperative of reconfiguring previous
patterns of relationships and creating a community of interdependence,
mutuality, reciprocal love, and self-sacrifice (LaCugna 1989a:247, 238). Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God Venter The mystery is to be
located – consistent with her methodological principle! – in the economy
of salvation. The economy is as ineffable as the eternal mystery of God
(LaCugna 1991a:322); “the mystery of God in the economy permanently
remains ineffable mystery” (LaCugna 1991b:155). The reason for this is
that God is personal. A person is, according to her, an ineffable mode
of existence. To speak of God as mystery is another way of saying God
is personal (LaCugna 1991b:156). A balance between apophatic and 57 Battaglia, V.
2007. An examination of Karl Rahner’s trinitarian theology. Australian eJournal
of Theology 9:1-18. Venter At present, the baptism could be a symbol of liberating relationships,
enabling the church to be an icon of the triune God. A trinitarian spirituality
emphasises that one should become what God’s personal reality already is
(LaCugna 1991b:189). Socially, the Trinity functions as a critical theological
principle (LaCugna 1991a:17), and the basic principle to be followed is:
“Divine relationality becomes the paradigm for every type of relationality
in creation” (LaCugna 1991a:17, 168). Her sociopolitical perspectives 58 Acta Theologica Supplementum 34 2022 are derived from the reconstructed model of divine monarchy which she
calls “trinitarian politics”, as it conveys the basic principle of communion
among equals (LaCugna 1991a:388-400). The Trinity is a theology of “right
relationships” (LaCugna 1992:681). The sentiments of feminist theology figure prominently in LaCugna’s
work and one is struck in her response by a balanced attitude to respect
both tradition and contemporary experience. Because of its direct bearing
on liturgy, i.e. how people worship, the feminist concern for adequate
language for God, is no passing fad (LaCugna 1989b:9). Trinitarian language
and specifically the baptism formula need not be inherently sexist and
patriarchal. She encourages the depatriarchalising of the concept of God
by using a variety of images and names, provided they reflect the relational
nature of God and match some aspect of the pattern of salvation history
(LaCugna 1993:106,109; 1989a:246). She believes the most effective
response to the oppression of women and the contemporary feminist
theological engagement is to argue that the best defence against sexism is
to argue ontologically that “the being of God is utterly antithetical to every
kind of subordination and subservience” (LaCugna 1991:287). For her, the
deeper issue at stake is not language as such, but patterns of relationships
in the church. By virtue of their baptism, believers are equal in the church;
inclusive language must be matched by inclusive community. There is something deeply attractive about LaCugna’s life work. She
enriched trinitarian scholarship with her penetrating reading of the history
of interpretation and her innovative constructive proposals. In light of her
work, one cannot consider the Rahner axiom a closed matter. Her emphasis
on liturgy, apophatism, baptism, and practical social implications are
enduring contributions that any trinitarian construal will have to consider,
even if one does not follow her in all detail. Her specific contribution is
the contraction of major insights in a coherent project that continues to
intrigue and stimulate. Baik, C-H.
2011. The Holy Trinity – God for God and God for us: Seven positions on
the immanent-economic Trinity relation in contemporary theology. Eugene,
OR: Pickwick. 49
Holmes (2012a) gives a good overview of the history. He aptly describes an attribute as “any term
that adequately completes the sentence ‘God is…’” (2012a:48). Sadly, he (2012a:65), having
acknowledged that the attribute traditions have been “subject to a ‘perfect storm’” (2012a:64)
over the past two hundred years, and that considering Jesus Christ as part of the internal history
of God should lead to a redefinition of the attributes, still prefers to hold to the scholastic forebears. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Groppe, E.T. 2002. Catherine Mowry LaCugna’s contribution to trinitarian theology. Theological Studies 63:730-763. https://doi.org/10.1177/004056390206300404 Hilkert, M.C. 1998. The mystery of persons in communion: The trinitarian theology of
Catherine Mowry LaCugna. Word & World 18(3):237-243. LaCugna, C.M. & McDonnell, K. 1988. Returning from “the far country”: Theses for a contemporary trinitarian
theology. Scottish Journal of Theology 41:191-215. https://doi.org/10.1017/
S0036930600040783 Recommended further reading The recommendations focus on treatments of the Rahner axiom, on
discussions of LaCugna’s project, and a major general discussion by her
and a collaborator on trinitarian theology. 59 50
Recently, a somewhat idiosyncratic approach is found in the 2-volume Systematic Theology
by Sonderegger. In volume 1 (2015), she discusses omnipresence, omnipotence, and
omniscience at length. In volume 2 (2020), she submits her detailed trinitarian theology and
links holiness thereto.
51
For a discussion of the problematic with source referencing, see Venter (2019:2). 3.2.1 The attribute tradition –
Approaches and challenges Basically, in all doctrines of God, one finds treatments of divine attributes. Theologians are clearly inclined to describe the God worshipped by
Christians, to indicate the difference between the deity and creation. It is
possible to write an entire history of the attribute tradition in the various eras
of history. Structurally, it has become conventional to classify the attributes
as, for example, communicable and incommunicable. One pervasive and
longstanding tradition has been the separation of a discussion of the One
God together with the attributes from one of the Trinity which typically
comes thereafter. This divorce has far-reaching consequences. Another
feature is the neglect of obvious descriptions such as beauty and justice. A
genealogy of the attribute traditions betrays deep philosophical influences
and – this is crucial – the operative historical conceptions of divinity of
the time under consideration. A history could be written of how people,
over various periods of human civilisation, considered and conceptualised
Ultimacy, or the Sacred. In this brief and limited section, the interest lies
basically in one major shift that has taken place since the contribution of
Barth – to consider attributes in a trinitarian way. At stake, in this instance,
is the resistance to what Gunton (2002:2) describes as the “hybrid of two
organisms”, and the retrieval of the Trinity to determine the interpretation
of attributes. Only a few historical milestones will be mentioned.49 60 52
Unfortunately, this is not a trinitarian approach. Great strides have been made to re-envision the
attributes. Berkhof (1993:326) understands the doctrine of the Trinity as a concluding description
and not as a fundamental optic, which finally impacted on this treatment of attributes.
53
Van den Brink, who established himself in several excellent articles, is thoroughly acquainted with
the trinitarian discourse and is a solid thinker in this regard.
54
For a major text on this theme by well-known scholars, see Keating and White (2009).
55
See the introduction by Levering and Kalantzis (2019:411-417) to the discussion by various
theologians on simplicity in Modern Theology (2019[35/3]). Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 2022 The influence of post-Reformation scholastic theology, by separating
the “what” question from the “who” question (see Beck 2016:196-204),
and by prioritising the One God over the Triune God is pervasive. The
formalisation of this approach was already present in the Belgic Confession. A theologian such as the South African Heyns (1992:40, 113), in his last
book, an introduction to Dogmatics based on this confession, discusses
the attributes in article 1 and the Trinity in article 8 – seventy pages further
in the book. The “who” and “what” questions are not integrated.50 With Schleiermacher, one encounters a revolutionary change in his
Christian faith (2016). He locates his discussion in three places throughout
the work (§§ 50-56, 79-85, 164-169) and correlates the attributes with
various experiences – of a general religious consciousness of the
relationship between God and world (eternity, omnipresence, omnipotence,
omniscience); of a consciousness of sin (holiness, justice, mercy), and of
a consciousness of redemption (love and wisdom). His approach raises
many questions. What is the referent in this instance? Is it still in some
way the being of God, or are the attributes mere cyphers for human
experiences of divine causality?51 They are definitely not expressions of
the divine triune reality, as only sections §§170-172 focus on this doctrine. With Barth, one comes across a major innovation with regard to an
exposition of the attributes. This was indicated earlier in section 3.1.1,
with the description of his contribution to trinitarian development. One
can merely mention his achievement. The positioning of the attributes,
after attending to the trinitarian identification of God, and the consistent
rethinking thereof should be considered one of the major developments
in the doctrine of God. Furthermore, his pairing of attributes dialectically
reinforces the understanding of the complexity of describing divine
“perfections”. Finally, his creative emphasis on divine space and beauty is
a particularly fine theological insight. Wise theologising should pay careful
attention to Barth’s interpretation. In this short section, it may be productive to refer to the particularly
rich tradition of developing divine attributes in Dutch Calvinistic theology
and in Dutch philosophy of religion. The dogmatist H. Berkhof (1993:121-
149) submits an excellent and attractive proposal developed from the
perspective of the encounter (“ontmoeting”) and grouped dialectically: 61 Considering the doctrine of God Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 2022 – hospitality56 and gifting.57 These capture adequately what divine economy
is about – action of sheer generosity to and embrace of the other. At the
same time, the two notions interact easily with wider philosophical trends
and social dilemmas. – hospitality56 and gifting.57 These capture adequately what divine economy
is about – action of sheer generosity to and embrace of the other. At the
same time, the two notions interact easily with wider philosophical trends
and social dilemmas. In future, reflection on divine attribute would have to carefully consider
the structural design of a doctrine of God. It is unlikely that one could argue
for a treatment separate from a consistent trinitarian view. Ultimately,
the “what” of divinity, to what extent that could be intimated, should be
informed by the very identity of God, the “who” of God. It is obvious that
divine life is totally “saturated” and one cannot truncate that in any way. A
plethora of descriptions may be justified and required. What one ultimately
identifies should obviously be adjudicated by a conversation between
Scripture, tradition, and the horizon of time. The critical question would
be: How should one speak in a trinitarian way under these specific current
conditions? In the final chapter, some intimations will be given about some
form of naming the present. 57
The work by Webb (1996:139-158) could be mentioned in this regard. God is understood as
Giver, Given, and Giving. 56
The space accorded by a trinitarian thinker such as Kärkkäinen (2014:310-339) to hospitality
is noteworthy. Venter God is viewed in terms of “heilige liefde”, “weerloze overmacht”, and
“veranderbare trouw”.52 The Utrecht School of Philosophy of Religion, with
Brümmer as mentor, published a major volume of essays re-imagining the
attributes – Hoe is uw naam? (see Van den Brink & Sarot 1995a). The key for a
reinterpretation is to be found in an explicit personalistic conceptualisation
of the divine (Van den Brink & Sarot 1995b:20). The systematic theologian
Muis (2016) published a major doctrine of God and applied a unique
approach by departing from the Lord’s Prayer, and consequently focusing
on notions such as love, holiness, righteousness, might, and eternity. In
his extensive doctrine of God, the scholar Van de Beek (2017:203-221)
surprises by giving a fairly truncated treatment of attributes before treating
the Trinity and focusing merely on incomprehensibility and omnipotence. The strange role of the Trinity in twentieth-century Dutch theology cannot
be missed. In a fascinating article, Van den Brink & Van Erp (2009) raised
the question as to whether the Dutch theologians have not missed
the trinitarian renaissance. In the outstanding Systematic Theology –
Christelijke Dogmatiek (2012) – Van den Brink53 & Van der Kooi redressed
this weakness with two good chapters (namely, 3 and 4) on God. In this
approach, the Trinity enjoys a primary place, and the attributes are treated
in light thereof (see specifically 2012:139). They still find it necessary to
classify transcendence and condescension. Two trends in contemporary discourse should be mentioned. There
are clearly two camps in the God discourse – those who are willing to re-
imagine the divine and those who strongly defend traditional positions. This can be clearly observed in the continuing and unresolved debate on
the impassibility54 and simplicity55 of God. For some, God and suffering are
untenable and, despite the Rahner rule, God’s being is purely simple. For
others, a consistent trinitarian conceptualization renders these attributes
open for re-envisioning. This difference is most likely to persist in Christian
thinking about the God being worshipped. A second trend moves into a different direction – seeking descriptions
that might express the unique life of the Christian God in categories
relevant to new, maybe postmodern, sensibility. Two may be mentioned 62 58
See the good treatment in the Stanford Encyclopedia of Philosophy – Howard-Snyder & Green
(2022). The views of Schellenberg and Rea are particularly relevant. The context of interpretation
is that of non-belief.
59
Brueggemann’s (1997:333-358) interpretation is exceedingly important. His interpretation
focuses on YHWH as the “guarantor of order”.
60
All references in this paragraph will be to this text. Recommended further reading The recommendations focus on two volumes of essays that deal with
various attributes, specifically with traditional and contemporary antennas,
and on three explorations of trust, vulnerability, and love, respectively. The recommendations focus on two volumes of essays that deal with
various attributes, specifically with traditional and contemporary antennas,
and on three explorations of trust, vulnerability, and love, respectively. Chalamet, C., Mazzocco, M. & Vial, M. (Eds)
2018. Naming God today: Contemporary approaches to the divine attributes. Special theme issue of Modern Theology 34(3):320-491. https://doi.org/10.1111/
moth.12418 McCormack, B.L. (Ed.)
2008. Engaging the doctrine of God: Contemporary Protestant perspectives. Grand Rapids, MI: Baker Academic. Reichel, H. 2017. The God who trusts. In: H. Springhart & G. Thomas (eds), Risiko und
vertrauten. Risk and trust (Leipzig: Evangelische Verlagsanstalt), pp. 23-36. Thomas, G. 2017. Divine disaster management: Cross and resurrection as an event of
creative vulnerability. Dialog 56(4):373-381. https://doi.org/10.1111/dial.12357 Vanhoozer, K.J. 2001. The love of God – Its place, meaning, and function in systematic theology. In: K.J. Vanhoozer (ed.), Nothing greater, nothing better: Theological essays on
the love of God (Grand Rapids, MI: Eerdmans), pp. 1-29. 63 Considering the doctrine of God Venter Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 He also connects it to revelation: revelation is the revelation of the
hiddenness of God (1983:124). The older dogmatic tradition emphasised
the incomprehensibility of God and one encounters a twofold narrowing,
in this instance: the focus is on epistemology and on human inability to
understand God, and one finds a reference to mysteries in the plural,
and not an emphasis on the one mystery of God’s salvation (1983:126). Kasper (1983:128) links the interpretation of hiddenness to mystery and
makes three crucial points. First, it refers to something theological and
not epistemic. It is about the revelation of God’s mystery, and it enables
one to speak. Secondly, it is about the revelation of the mystery of God’s
free self-communication in love. Ultimately, hiddenness is a theology of
the cross (1983:129). Thirdly, the revelation of God’s mystery is a message
of the mystery of one’s salvation. The constituting elements of Kasper’s
interpretation are clear: It is about God: God’s revelation, the mystery of
God’s love, and one’s salvation. The trinitarian profession of faith is the
“concrete exposition of the hiddenness of God” (1983:128). Jenson (2000:9) developed a captivating interpretation of hiddenness,
which he connects explicitly to the Trinity – “the locus of God’s hiddenness
is the very substance of all trinitarian teaching”. Hiddenness is not to be
understood in terms of human epistemic weakness or God’s ontological
uniqueness, but in terms of God’s impenetrable reality as moral agent. It
is a root error to view God’s hiddenness as metaphysical distance; the
exact opposite is rather true: God is hidden by his offensive availability
(1997:233). It is not that God holds something back from one; but that one
cannot keep up with God: “it is that we cannot at any moment of his life
with us fully understand what he is up to next” (1997:233). Jenson finally
explains hiddenness with reference to each one of the triune Persons, and
this is extremely crucial. For Jenson (2000:7), a divine attribute is properly
construed when the different roles of each divine identity is told with
regard to this aspect of their mutual life. Because the Father is the ultimate
Source, theodicy is finally impossible; his ways will remain unsearchable. In light of Jesus’ cross, the real God has the face of a suffering servant;
God will always come in suffering and rejection. Any other way will amount
to idolatry. 3.2.2 Hiddenness as the beginning of the knowledge
of God By assigning hiddenness a space in a doctrine of God carries with it basic
considerations. On the one hand, it counters a hubris that speaking about
the divine has been settled, as found in classical theism or in the relational
paradigm, and, on the other, a resignation that speaking is not possible or
no longer makes sense. In his excellent discussion of the hiddenness of
God, Peterson (2005:208) perceptively refers to a “middle way” between
hubris and atheism. Exploring the hiddenness of God could be a way
to navigate options in the current horizon of engaging the divine. It is
interesting to note that this thematics attracts a fair amount of attention
from diverse disciplines; one finds extensive discussions in Philosophy of
Religion,58 Old Testament scholarship,59 and Systematic Theology. It is also
clear that some surplus of meaning is at play, in this instance; no simplistic
interpretation is available. This brief section addresses the understanding
of four systematic theologians. Barth (1957:179-204)60 situates his treatment of “hiddenness” as part
of the knowledge of God. Knowledge of God begins with the knowledge of
God’s hiddenness. It is important to note that he considers hiddenness a
property of God. In the older dogmatics, it was coupled with the idea of the
incomprehensibility of God (1957:184). Something fundamental is at stake,
in this instance. Hiddenness wants to convey that God’s nature is not in
the sphere of human power; one cannot comprehend God (1957:187). Strictly, “we do not really know what we are saying when we say ‘God’”
(1957:189). This, however, does not render one silent. The hiddenness of
God is the terminus a quo of one’s knowledge of God; the moment one
confesses God’s hiddenness, one has begun to know God (1957:192). This
is a statement about God, and only in a secondary sense, one of human
incapacity. Hiddenness is the inconceivability of the Father, the Son, and
the Spirit (1957:197), and directs one to the revelation of God in which God
does not dissolve God’s hiddenness, but makes himself apprehensible. In his great work, The God of Jesus Christ (1983), Kasper submits
an excellent exposition of hiddenness. His interpretation lies in the
extension of Barth and Rahner; it conveys rich theological textures. 64 2022 Acta Theologica Supplementum 34 Venter especially in the suffering of the marginalised. The hidden God is the “deus
crucifixus, the crucified God” (Tracy 1996:9). Tracy’s contribution is that
the hiddenness should be rendered in historical-political terms (1996:9,
13). The categories of otherness, difference and marginality are important
for him. A second unique emphasis in Tracy’s approach is borrowed from
Luther: there is a radical form of hiddenness beyond the Word of the cross,
which can only be signified by the metaphors of abyss, chasm, chaos,
and horror (1996:10). This radical form of hiddenness is part of the biblical
portrait and of human experience. Tracy aligns himself with a Marcan view
of history: God is genuinely present through absence (1996:14) and is
disclosed as the God of life in conflict, struggle, and suffering. These few interpretations demonstrate the importance of the notion
“hiddenness”, but also what reservoir of meaning it encompasses. In
future, theology will inescapably have to listen to the rich interpretations
and continue making sense of these in each new horizon. The reality of the
divine meets one as an inexhaustible source of meaning. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 As God’s own future, the Spirit frees God “to surprise Himself”
(1997:12) and involves one in a play of infinite freedom. With Tracy’s interpretation, one meets innovative ideas and novel
directions. His view combines several strands of thinking: the turn to
the face of the other by Levinas; the critique of modernity’s violence; the
twofold notion of hiddenness by Luther, and the Gospel of Mark’s view
of God and history. Tracy incorporates the “turn to the other” seriously. God reveals Godself in hiddenness, in the cross, and in negativity, and 65 Recommended further reading Various interpretations are represented by the recommendations:
positions from Philosophy of Religion, from the Old Testament, and from
major individual thinkers from the Reformation to the twentieth century. Gericke, J. 2015. A comparative-philosophical perspective on divine hiddenness in the
Hebrew Bible. Verbum et Ecclesia 36(1), Art. #1400, 8 pages. http://dx.doi. org/10.4102/ve.v36i1.1400. Gericke, J. 2015. A comparative-philosophical perspective on divine hiddenness in the
Hebrew Bible. Verbum et Ecclesia 36(1), Art. #1400, 8 pages. http://dx.doi. org/10.4102/ve.v36i1.1400. Gerrish, B.A. 1973. “To the unknown God”: Luther and Calvin on the hiddenness of God. The
Journal of Religion 53(3):263-292. https://doi.org/10.1086/486347 Gerrish, B.A. 1973. “To the unknown God”: Luther and Calvin on the hiddenness of God. The
Journal of Religion 53(3):263-292. https://doi.org/10.1086/486347 Holmes, C.R.J. 2006. Disclosure without reservation: Re-evaluating divine hiddenness. Neues
Zeitschrift für Systematische Theologie und Religionsphilosophie 48:367-380. (Note – On Jüngel). https://doi.org/10.1515/nzst.2006.48.3.367 Norman, M. 2019. Luther, Heidegger, and the hiddenness of God. Tyndale Bulletin
70(2):291‑316. https://doi.org/10.53751/001c.27726 Rea, M.C. 2018. The hiddenness of God. Oxford: Oxford University Press. https://doi. org/10.1093/oso/9780198826019.001.0001 66 Acta Theologica Supplementum 34 2022 61
See also Moltmann’s discussion (2010:117-126) of this Psalm.
62
For a good discussion of the material situation of these groups, see Jacobson (2010). 62
For a good discussion of the material situation of these groups, see Jacobson (2010). 61
See also Moltmann’s discussion (2010:117-126) of this Psalm. 63
For a balanced treatment of the problems involved with such an approach, as well as the
fruitfulness, see Barton (2007).
64
For a discussion, see Feldmeier & Spieckermann (2011:291).
65
For a thought-out discussion, situating dikaiosynē contra competing understandings of the time
and considering it the new ethos of the household of God’s kingdom, see Crosby (2005:100-118).
66
The memorable phrase by Stendahl (see his seminal article 1976:78-96).
67
His targets are punitive notions of the divine and understandings of righteousness as imputation. Considering the doctrine of God Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God Venter 3.2.3 Is there justice in the Trinity? The key interest of this section is the question of how to think justice in the
most fundamental way possible, in order to make it socially productive. The argument to be developed will attempt at making justice inherent to
the nature of Ultimacy, or, in a specific Christian sense, to the character
of the triune God. The first part of the argument will pursue a brief and
cursory genealogy of the Christian concept of God, beginning in the Old
Testament and moving to the articulation of a trinitarian exploration from
the perspective of justice. The focus will be on developing a proposal
to think divinity in a Christian sense in terms of justice. The final part of
the argument will intimate some implications of a trinitarian approach for
understanding justice. This section aims to retrieve justice as primary
cypher to speak the Christian God; to rethink justice along relational lines,
and to highlight the socio-ethical function of the attribute tradition. Justice is a central theme in Old Testament religion, especially in the
later strata of the tradition. Texts such as Isaiah 61:8 (“For I, the LORD, love
justice”) and Psalm 89:14 (“Righteousness and justice are the foundation
of your throne”) point clearly in this direction. C. Wright (2004:258)
summarises this well: … for Israel the whole idea of justice was wrapped up with the
qualities and characteristics of the LORD, their God … . One remarkable text should be referred to, namely Psalm 82. Two
crucial insights should be mentioned. This Psalm answers the question
as to what “constitutes ‘godness’” (Brueggemann 1997:143) and about
the importance of justice in society. Miller’s discussion is worth noting. According to his reading (Miller 1986:4), “justice is the cornerstone of the
universe” and “justice is the issue on which the very claims of deity are
settled”. These comments obviously have far-reaching ramifications for
systematic theological thinking of the nature of Ultimacy, of the divine. Divinity as such should be construed in terms of justice.61 Not only is
justice central in Israel’s theological imagination; it is also informed
by a conspicuous concrete social antenna. God’s justice is directed to
the material conditions of the stranger, the widow, the orphan, and the
poor; Wolterstorff (2013:77) refers to the “quartet of the vulnerable”.62
Two additional connotations should also be mentioned: Old Testament 67 68
For a succinct discussion, see Sanders (1991:74-76) who gives six reasons why he believes a
participation mode of thinking should be preferred to a legal one. Venter social ethics could hardly be thought of without an imitatio Dei dynamic.63
The character of the divine is the ultimate source of the moral order. The second connotation is crucial for the link with the New Testament:
salvation. Especially in Deutero-Isaiah (see especially chapter 46:12-13),
the mediation of God’s justice is experienced as salvation.64 When engaging the New Testament world and traditions, one has no
difficulty in encountering a similar sensitivity to social justice. Especially in
Matthew and Revelation, justice is valued as central virtue. One can only
think of the beatitudes, for example Matthew 5:10, which conveys blessings
to those who are persecuted for the sake of justice.65 Matthew 25:31-46
can rightly be called the “grand charter for doing justice” (Wolterstorff
2013:102). The book of Revelation is an impressive re-imagining of power
amidst pervasive Roman injustice. Utilising the throne as central symbol,
the author envisions an alternative symbolic world. Schüssler Fiorenza
(1991:117) is correct when stating that “Revelation’s central problem and
topic is the issue of power and justice.” The intellectual question when
reading the New Testament is not whether justice is prominent, but
whether a major thinker such as Paul has not shifted the focus of the Old
Testament, especially in the letter to the Romans. Has a social orientation
not been eclipsed by a metaphysical occupation situating the individual
struggling with “an introspective conscience”66 to the centre? At stake is
obviously the long and immensely intricate history of interpretation of both
the Letter to the Romans and the concept of “dikaiosynē”, and the actual
effect thereof (Wirkungsgeschichte). Waetjen (2012:198), for example,
critiques Reformational interpretations, as they have a specific result: “a
gospel of individual salvation without commitment to the actualization of
God’s justice”.67 To interpret Paul as not superseding or eclipsing but rather as
radicalising Old Testament sentiments, one should undertake at least two
reading strategies: contextualising Romans and relaxing a consistent and
exclusive forensic thrust. Both these lenses do have some respectable
proponents. In his theology of Romans, Haacker (2003:116-124)
emphasises the Roman understanding of righteousness as background
to the letter: “the peculiar emphasis on righteousness in Paul’s letter to 68 Acta Theologica Supplementum 34 2022 Acta Theologica Supplementum 34 Venter (1999:35) thinks along similar lines. God is a “Trinitarian community of
reciprocal otherness” and emphasises that (1999:35) thinks along similar lines. God is a “Trinitarian community of
reciprocal otherness” and emphasises that [t]his is the decisive viewpoint from which to consider God’s
righteousness: God does not exist in splendid isolation as a solitary
being, but in God otherness is affirmed. (1999:35). The important insight, in this instance, is that God ís righteousness in
God’s being as triune, as affirmation of reciprocal otherness. On this
basis, God communicates this ad extra, making others also righteous. Jüngel’s relational mode of thinking should not escape notice: sin is
relationlessness, and salvation entails the establishing of “new relations,
new life-connections” (1999:38). The train of thought expressed by Jenson and Jüngel is also found in
the work of the philosopher Wolterstorff. His views are important, as he
not only engages a wide scope of contemporary discourses on justice, but
prioritises social justice, and explicitly advocates a “theistic grounding of
justice”. He is critical of justice as right order, a view found in the seminal
work by Rawls – A theory of justice – and he proposes an alternative
conception: justice as natural rights based on human worth: The theory of claim-rights that I have articulated is an inherent rights
theory; I hold that it is on account of our worth, our dignity, that we
have rights (Wolterstorff 2013:121). This theory of rights is based on the distinction between primary justice
and reactive/rectifying justice (see 2008:71; 2013:22). This worth is
grounded in God’s love for human beings – “What does ground human
rights … God’s love for each and every one of God’s human creatures
(2013;137; see also 2008:360). Wolterstorff’s essay (2006) in the Moltmann
Festschrift – “Is there justice in the Trinity?” – is of relevance to the present
argument. Basic to this view is that rights are social (2006:184; 2008:286;
2013:44). His notion of primary justice is axiomatic to arguing for justice
in the Trinity: the way persons treat one another with due respect for their
worth. With this framework as background, he concludes: Not only is there justice in the relation of the persons of the Trinity
to each other, justice in their relationship is caught up within love for
each other (2006:187). Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 the Romans cannot be haphazard”. A de-contextualised interpretation
of dikaiosynē would not be possible. The polemical thrust of notions
such as “impartiality” and “peace” should be noted. Haacker (2003:124)
observes that one of the rare instances of “kingdom of God” in Romans
14:17 is not without reason connected to “righteousness, peace and joy”. Such a reading of Romans de-stabilises an a-historical interpretation of
righteousness/justice. The well-known Pauline scholar Sanders (1991:74)
is of the opinion that “the deeper levels of Paul’s thought are not found in
the juridical categories”. He believes that another mode of thinking takes
place, namely “participation in a new being”. This participation in Christ
produces real transformation.68 The work by N.T. Wright is also important
in this regard. He acknowledges that there is no word in English that
adequately translates the relational aspects of dikaiosynē with its juridical
overtones. In this instance, Wright (2013:801) identifies a “web of meaning”,
comprising the law court, covenant, and cosmic eschatology. Important in
his detailed and multi-levelled interpretation is the view that dikaiosynē
is “an attribute of God himself”, “an attribute of covenant faithfulness”
(2013:804). The fundamental question, for the present argument, is: What
kind of image of God crystallizes in the Letter to the Romans? Is it one
that stands in some continuity with the Old Testament with regard to
justice? In a construal such as this, one could obviously follow various
approaches. In her comprehensive study, Bassler (1982) highlights the
notion of impartiality. Hays (2002:124) emphasizes the narrative structure
of the God of Romans, which amounts to “an epistemological revolution”
(2002:125). Despite a magnificent discussion, one cannot escape the
impression that something is amiss in his treatment – a specific framing of
the question: What does the prominence of dikaiosynē imply for the very
nature of God self? Two Lutheran systematic theologians – Jenson and Jüngel – may be
of some assistance to take the argument further than mere covenantal
thinking. Jenson (1995a:426) raises the pertinent question: “Why is God
himself righteousness? What is the actuality of God’s own righteousness?”
As trinitarian thinker, Jenson (1995a:425)
identifies
patrological,
Christological, and pneumatological dimensions: The Father “sets”
righteousness; Jesus Christ is the “event” of righteousness, and the Spirit
is the “movement” of righteousness. Jenson understands righteousness
as “the underived event of communal faithfulness in God”; it “occurs in
God” as perichoresis (1995a:426, 427). This is the critical point. Jüngel 69 Venter Love as attachment and love as acknowledgement of worth of the other
are both to be found in the Trinity; thus, justice is “a constituent of love
within the Trinity”. He does not hesitate to refer to human beings called to 70 Acta Theologica Supplementum 34 2022 reflect the life of the Trinity; see, for example: “We mirror the inner life of
the Trinity” 2006:187; also 178). reflect the life of the Trinity; see, for example: “We mirror the inner life of
the Trinity” 2006:187; also 178). The brief discussion of the three trajectories conveys an impression
that it is possible to think justice and God in a Christian construction of
divine identity in an intimate manner; there are ample resources available
for that. In the genealogical journey, justice could be found as central to
the nature of the divine, and there are subsequent attempts to radicalise
the association that culminates in thinking of the being of God in terms of
justice. A thorough and systematic exploration of this relationship between
God and justice will have to be situated in the long tradition of divine
attributes. It remains an outstanding task to retrieve justice as central to
God’s being and to re-construct this in a consistent, trinitarian manner. At
least two tasks need to be completed in a project of this nature: to think
attributes along relational lines, and to explore the ethical ramifications of
such a vision. If the identity of the Christian God is relational, such an optic
in the treatment of the attributes becomes the hermeneutical key.69 One’s
naming of the attributes is not epistemologically neutral; the theologian
makes choices. What is emphasized and what is marginalized or even
neglected mask values and interests. The construction of a theology of the
attributes does not only require intricate theological discernment, but also
sociopolitical responsibility.70 A trinitarian re-imagining of justice will prioritise sociality as key
to understanding justice. Justice is a quality of social relations. Such
an understanding will consciously attempt to de-privatise, or even
de-interiorise justice. The intellectual framework for thinking justice
is not primarily a legal one, but rather a relational one. The trinitarian
confession provides theologically a much fruitful and productive key for
conceptualising this. In the so-called trinitarian renaissance of the last half
a century, a host of central motifs emerged, for example, mutuality, gifting,
hospitality. 69
The work by Krötke (2001) – Gottes Klarheiten – should be mentioned. God’s self-relatedness
forms the basis for the doctrine of the divine attributes. As God is an event of relatedness, the
attributes should be understood as “concretisations” of this relation to the self. “Glory” plays an
important role in his entire attribute theology; it represents the relationally rich reality of God. For a
discussion of this aspect of Krötke’s work, see Holmes (2007:162-165).
70
Power and Downey (2012:53) are aware of this. They correctly refer to “Naming God as an
ethical project”. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Although a trinitarian imagination has virtually an inexhaustible
hermeneutical potential to make sense of intellectual challenges and
questions, much will depend on how one understands the approach. The
previous insight about sociality relies on an argumentative move based
on the immanent Trinity. A rich array of interpretative associations comes
to the fore when one uses the traditional doctrine of appropriations. For
example, a patrological perspective will shed light on otherness; the
Father provides the fecund foundation for the possibility of alterity, and
constitutes an ontological basis for this in creation. A Christological view
could open vast possibilities to think materiality, narrativity, and dialectic
in light of the incarnation, the story of his earthly life, and the event of
the cross/resurrection that could lend much texture to any treatment of
justice. A pneumatological exploration could highlight agency and the
reality of a novum amidst historical hopelessness. These suggestions
obviously require further explanation. Suffice it to suggest that a trinitarian
approach could generate meaningful avenues to understanding justice in
a Christian sense. A consistent trinitarian understanding of justice creates the possibility
of a full and coherent Christian theology of this most important social and
existential yearning. Imagining the life of the divine in terms of justice,
opens avenues for thinking the moral order in ontological terms as well
as the character thereof. Justice belongs to the fabric of being and
is fundamentally a relational reality. At the same time, a trinitarian view
conveys hope for the righting of injustice and the ultimate rehabilitation of
victims and the innocent; it provides a powerful motivation and direction
for human moral action. Moberly, R.W.K.
2020. The just God: The nature of deity in Psalm 82. In: Idem., The God of the Old
Testament (Grand Rapids, MI: Baker Academic), pp. 93-123. Kevern, J.R.
1997. The Trinity and social justice. Anglican Theological Review 79(1):45-54. Johnson, E.A.
2010. Female symbols for God: The apophatic tradition and social justice.
International Journal of Orthodox Theology 1(2):40-57. Venter These terms all try to convey something of the exceptional
quality of the relationship of the three divine persons in their equality,
unity, and otherness. A trinitarian re-envisioning of justice could employ
this promising scholarship. 71 Considering the doctrine of God Acta Theologica Supplementum 34 2022 Muis, J. 2018. The justice of God. Modern Theology 34(3):356-367. https://doi.org/10. 1111/moth.12422 Van Wyk, T. 2019. Redressing the past, doing justice in the present: Necessary paradoxes. HTS Teologiese Studies/Theological Studies 75(4), a5625. https://doi. org/10.4102/hts.v75i4.5625. Recommended further reading The recommendations entail a detailed discussion of the fascinating
Psalm 82, a general systematic discussion of “justice” as attribute, and
various interpretations of how thinking about the divine and justice could
be construed. Moberly, R.W.K. 2020. The just God: The nature of deity in Psalm 82. In: Idem., The God of the Old
Testament (Grand Rapids, MI: Baker Academic), pp. 93-123. 72 Acta Theologica Supplementum 34 71
Groothuis’ review evidences this. He opines that a “misnomer is an understatement” for labelling
the book! Venter this way neither requires God to be an extra-literary character, nor does it
require or preclude faith. this way neither requires God to be an extra-literary character, nor does it
require or preclude faith. It is not possible to summarise the nuances and subtleties of Miles’
interpretation and argumentation for claims made. Only a few of the more
prominent perspectives can be conveyed in this instance. The books of
Genesis and Exodus portray God’s “childhood”; the book of Isaiah, the
middle years. The turning point is reached by Job, and the final years are
described by books such as Esther, Daniel, Ezra and Nehemiah. • The creation of humankind in the image of God is the plot that drives
the narrative. The story of God’s life is the story of his life work. The
quest for a self-image, the sole stated motivation for the creation, is
God’s indispensable tool for self-understanding. At the beginning of
the Bible, God does not know who he72 is. His only way of knowing
himself is through mankind as an image of himself (Miles 1996:187).73
The protagonist receives his life story from the antagonist. God comes
to full self-consciousness only after a tumultuous history. • The flood story reveals “the deepest of all fault lines in the divine
character” – he could be an outright destroyer (1996:42). This action
proves that the creator has the capacity to destroy and renders him
dangerously unpredictable. • Miles returns time and again to the divine ambivalence towards human
sexual fertility, and points out how the narrative – especially in the
first few books – is obsessed with reproduction as the image of divine
creativity. The covenant with Abraham and circumcision are placed in
this interpretive context and understood as the surrender of generative
autonomy (1996:90, 101). • The Exodus and the conquest of the land profoundly change God’s
identity. He is drawn into battle; war transforms him, and he becomes
a permanent divine warrior (1996:92). The Baal element, although
desexualised, dominates in God’s life at this moment (1996:189). • Deuteronomy stabilises God’s character to some extent and
synthesises the elements of the portrait up to this stage: God as
creator, destroyer, warrior, lawgiver, and the God who can enter into
a personal relationship. 73
All references in the text will be to Miles’ book (1996). Considering the doctrine of God Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God 72
Pronoun conventions by Miles are followed. 72
Pronoun conventions by Miles are followed.
73
All references in the text will be to Miles’ book (1996). 3.2.4 Narrativity and a biography of the divine
as character A radical question arises as to whether one should not investigate an
alternative to the attribute tradition for assigning descriptive qualities to
the divine, a less reductionist, essentialist, and a-historical approach. If the
God encountered in the Bible is an active Agent, a Person in a great drama,
one candidate for such an alternative approach could be characterised
as part of narrativity. In the state of scholarship, one finds intimations of
such a method. One fascinating example will be given, in this instance, by
way of an extended summary. The rationale for this inclusion is multiple:
it simultaneously showcases a narrative character approach and re-
emphasises the plurality of representation in Scripture and the often-
problematic nature of the profile(s) drawn. Jack Miles’ Pulitzer prize-winning work, God: A biography (1996)
can be predicated in divergent ways: imaginative, courageous, novel,
blasphemous. Whatever the verdict, the approach followed and the
conclusions drawn linger long after one has read the book and pose
a challenge not easily dismissed. God is studied as a character, the
protagonist of a classic of world literature. By reading the Bible as literature
and God as major character, one encounters an approach different from
the typical dogmatic attribute tradition one. The author made one crucial
decision that ultimately influenced the final portrayal: the text of the Hebrew
Bible (Tanakh) is used, thus placing the prophetic corpus before the so-
called writings, and not the Old Testament with its well-known order of
torah-writings-prophets. This changed order of the books ultimately has
far-reaching implications for the character of God. As part of his method,
Miles follows a sequential reading of the Hebrew Bible, which allows for
the detection of character development, as well as apparent conflicts and
contradictions. He is interested in the effect of the whole Tanakh on the
reader, which he unsettlingly achieves.71 To write a biography of God in 1
Groothuis’ review evidences this. He opines that a “misnomer is an understatement” for labelling 73 Acta Theologica Supplementum 34 2022 • The establishment of the monarchy causes a fateful change in God’s
self-understanding: he starts to refer to himself as Father of the king
(1996:168). This will allow him to escape from the dilemma in which
the covenant with Israel has placed him. He cannot change the
covenant, but he can change himself (1996:172). This change happens
concurrently with a concern on his part for the poor and the needy that
will become more prominent during the exile. “The new Lord God” emerges in the Book of Isaiah and it presents a
massive synthesis of divine personalities (1996:203). The exile allows
God to emerge as international arbiter, a role which he has hitherto not
assumed to this extent. Especially in Deutero-Isaiah, one encounters
a “miraculous benignity” (1996:213), an adoption of a tone of tender,
almost maternal solicitude that is without precedent (1996:220). Up
to this stage, the notion of comfort has never figured in connection
with God. Miles is emphatic that God has never loved until this point
in history (1996:237). God was previously wrathful, vengeful, and
remorseful, but the Bible, from Genesis through 2 Kings, is silent about
joy, happiness, or pleasure. A clear emotional evolution has taken
place in the life of the protagonist. In addition to these perspectives,
his uniqueness is exceptionally emphasised: he is the one and only
God. The source of this feature is not so much his power than his
mystery. God’s unknowability is the central novelty in the Book of
Isaiah (1996:226) and is a way to escape the all-too-imprisoning clarity
of Deuteronomy. Mystery opens the door to novelty. The underlying
question of the exile – Is a new start possible? – finds an answer in
the mysteriousness of God (1996:228). Incomprehensibility is a feature
introduced into the idea of God under a particular set of circumstances
(1996:235). The juxtaposition of the notions of benignity and mystery
has remained the defining incongruity at the core of the word “God” as
understood by the West, and Isaiah’s role in creating this incongruity
can scarcely be exaggerated (1996:236). • As to the question whether a Goddess figure is to be recognized among
the personalities that fuse in the character of God, Miles believes that
one should think along the lines of deliberate exclusion rather than mere
absence. The exclusion of Asherah is nothing less than a “violently
emotional revulsion” (1996:265). Venter According to Miles, this is the God, who has
remained God for Christians and Jews up to modern times, but who
was also rejected by Western atheism (1996:141). 74 Acta Theologica Supplementum 34 Venter voice in the whirlwind and the answer by Job should be understood
as irony responding to sarcasm. God subsumes justice to his power,
by refusing to address the question of justice and by only referring to
his power. Job refuses to accept mere power as the criterion of moral
integrity; “it is the simultaneity of justice and power that makes God
God” (1996:315). Job’s response is a refusal to respond and withholds
everything that can be withheld. Job has won, and the Lord has lost. After the encounter with Job, God never speaks again in the Tanakh. Job has reduced God to silence (1996:329). What does the reading of the various books amount to überhaupt about
God for Miles? The following conclusions can be gleaned from the book: • The movement in the Tanakh is from activity through speech to silence:
“The Lord God’s life in the Tanakh begins in activity and speech and
ends in passivity and silence” (1996:402). After the response of Job,
there is a long twilight of God, a long decrescendo to silence. • The development is from an image of God the creator at the beginning
to one of the Ancient of Days towards the end. One encounters God in
Genesis with no history, no genealogy, no past, and no social and private
life. The narrative is one great struggle of God with himself towards
full self-consciousness and self-knowledge (1996:21). The course of
the Lord God’s life runs from omnipotence to relative impotence, from
ignorance to relative omniscience (1996:402). But why does the story
end in silence? It seems as if the desire for a self-image carries a tragic
potential: self-knowledge resulted in a loss of self-interest (1966:404). • After the study, the central insight for Miles may be captured in the
following words: • After the study, the central insight for Miles may be captured in the
following words: God is at once unity (one character) and multiplicity (different
personalities). He is an amalgam of several personalities in one
character (1996:6). One example illustrates this crucial insight of Miles;
relatively early in the narrative, he raises the question as to what makes
God Godlike; his answer (1996:93): creator (yahweh/elohim) + cosmic
destroyer (Tiamat) + personal God (God of the fathers) + warrior (Baal)
= God. Considering the doctrine of God Considering the doctrine of God Venter Considering the doctrine of God Venter Acta Theologica Supplementum 34 The female in the divine male was
suppressed far more thoroughly than the destroyer in God (1996:405). • As to the question whether a Goddess figure is to be recognized among
the personalities that fuse in the character of God, Miles believes that
one should think along the lines of deliberate exclusion rather than mere
absence. The exclusion of Asherah is nothing less than a “violently
emotional revulsion” (1996:265). The female in the divine male was
suppressed far more thoroughly than the destroyer in God (1996:405). • The Book of Job forms the climactic moment in the biography of
God (1996:354) and his destructive and demonic side is brought
to full consciousness (1996:303). For Miles, in his provocative and
idiosyncratic interpretation, the book is about justice and power. The • The Book of Job forms the climactic moment in the biography of
God (1996:354) and his destructive and demonic side is brought
to full consciousness (1996:303). For Miles, in his provocative and
idiosyncratic interpretation, the book is about justice and power. The 75 Considering the doctrine of God Venter All of these result in an impression of God being incongruent
(1996:70), inherently unstable (1996:197), ambiguous (1996:328), and
contradictory (1996:408). The Book of Job radically underscores this: 76 Acta Theologica Supplementum 34 74
For Duquoc (1992:8), the issue is one of the “localization” of the divine and he suggests that
the outcast has become a pointer to the presence of God, who “sets up his tent where all hope
seems to have been abolished”. Recommended further reading The work recommended represents two examples from the Old Testament
and two from the New Testament, and one more fundamental reflection. The work recommended represents two examples from the Old Testament
and two from the New Testament, and one more fundamental reflection. Gericke, J.W. 2017. Metaphysical perspectives on YHWH as a fictional entity in the Hebrew
Bible. HTS Teologiese Studies/Theological Studies 73(3), 4566. https://doi. org/10.4102/hts.v73i3.4566. Lasine, S. 2016. Characterizing God in his/our own image. In: D.N. Fewell (ed.), The Oxford
handbook of biblical narrative (Oxford: Oxford University Press), pp. 465-477. https://doi.org/10.1093/oxfordhb/9780199967728.013.40 Moore, S.D. 1995. The beatific vision as a posing exhibition: Revelation’s hypermasculine
deity. Journal for the Study of the New Testament 60:27-55. https://doi. org/10.1177/0142064X9601806002 Patrick, D. 1981. The rendering of God in the Old Testament. Philadelphia, PA: Fortress. Tolmie, D.F. 1998. The characterization of God in the Fourth Gospel. Journal for the Study
of the New Testament 69:57-75. https://doi.org/10.1177/0142064X9802006903 Patrick, D. 1981. The rendering of God in the Old Testament. Philadelphia, PA: Fortress. Tolmie, D.F. 1998. The characterization of God in the Fourth Gospel. Journal for the Study
of the New Testament 69:57-75. https://doi.org/10.1177/0142064X9802006903 Venter Recommended further reading Acta Theologica Supplementum 34 2022 good and evil are to be found simultaneously in him (1996:327). The
demonic strand, though never finally dominant, cannot be excised from
his character (1996:333). good and evil are to be found simultaneously in him (1996:327). The
demonic strand, though never finally dominant, cannot be excised from
his character (1996:333). • “Nothing that once appears in God’s personality ever quite disappears”
(1996:281). God will never not be a destroyer and never not be a warrior. The overwhelming impression is one of anxiety and unpredictability. The all-defining question is: When will the Lord God act, or will he ever
act? (1996:402). God is finally friend and foe (1996:352). • Read sequentially, one cannot escape to notice the definite retreat from
demonstrated power to attributed power (1996:378). The Tanakh ends
with the mind of God objectified in law, the action of God incarnated in
leadership, and the voice of God transferred to prayer (1996:396). Miles’ book was written in the form of one massive intercalation; the
extended biography of God is bracketed by a concern about the self-
image of the West. Imitatio Dei has always been the ideal of character
building. The God of the Bible has been the mirror of the West. The divided interior life of God is the divided interior life of the West
(1996:408). The divided interior life of God is the divided interior life of the West
(1996:408). The divided interior life of God is the divided interior life of the West
(1996:408). Miles’ book is exceedingly important; one cannot question his creative
and careful reading of texts. The total non-reception of his approach and
findings by Systematic Theology tells a great deal about how the dynamics
of truth and method functions. The moment a reading strategy changes,
a new world crystallises. A conventional and non-pluralistic Systematic
Theology requires a typical atomistic proof-texting approach; this can
control and domesticate the divine. When the diversity of text types in the
Bible is recognised as literature, exciting possibilities emerge to encounter
profiles of the divine that may speak to our time with a fresh urgency and
relevance. The engagement with Miles’ work, which is over twenty-five
years old, is still a future task. 77 77 Considering the doctrine of God Venter 3.3 In his seminal article, Duquoc (1992:4) asserts that the theological question
is no longer “Who is God?” or “What is God?”. Rather, the question is
“Where is God?”. Identity and essence issues have lost their relevance;
the central issue is: Where does God act?74 Conventionally, the matter
is addressed by Systematic Theology in the doctrine of providence, and
then often under the rubric of theodicy. In this instance, it is proposed that
the doctrine of God should suggest some guidelines and direction in this
regard. divine action, in light of its sheer urgency, is a constituent element
of a theo-episteme. The background to the question about divine action is fairly simple to
profile. The occurrence of immense historic suffering and the achievements
of science and technology have assigned any claim to divine presence and
action to an embarrassingly obscure position. To make claims about divine
perseverance and governance has become increasingly problematic. The 78 Acta Theologica Supplementum 34 2022 Acta Theologica Supplementum 34 Acta Theologica Supplementum 34 anxiety about the eclipse of the divine presence in our world has become
a theological datum that warrants serious attention in any doctrine of God. It may have been a question in the life of faith all along. One encounters
prominent traces of this struggle already in the Old Testament. This was
noted in Miles’ controversial work (1996). More work points to this fact. For example, Friedman (1995) talks about the “disappearance of God in
the Bible”. Recent scholarship has started to take this as a serious field
of research.75 During the twentieth century, this quest and this growing
despondency about divine action have become acute. Only five seemingly
disparate trajectories may be mentioned: post-Holocaust thought; the
controversy on impassibility; the Divine Action Project; weak thought in
Philosophy of Religion, and Covid theology. The Jewish thinker Jonas
(1987:3, 6, 9) claims that Auschwitz has called the traditional concept of
God into question. It is no longer possible to continue with a notion of
divine omnipotence; there is only space for a suffering God. The work of
the Christian theologian Moltmann, especially his The crucified God (1974),
inaugurated a serious debate on divine suffering during World War 2. The
critical fact to be borne in mind is Moltmann’s own experience of suffering. 75
See, for example, Melton (2018:13-20) on present scholarship in this regard.
76
For an excellent overview and discussion, see Wildman (2004).
77
See the major work by Korpel and De Moor (2012) on this theme. 3.3 In a totally different context – one of multi- and interdisciplinary dialogue
between faith and science – the cardinal and problematic question of divine
action became the focus of a major research project by Vatican Observatory
and the Center for Theology and the Natural Sciences in Berkeley.76 This
Divine Action Project emphasised the importance of the issue of divine
agency and the immense complex undertaking of addressing this. In yet
another discourse – that of Continental Philosophy of Religion – one finds
a farewell to the Omni God of traditional thought and of all attempts at
theodicy; what remains are various proposals for a weak theology; at most,
one can talk, in Caputo’s words (2020:18), of the “subjunctive power” of
God. There is a similar struggle in the emerging discourse on God and the
pandemic. Oberg (2021:140) aptly refers to a “reduced idea of the divinity”,
or “enervating the divine”. In all these discourses, there is no abandonment
of God, but an intense occupation and anxiety to consider divine agency
afresh in line with historical and social experience. Classical theism, with
its uncritical and optimistic notions, sounds increasingly hollow. anxiety about the eclipse of the divine presence in our world has become
a theological datum that warrants serious attention in any doctrine of God. It may have been a question in the life of faith all along. One encounters
prominent traces of this struggle already in the Old Testament. This was
noted in Miles’ controversial work (1996). More work points to this fact. For example, Friedman (1995) talks about the “disappearance of God in
the Bible”. Recent scholarship has started to take this as a serious field
of research.75 During the twentieth century, this quest and this growing
despondency about divine action have become acute. Only five seemingly
disparate trajectories may be mentioned: post-Holocaust thought; the
controversy on impassibility; the Divine Action Project; weak thought in
Philosophy of Religion, and Covid theology. The Jewish thinker Jonas
(1987:3, 6, 9) claims that Auschwitz has called the traditional concept of
God into question. It is no longer possible to continue with a notion of
divine omnipotence; there is only space for a suffering God. The work of
the Christian theologian Moltmann, especially his The crucified God (1974),
inaugurated a serious debate on divine suffering during World War 2. The
critical fact to be borne in mind is Moltmann’s own experience of suffering. 77
See the major work by Korpel and De Moor (2012) on this theme. 76
For an excellent overview and discussion, see Wildman (2004). 75
See, for example, Melton (2018:13-20) on present scholarship in this regard. Venter with the divine; there are only persons “in search of God”.78 Human beings
have become increasingly naturalistic; engulfed by a closed universe. Thaumaturgical experiences have become obsolete; occurrences found
a direct link between cause and effect. The frontier between this material
natural work and a metaphysical one has closed. How the divine is present,
how the divine works have become immeasurably obscured. This shift can
be told in various narratives: it could be the story of the move from pre-
modernity to modernity; it could be the story of the evolving self, from fear
to optimism to cynicism. The question is: How to think about divine action in our time, against
the background indicated? In line with the intended format of the text, one
cannot venture into a detailed exposition; five signposts for theological
thinking should be identified. • The struggle to gauge God’s presence in history in the Bible deserves
greater attention. These theologies of divine presence and movement
could become a major optic for thinking about action. The reality of
these theologies and their plurality are already a significant datum.79 In
the New Testament, the Book of Revelation is an outstanding instance
of sense-making that gives a glimpse of the sophisticated interpretation
operative. Some-one sits on the throne (Rev. 4:3), and when the Lion of
Judah should open the scroll, a slain Lamb (Rev. 5:5, 6) appears. The
use of the apocalyptic form and of paradox, of the dialectic of suffering
and the triumph of justice convey a sense of nuanced thinking. • In traditional Systematic Theology, the action of God has been
scattered over several compartmentalised loci – creation, providence,
salvation, and eschatology. The question should be raised about
a more integrated dramatic approach. Obviously, a large number of
historically formed ideas acquired settled status, and aversion to others
became ossified. The renewed interest in creatio continua, the viability
of panentheism, and the placement of eschatology at the beginning
of thinking deserve careful attention. The engagement of the Ultimate
with reality cannot be domesticated in compartments of thought. • In traditional Systematic Theology, the action of God has been
scattered over several compartmentalised loci – creation, providence,
salvation, and eschatology. The question should be raised about
a more integrated dramatic approach. Obviously, a large number of
historically formed ideas acquired settled status, and aversion to others
became ossified. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 79
For the diversity of interpretations of divine intervention in history, see, for example, Schmid
(2019:327-350). One finds, for example, deuteronomistic, prophetic, and sapiential interpretations
of history. 78
In his fascinating book, Kugel (2017) explores this “great shift” already observed in the Bible: God
no longer appears to people, telling them what to do; there are only people reaching out to a
“distant God” in the later traditions. 3.3 In a totally different context – one of multi- and interdisciplinary dialogue
between faith and science – the cardinal and problematic question of divine
action became the focus of a major research project by Vatican Observatory
and the Center for Theology and the Natural Sciences in Berkeley.76 This
Divine Action Project emphasised the importance of the issue of divine
agency and the immense complex undertaking of addressing this. In yet
another discourse – that of Continental Philosophy of Religion – one finds
a farewell to the Omni God of traditional thought and of all attempts at
theodicy; what remains are various proposals for a weak theology; at most,
one can talk, in Caputo’s words (2020:18), of the “subjunctive power” of
God. There is a similar struggle in the emerging discourse on God and the
pandemic. Oberg (2021:140) aptly refers to a “reduced idea of the divinity”,
or “enervating the divine”. In all these discourses, there is no abandonment
of God, but an intense occupation and anxiety to consider divine agency
afresh in line with historical and social experience. Classical theism, with
its uncritical and optimistic notions, sounds increasingly hollow. Corresponding to an experience of a “silent God”77 is arguably a Corresponding to an experience of a “silent God”77 is, arguably, a
reconfigured self, also long in the making. There are no direct encounters 79 Considering the doctrine of God 78
In his fascinating book, Kugel (2017) explores this “great shift” already observed in the Bible: God
no longer appears to people, telling them what to do; there are only people reaching out to a
“distant God” in the later traditions.
79
For the diversity of interpretations of divine intervention in history, see, for example, Schmid
(2019:327-350). One finds, for example, deuteronomistic, prophetic, and sapiential interpretations
of history. Venter The renewed interest in creatio continua, the viability
of panentheism, and the placement of eschatology at the beginning
of thinking deserve careful attention. The engagement of the Ultimate
with reality cannot be domesticated in compartments of thought. • The imperative of adequate and appropriate categories of thinking can
hardly be stressed enough. To capture the presence and action of God, • The imperative of adequate and appropriate categories of thinking can
hardly be stressed enough. To capture the presence and action of God, 80 Acta Theologica Supplementum 34 2022 stretch human conceptualisation. Luther intuited this with his notion
of sub contrario specie; Paul with his power in weakness. To think
in terms of paradox, of irony, of fragment, of the subjunctive are all
possibilities to deconstructing the theological propensity for mastery of
the divine. The way one thinks and speaks should be commensurable
to the subject matter one approaches with hesitation. Substantially, the way forward for making sense of divine action could be
one of creative and consistent trinitarian imagining. Such an approach
has the advantage of assembling the multiple Scriptural voices, and
of establishing some form of coherence. The work of the Persons
of the Trinity is both distinguished and in unison. In the traditional
appropriation doctrine, the Father is associated with creation, the
Son with salvation, and the Spirit with perfection. These should be
interpreted in a hermeneutical account. Patrologically, one is justified
to think in terms of sheer fecundity, of life, of meaning. Christologically,
one is confronted with suffering, duration of death, and the promise of
new life – a dialectic wrought through time. Pneumatologically, one is
astounded by new and surprising possibilities of the future breaking into
this life. The promise of a trinitarian approach is the profiling of a certain
direction for thinking. There is a forward movement, an emergence
in reality; a time for explicable darkness and the dawn of justice,
and there is the possibility of a novum. A trinitarian understanding
could potentially address some of the fundamental questions of life
and engage with typical lines of thought in contemporary discourse. A trinitarian directionality does not provide simplistic answers. It
creates space for affirming meaning in life, and for the evolution of
life. It simultaneously confirms the inexplicability of suffering and of the
overweight of justice. And it celebrates the power of the future. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter TÖnsing, D.
2013. Cornel du Toit’s science and religion contribution in the lens of the Divine
Action Project: The advantage of limited aims and epistemological diversity.
Studia Historiae Ecclesiasticae 39(1):7-21. Venter • A trinitarian approach is accompanied by an explicit acknowledgement
of the intra-canonical plurality of theologies. A full theology of divine
action should attend to a host of motifs that are related to the Father,
the Son, and the Spirit. For example, the Old Testament notion of
“blessing” has disappeared from Systematic Theology. Seldom does
one come across a respectable treatment of the “will of God”, or the
continued priestly work of the risen Lord, or the disruptive work of
the Spirit. Theological sense-making is a never-ending process. Discerning,
naming the presence and the work of the triune God can never be captured
in schemata. The very process of struggling to perceive, to interpret and to
articulate is part of being human. At least, there are rudimentary maps that
enable one to navigate. 81 80
Kaufman (1993:306, 309, 315) points out that the symbol of God provides a focus for human
consciousness, devotion, and activity. His own project focuses on constructing a new conception
of God that resonates with the modern experience and understanding of the world, and that he
finds in a concept “of the humanizing and relativizing God”.
81
In this highly developed field, only one overview may be referred to, namely Pals (2021), who
discusses influential views such as those by Durkheim and Geertz.
82
Nicholls’ (1989) study on the political effects of God images is fairly well-known in this regard. Recommended further reading The work recommended comprises two major monographs on divine
providence and theodicy. The remaining three – by South Africans – focus
on the Divine Action Project and the complexity to discern God’s work
in history. Conradie, E.M. 2010. Lewend en kragtig: In gesprek oor … God se handeling in die wêreld. Wellington: Bybel-Media. Fergusson, D. 2018. The providence of God: A polyphonic approach. Cambridge: Cambridge
University Press. https://doi.org/10.1017/9781108683050 Smit, D.J. 2012. Trinity, history – and discernment? NGTT 53(3&4):337-352. https://doi. org/10.5952/53-3-4-273 Southgate, C. 2008. The groaning of creation: God, evolution and the problem of evil. Louisville,
KY: Westminster John Knox. TÖnsing, D. 2013. Cornel du Toit’s science and religion contribution in the lens of the Divine
Action Project: The advantage of limited aims and epistemological diversity. Studia Historiae Ecclesiasticae 39(1):7-21. 82 Acta Theologica Supplementum 34 2022 4. THE TRINITY AS A WAY OF LIFE 4.1
Divine performance and a trinitarian self 84
See the interesting work by Elliott (2020) on the various concepts of the self – for example
postmodern, algorithmic, and mobile selves. 83
Taylor identifies three elements: inwardness, the affirmation of ordinary life, and nature as
moral source. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 4.1
Divine performance and a trinitarian self In a theo-episteme, one should attend to the “fatal separations” of
modernity, to which Tracy (2005b:47) refers, those between theory and
practice, feeling and thought. The fragmentation of the disciplines,
especially between Systematic Theology, Ethics and Spirituality, should
converge in the doctrine of God. To recover the meaning of the Trinity for
the life of faith, Lacugna (1991a:1) justifiably refers to it as “ultimately a
practical doctrine”. That was also Rahner’s main motivation for formulating
his axiom. The work of the great scholar of Greek philosophy Hadot (see,
for example, 2002:55-233) has been most influential in contemporary
thought, especially his key insight that ancient philosophy was finally a
“way of life”. This idea should be embraced and developed in Systematic
Theology and in the treatment of a doctrine of God. One way of doing so is
to address the issue raised by Kaufman (1993:301-321) about the “function
of the symbol ‘God’”.80 The basic contention, in this instance, is that one should think in terms
of performance. When believing in God, when developing a doctrine of
God, one is doing something; but even more crucial: there is something
reciprocal. God is affecting us. Visions of the divine do function, they
perform. This can be studied in many ways. Three can be referred to and
a fourth will be explained as the preferred approach. The first approach
is that one can study the functions of religion as such. The discipline of
Religion Studies abounds with proposals as to why people are religious
and what potential effect that may have on human society.81 A second
approach is often found in psychology or political theory – a study of the
impact of God images.82 Often, these could be fairly pathological. A third
approach does come closer to the focus of this study – the Trinity itself
– and raises the question about the way it functions in discourses. The
specific function carries with it particular effects. If one emphasizes the
agency of the Trinity, one could think of grace being extended and the
consequence would be comfort. If one highlights the mimetic function
of the Trinity, the consequence could be a relational one, as has been 83 Venter argued in social trinitarianism. This third approach is important, and the
various functions should be identified. This is part of doing trinitarian
theology. In this section, a fourth possible approach will be suggested and
eventually be followed: divine performance as self-formation. A particular
representation of the divine has as a consequence that a corresponding
human self is nurtured and formed. The notion of the human self has become a fruitful category to employ
in thinking. The sociologist Joas (2013:146) credits William James for
the shift from soul to self. One cannot refer to the notion of self, without
alluding to the magisterial work by the philosopher Taylor (1989) – Sources
of the self – on the long making of modern Western identity.83 At present,
the study of the human self has become a fascinating and burgeoning field
of research.84 Inevitably, the theologian should raise the question: What
self is being formed by a trinitarian rendering of the divine? The bridge or connecting point between the triune God and the believing
community is arguably provided by the sacrament of baptism. An identity
is conferred to the child or the believer. Butin (2001:3) insightfully started
his short book on the Trinity with a discussion of baptism and considered
the triune formula as “performative words”. Baptism is a rite of identity
establishment: “The words of my baptism tell me who I am by telling
me Whose I am” (2001:3). To unpack that identity could move in various
directions; all would depend on one’s trinitarian theology. A particularly
wide spectrum of associations is evoked the moment one is set to identify
contours of a possible emerging trinitarian self. A rich dramatic narrative
comes into focus of the action of the triune God and a host of motifs
generated by biblical and patristic traditions. Only ten possible cyphers
could be mentioned that may function for delineating a baptismal identity. The trinitarian self is a/an - • Thinking self – the identity of the triune God was born in a history of
intense debate and argumentation. • Thinking self – the identity of the triune God was born in a history of
intense debate and argumentation. • Relational self – the one God is a community of three Persons. • Faithful self – the God of the Covenant was especially honoured by
Israel for faithfulness to promises. Acta Theologica Supplementum 34 2022 • Dialectical self – the cross and resurrection of Jesus Christ manifest
the most radical dialectic ever seen, that between death and life. • Dialectical self – the cross and resurrection of Jesus Christ manifest
the most radical dialectic ever seen, that between death and life. • Kenotic self – the self-giving of the triune God exemplifies this amply. • Carnal self – the incarnation, the bodily assumption of Jesus Christ,
elevated materiality against all cultural degradation. • Hopeful self – the Spirit as eschatological gift continuously opens new
horizons for agency amidst trauma and despondency. • Hopeful self – the Spirit as eschatological gift continuously opens new
horizons for agency amidst trauma and despondency. • Evolving self – the possibility of forgiveness and the promise of the
ultimate establishment of justice keep life essentially open. • Evolving self – the possibility of forgiveness and the promise of the
ultimate establishment of justice keep life essentially open. Worshipping self – the triune God is worthy of adoration. • Worshipping self – the triune God is worthy of adoration. Each one of these elements could obviously be argued and supported
by extensive source referencing. Most, if not all of these elements are
self-evident and form part of rudimentary trinitarian theology. The specific
point is that the confession of the trinitarian doctrine furnishes an immense
rich corpus of ideas that could be developed into a textured notion of the
self; a notion born from Scriptural and theological tradition. Furthermore,
in a multidisciplinary engagement with secular disciplines, a trinitarian
contribution would not embarrassingly retreat into insularity. A trinitarian
self could, by virtue of its Ultimate Source, embody or exemplify a self
deeply centred in a world of tragic disorientation. The ethical dimension inherent in this task of thinking the self before the
divine should be stressed. Constructing a doctrine of God is a thorough-
going ethical task, because of its performative function. Several studies
have been done on the rise of atheism and the contributing factors of
specific (distorted) conceptualisations of the divine. Kasper (1983:315)
concludes his great study on God with the hope that the trinitarian
confession would be the answer to modern atheism, asserting that the
triune God is the “definitive determination of the indeterminate openness of
man”. The fundamental question is: How is that triune identity interpreted? Venter • Faithful self – the God of the Covenant was especially honoured by
Israel for faithfulness to promises. • Anxious self – the counter-traditions of the Old Testament and the
hiddenness of God resist any domestication. • Anxious self – the counter-traditions of the Old Testament and the
hiddenness of God resist any domestication. 84 Acta Theologica Supplementum 34 Recommended further reading In the following list, two general studies on the notion of a “way of life” are
recommended, one investigation on the idea of the Trinity as a practical
doctrine and then, finally, two on the ethical implications of God images and
on the multiple ways in which the Trinity could be employed discursively. Neder, A. 2019. Theology as a way of life: On teaching and learning the Christian faith. Grand Rapids: Baker Academic. Neder, A. 2019. Theology as a way of life: On teaching and learning the Christian faith. Grand Rapids: Baker Academic. Sharpe, M. & Ure, M. 2021. Philosophy as a way of life: History, dimensions, directions. London:
Bloomsbury. https://doi.org/10.5040/9781350102170 Sigurdson, O. 2005. Is the Trinity a practical doctrine? In: W.G. Jeanrond & A. Lande (eds), The
concept of God in global dialogue (Maryknoll, NY: Orbis), pp. 115-125. Venter, R. 2008. God images, ethical effects, and the responsibility of Systematic Theology. Acta Theologica 28(2):146-164. https://doi.org/10.4314/actat.v28i2.48889
2010 Doing trinitarian theology: Primary references to God and imagination. In
die Skriflig 44(3&4):565-579. https://doi.org/10.4102/ids.v44i3/4.162 2010 Doing trinitarian theology: Primary references to God and imagination. In
die Skriflig 44(3&4):565-579. https://doi.org/10.4102/ids.v44i3/4.162 85
One can refer to two excellent recent volumes exploring this problematic – Banner & Torrance
(2006), and Renaud & Daniel (2018). Acta Theologica Supplementum 34 It is unlikely to continue with a claim of the centrality, for example, of the
omni-tradition, as Sonderegger (2015) does. The next two sections will
explore two of the elements – the ethical and the mystical. The choice
for this decision is mainly based on the prominence given to these in
contemporary trinitarian scholarship. 85 Considering the doctrine of God Considering the doctrine of God 4.2
The Trinity and an ethical self One of the primary reasons for the enthusiasm for the Trinity since the
1970s is the link established between a social interpretation of God and
the concrete ethical life. The notion of a theonomous ethic is very old and
many studies have investigated the relation between God and morality.85
To approach the ethical as such, explicitly from a trinitarian perspective, is
of fairly recent interest. The theologian Cunningham could be mentioned
in this regard. In his major trinitarian work (1998b:3), Cunningham is
particularly interested in trinitarian “practices”: “The doctrine of the Trinity
becomes meaningful only in the context of Christian practices.” In his
ethics (2008:167), he gives a clear space to the Trinity; of value, in this
instance, is his notion of “trinitarian virtues”; these are “dispositions that
God has by nature, and in which human beings can participate by grace”. He (2008:269) identifies three such virtues – faithfulness, peaceableness,
and courage. A trinitarian-oriented ethic, especially one informed by a
social or relational paradigm, succeeds more effectively in bridging the
divide between confession and life than a classical theistic one. Much of 86 Acta Theologica Supplementum 34 2022 Acta Theologica Supplementum 34 the enthusiastic embracing of social trinitarianism has been based on a
form of mimesis. One gets the impression that God, as example to be
imitated or “echoed”, is a powerful avenue into linking God and life. Obvious
banal and crude analogies could be claimed and should be questioned. In
the trinitarian scholarship, one also finds nuanced arguments that have
enduring value. Two proponents will be briefly described, namely Gunton
and Volf. Their visions transpose the human self from a privatized sphere
to a social public space with their specific emphases. Colin Gunton (1941-2003) had a clear understanding of the task of
theology (1997:7). It is the enterprise that seeks to express conceptually
the being of God and then the implications for human life. The Trinity
was about life (2003:11) – “life before God, with one another and in the
world”. The fragmentation of modern culture was of particular concern for
the late Gunton in his work The One, the Three and the Many (1993). This
outstanding work86 offers a theological account of the shape and failure of
modernity. The emergence of this cultural epoch has resulted in deficient
notions and practices with regard to relationality, particularity, temporality,
and truth (p. 123). 86
The rest of the references in this paragraph is to this study. Venter (p. 169). At the heart of the being of God, we find particularity by virtue
of the three persons’ relationality to one another (p. 194). The next step
in Gunton’s argument is crucial: he extrapolates from this conception
of the Trinity a specific understanding of the creation marked by its
relatedness to the creator (pp. 124, 166, 167). A specifically construed
Trinity leads to a concomitant ontology: an ontology of communion that
connects being and relation (p. 214). Three open transcendentals result
from this: perichoresis, substantiality, and relationality. The universe is
perichoretic, meaning that everything in the universe is what it is by virtue
of its relatedness to everything else (p. 172). Such an ontology honors
uniqueness, distinctiveness, and diversity within unity. Gunton’s project is
most ambitious; he intends to heal the fragmentation of the human cultural
enterprise (pp. 7, 151). The second proposal for a link between Trinity and social ethics is
that of the Croatian theologian Miroslav Volf, a teacher at the Yale Divinity
School. In a seminal essay (1998), he interacted with the notion of “the
Trinity is our social programme” expressed by the nineteenth-century
Russian theologian Fedorov. Both his affirmation of the positive relation
between the Trinity and society and his careful qualification of this
relation renders this article important.87 Volf justifies some form of analogy
between the triune God and society with the biblical warrant of the human
creation in God’s image. The following question must be answered: In
which respects and to what extent should the Trinity serve as a model for
human community? (p. 405). He explicitly identifies two basic limitations
to any form of modelling: there is an ontic difference between the divine
and humanity, and human life is marred by sin and finitude. The road from
the doctrine of the Trinity “is long, tortuous and fraught with danger” and
requires a process of complex translation (pp. 406, 414). Volf prefers to
speak about a “social vision” instead of a “social programme”, as the Trinity
represents “the contours of the ultimate normative end towards which all
social programme[s] should strive” (p. 406). It is crucial to attend carefully
to the precise point of comparison in his argument. The Trinity serves a
heuristic function to disclose “the shape of the social self” (p. 406). Central
in Volf’s thesis is the “trinitarian construction of identity” (pp. 420, n 210). Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 87
All references will be to this article, unless otherwise specifically indicated. 4.2
The Trinity and an ethical self The relentless pressure for homogeneity is the real threat
of modernity (p. 39). The rights of the many are subverted by new and
demonic versions of the one: either the many become an aggregate of one,
or the many become homogenised (p. 33). The predicament of modernity
is directly related to the displacement of the Christian God, according to
Gunton (p. 38): “My contention is that the distinctive failures of our era
derive from its failure of due relatedness to God.” Traditional theism shares
the blame for the emergence of modernity. The prime culprit is Christian
theology’s tendency towards a monolithic concept of God. Understanding
God as single, simple, and unchanging contributes to the rebellion of the
many against the repressive one. Trinity and creation become the foci
of his theological project. The idea of “open transcendental” is central
in his argument; it is a notion basic to the human thinking process that
enables the exploration of the universal marks of being (p. 142). Gunton
responds to the perceived crisis with a specific understanding of God: a
trinitarian God functions as an idea that generates transcendentals. The
underlying assumption is that all of creation reflects the being of God, the
source of meaning and truth (p. 145). The concept perichoresis emerges,
in this instance, as crucial: the one and the many in dynamic interrelations:
“God is what he is by virtue of the dynamic relatedness of Father, Son,
and Spirit” (p. 165). This notion of relatedness safeguards particularity
as persons are constituting each other, making each other what they are 86
The rest of the references in this paragraph is to this study. 87 Considering the doctrine of God Social analogies remain indispensable.88 Gunton’s treatment of the fundamental question of the one and the
many, his notion of “trinitarian transcendentals”, as well as Volf’s carefully
argued understanding of identity based on the Trinity should have enduring
significance: The way in which they configure their understanding of
God, their discernment of social exigencies, and the link to the ethical
are constructive. 88
It is interesting to note that he has become more suspicious of relations of origin (2021:413) – “we
don’t need relations of origin”. Venter A specific notion of identity is inscribed in the character and relation of the
divine persons. For example, the identity of the Son is shaped through a
twofold relationship to the Father and Spirit. To avoid the charge directed
against much of recent talk about the Trinity and society, Volf situates the
construction of identity in the “narrative of divine self-donation” (p. 412);
this is the crux of his argument. The doctrine of the Trinity is the “doctrinal 87
All references will be to this article, unless otherwise specifically indicated. 88 Acta Theologica Supplementum 34 2022 expression of the narrative of the triune God’s engagement with humanity”
(pp. 423, n 59). In this narrative of self-donation, the cross of Jesus Christ
takes central position. A social position based on the doctrine of the Trinity
should take this downward movement seriously. Divine self-donation is an
act of love that involves suffering and risk. This should be imitated (p. 413). In concrete terms, this implies an embrace of the other after truth has been
told and justice has been done. In another article, Volf (2006:7) expands
the content of the imitatio Trinitatis: to identity and donation, he adds
creativity, generosity, and reconciliation. In a recent article, Volf (2021:410,
411, 419) confirms his position despite critique by anti-social trinitarians. The Trinity remains the ultimate source of a Christianly inflected social vision with its
account of the self, social relations, and the good. the ultimate source of a Christianly inflected social vision with its
account of the self, social relations, and the good. Social analogies remain indispensable.88 4.3
The Trinity and a mystical self 4.3
The Trinity and a mystical self
To integrate reflection and life practices, and to argue for the Trinity
way of life, one should inevitably interact with a set of discourses that
become fairly fashionable in theology and even in religion studies –
of spirituality and mysticism. To place thinking about God in a Chr
sense in this orbit requires the knowledge of a number of perspec
of which four can be referred to, in this instance. The so-called “tu
spirituality” has become a widely accepted cypher for understandin
sociology of religious development of contemporary life. The aversi
institutionalised religion, but not necessarily to the embrace of a sp
journey, is a complex phenomenon and should be understood w
the context of the transformations of (post-)modern society (see K
2006). Trinitarian thinking cannot dismiss this. The pertinent que
unavoidably surfaces: What exactly is “spirituality”? Especially sinc
1960s, a significant effort was made to map the contours of spiritu
A scholar such as Schneiders played a definitive role in helping to c
this. She (1989:678) distinguishes three senses to the term: a fundam
dimension of being human, the lived experience that actualises
dimension, and an academic discipline that studies that experience
definition (1989:684), which has gained wide positive reception, is
relevant in this instance:
the experience of consciously striving to integrate one’s life in
terms of … self-transcendence towards the ultimate vale that
one perceives. The important aspects of the definition are experience, integration,
transcendence, and ultimate value. For a Christian interpretation
specific conceptualization of God as triune obviously moves into f
But that raises a further question: What exactly is the relationship bet
theology (read: Systematic Theology) and Spirituality? This has bec
an outright topic for discussion with various positions. It is no lo
tenable to perceive spirituality as deductively determined by doctrin
an overview of the problematic, Endean (2005:77) refers to the “or To integrate reflection and life practices, and to argue for the Trinity as a
way of life, one should inevitably interact with a set of discourses that have
become fairly fashionable in theology and even in religion studies – that
of spirituality and mysticism. To place thinking about God in a Christian
sense in this orbit requires the knowledge of a number of perspectives,
of which four can be referred to, in this instance. Venter Venter, R. 2012. Triniteit en etiek: Van ‘n relasionele God tot ‘n etiek van die Ander. In
die Skriflig/In Luce Verbi 46(1), Art. #52, 7 pages. http://dx.doi.org/10.4102/ids. v46i1.52 Volf, M. 2019. Exclusion and embrace: A theological exploration of identity, otherness,
and reconciliation. Revised edition. Nashville, TN: Abingdon. (see especially
the appendix). the appendix). 4.3
The Trinity and a m
To integrate reflection and life p
way of life, one should inevitably
become fairly fashionable in the
of spirituality and mysticism. To
sense in this orbit requires the
of which four can be referred to
spirituality” has become a widel
sociology of religious developm
institutionalised religion, but not
journey, is a complex phenom
the context of the transformatio
2006). Trinitarian thinking cann
unavoidably surfaces: What exa
1960s, a significant effort was m
A scholar such as Schneiders p
this. She (1989:678) distinguishe
dimension of being human, th
dimension, and an academic dis
definition (1989:684), which has
relevant in this instance:
the experience of consciou
terms of … self-transcend
one perceives. The important aspects of the de
transcendence, and ultimate v
specific conceptualization of Go
But that raises a further question
theology (read: Systematic Theo
an outright topic for discussion
tenable to perceive spirituality a
an overview of the problematic Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Recommended further reading The recommended works include two primary texts by Gunton and Volf,
two secondary sources on their oeuvre, and a general source establishing
a link between Trinity and the discourse on alterity. Gunton, C. 1990. Augustine, the Trinity and the theological crisis of the West. Scottish
Journal of Theology 43(1):33-57. https://doi.org/10.1017/S0036930600039685 Gunton, C. 1990. Augustine, the Trinity and the theological crisis of the West. Scottish
Journal of Theology 43(1):33-57. https://doi.org/10.1017/S0036930600039685 Gunton, C. 1990. Augustine, the Trinity and the theological crisis of the West. Scottish
Journal of Theology 43(1):33-57. https://doi.org/10.1017/S0036930600039685 Harvey, L. (Ed). 2010. The theology of Colin Gunton. London: T. & T. Clark. Santrac, A.S. 2016. Witness to life worth living: Reflections on Miroslav Volf’s ethics of
embrace. Eugene, OR: Wipf & Stock. Santrac, A.S. 2016. Witness to life worth living: Reflections on Miroslav Volf’s ethics of
embrace. Eugene, OR: Wipf & Stock. Santrac, A.S. 2016. Witness to life worth living: Reflections on Miroslav Volf’s ethics of
embrace. Eugene, OR: Wipf & Stock. 89 89
One can refer, in this instance, to the seven volumes published in the series The presence of God:
A history of Western Christian mysticism.
90
McGinn’s (1991:265-343) discussion of the “modern study of mysticism” is exceedingly important. 0
McGinn’s (1991:265-343) discussion of the “modern study of mysticism” is exceedingly important 4.3
The Trinity and a mystical self The so-called “turn to
spirituality” has become a widely accepted cypher for understanding the
sociology of religious development of contemporary life. The aversion to
institutionalised religion, but not necessarily to the embrace of a spiritual
journey, is a complex phenomenon and should be understood within
the context of the transformations of (post-)modern society (see Kourie
2006). Trinitarian thinking cannot dismiss this. The pertinent question
unavoidably surfaces: What exactly is “spirituality”? Especially since the
1960s, a significant effort was made to map the contours of spirituality. A scholar such as Schneiders played a definitive role in helping to clarify
this. She (1989:678) distinguishes three senses to the term: a fundamental
dimension of being human, the lived experience that actualises that
dimension, and an academic discipline that studies that experience. Her
definition (1989:684), which has gained wide positive reception, is also
relevant in this instance: the experience of consciously striving to integrate one’s life in
terms of … self-transcendence towards the ultimate vale that
one perceives. The important aspects of the definition are experience, integration, self-
transcendence, and ultimate value. For a Christian interpretation, the
specific conceptualization of God as triune obviously moves into focus. But that raises a further question: What exactly is the relationship between
theology (read: Systematic Theology) and Spirituality? This has become
an outright topic for discussion with various positions. It is no longer
tenable to perceive spirituality as deductively determined by doctrine. In
an overview of the problematic, Endean (2005:77) refers to the “organic 90 Acta Theologica Supplementum 34 2022 connection” between theology and spirituality. Nowadays, thinking moves
much more in a dialectical way – there is mutual enrichment. A final
perspective pertains to the mystical as such. Traditionally, in Protestant
circles, one comes across fairly dismissive and hostile positions. The tide
has clearly turned, and one can ascribe that again to many factors. One
major credit should go to solid scholarship on the nature of mysticism as
such. An outstanding intellectual such as McGinn has studied the history
of Western mysticism in several extensive volumes89 and his (1991:xvii)
understanding is crucial: the mystical element in Christianity is that part of its belief and
practices that concern the preparation for, the consciousness of,
and the reaction to what can be described as the immediate or
direct presence of God.90 What is at stake, in this instance, is an intensification of the experience
of God. Venter about the divine. When the grammar centres on personhood, relationality,
communion, intimacy, and ecstasis, a fairly profiled spirituality comes
into view. There is a particular rationale and a unique dynamic and
intersubjective quality. Mysticism has started to gain greater prominence in spiritual practice
and scholarship in our time. Rahner (1971:15), an outstanding theologian,
was fairly appreciative of spirituality, an exceptional statement to the effect
of the importance of mysticism: the devout Christian of the future will either be a ‘mystic’, one who
has ‘experienced’ something, or he will cease to be anything at all. One should not miss the entanglement of the yearning for greater intimacy
with the divine with the postmodern moment; it signifies the deep quest for
personal fulfilment. In his discussion of the orientation of the contemporary
mystical focus, Perrin (2005:454) points to the deconstruction of the classical
philosophical idea of God and the emphasis on the “God of extravagant
self-giving life”. This leads inevitably to the Trinity that is eternally fecund
in a threefold event of love – giving, receiving, and returning. That, in
turn, leads in the human-divine encounter to a “restructuring of the self”
(McIntosh 1998:34, 48, 225). In the giving away of the self, one finds the
constitution of the self; the critical insight is that “the trinitarian mutuality
of self-dispossession is an eternally affirming, life-constituting reality”
(McIntosh 1998:234, 235). It is crucial to note the kind of conception of the
self that transpires in this instance: identity lies in the bestowal of freedom
towards the other in embrace, in mutuality. Understandably, this movement into deeper intimacy with the divine
leads to greater silence; hence, the renewed appreciation for apophatism. The distinction between affirmation and negation in ways of speaking
about God was introduced in Christian theology in the sixth century.91 The
mysteriousness and incomprehensibility of God, who cannot be reduced
to rational categories, has returned to theology. The trinitarian discourses
have played no small role in a renewed sense of the inexhaustible depth of
the reality of God. A theologian such as Kilby (2010:65, 67), who became well
known for her resistance to the trinitarian renaissance and social versions
thereof, proposes an “apophatic trinitarianism” as a way to counter what
she labels as “trinitarian robustness”. 91
For a discussion of the history of this, see Louth (2012). Considering the doctrine of God Venter Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 4.3
The Trinity and a mystical self Thinking about the doctrine of God clearly takes place within a
horizon with many changes! What is at stake, in this instance, is an intensification of the experience
of God. Thinking about the doctrine of God clearly takes place within a
horizon with many changes! Against this background, one can move to a discussion about the
“mystical self”. The first pointer would be to draw attention to the growing
interest in a trinitarian spirituality, and the studies in this respect have
become extensive. One can refer briefly only to two good ones (LaCugna
& Downey 1993 and McIntosh 2005). Christian living is about ever-fuller
participation in communion with God and with one another. LaCugna
and Downey (1993:972-974) point out that a trinitarian approach would
understand “holiness” differently; it is much more about self-transcending,
ecstatic, and relational life. Personhood, communion, and relations become
central in this form of spirituality. Prayer is also more than conversation
and is about participation in the divine life. Union with God materializes in
communion and participation. As relational interdependence is a hallmark
of this spirituality (LaCugna & Downey 1993:980), the issue of social justice
is crucial, as spiritual life is about rightly ordered relationships. In McIntosh’s
reflection, one finds similar emphases; it is about ever-greater intimacy
with God. He (2005:179) identifies self-transcendence, a deepening love
for others, and a growing sense of freedom and agency as common marks
of this spirituality. He (2005:183) raises a critical perspective on alterity. In
light of otherness in the Trinity, there is no obliteration of human otherness
in the union with God – selfhood is constituted by that. In a trinitarian
approach to spirituality, fruitful avenues are opened by relational thinking 91 Venter 93 Considering the doctrine of God Considering the doctrine of God Venter Recommended further reading Venter, R.
2015. A trinitarian approach to spirituality: Exploring the possibilities. HTS
Teologiese Studies/Theological Studies 71(1), Art. #2952, 7 pages. http://dx.doi.
org/10.4102/hts.v71i1.2952 Venter She is obviously correct in her fear
of idolatry, projection, and overconfidence in speaking the divine, but is
questionable whether apophatism should lead to a hesitance of speaking
about the “immanent” Trinity at all or of contemplation of the divine
(2010:70-71). The relational turn in trinitarian discourse is not cancelled by 91
For a discussion of the history of this, see Louth (2012). 92 Acta Theologica Supplementum 34 2022 an increased sense of the hiddenness and incomprehensibility of the triune
God. Apophatism brings a specific quality to the relational understanding. The scholar Coakley should be recognised for his work in relating
theology and spirituality, Trinity, and mysticism. The kind of issues she
addresses are productive for the problematic in this instance. She accords
prayer an outstanding place in trinitarian reflection, referring specifically
to Romans 8 (1986:21; 1998:225). This also incorporates the notion of
experiencing the Trinity. There is a certain mutuality involved with prayer –
the Spirit praying in us, God receiving the prayer, and, in exchange, inviting
us into the divine life. Her third emphasis is on desire. Erotic language
becomes an indispensable mode of speaking of our intimacy with God. There is a mutual desire between God and us. In this trinitarian model
espoused by her, the Spirit receives a dominant role (see 2013:111). The
Spirit is the “primary instigator of transformative participation in God”. Interestingly, she continues and claims that prayer has, according to
Romans 8, social, cosmic, and political import (2013:114). In the work of Sheldrake, the renowned scholar of spirituality, one
encounters an admiring integration of spirituality, Trinity, and socio-
political sensibility. A trinitarian vision links theology and spirituality,
because at stake is about more than mere speaking about God, but a
desire for God (2010:82). Contemplation is not about self-preoccupation,
but about self-forgetfulness (2010:100). According to Sheldrake (2010:115),
the historical link between mysticism and subversion should be clearly
noted. He (2010:146) emphasises that a trinitarian anthropology “suggests
an inherently transgressive rather than bounded, individualized and
interiorized understanding of identity”. Coupling the Trinity with spirituality and specifically with the mystical,
accomplishes a remarkable feat; a rich array of motifs appears into focus. Identity, self, experience, intimacy, desire, prayer, self-giving, silence,
and society are all intertwined. The textures of a mystical self are virtually
inexhaustible, because the God being related to is the Ultimate Source
of Meaning. Recommended further reading The following are recommended: A historical study of mysticism during the
Reformation; a detailed exploration of the Trinity in the life of great mystics;
an investigation into the unique contours of Trinitarian spirituality, research
on apophatism in the theology of three woman theologians (Kilby, Coakley,
and Sonderegger), and finally a description of the mystical-prophetic in the
theology of Tracy. Hansen, L. 2010. Die mistieke en die profetiese, en die unieke van die mistiek-profetiese in
die teologie van David Tracy. Nederduitse Gereformeerde Teologiese Tydskrif
51(1&2):83-98. Hansen, L. 2010. Die mistieke en die profetiese, en die unieke van die mistiek-profetiese in
die teologie van David Tracy. Nederduitse Gereformeerde Teologiese Tydskrif
51(1&2):83-98. Hansen, L. 2010. Die mistieke en die profetiese, en die unieke van die mistiek-profetiese in
die teologie van David Tracy. Nederduitse Gereformeerde Teologiese Tydskrif
51(1&2):83-98. Hunt, A. 2010. The Trinity: Insights from thN: Liturgical Press. 5.1
A horizon with many names Thinking the divine obviously happens in a contingent horizon. Since
the mid-twentieth century, theology has become increasingly aware
of its own contextuality, and has also started to give more explicitly an
account thereof, although it had always been situated in concrete material
conditions. Nowadays, there is a heightened attentiveness, a finely tuned
sense of discernment of its own time in all its dimensions.92 Strictly, one
could or should have addressed a mapping of the landscape earlier in this
reflection. A decision was made to keep it back intentionally and relate it
to one important element of a theo-episteme, namely reconstruction. The
rationale of the project has not been to construe a creative doctrine of
God as such; such a venture would arguably assume a different shape. The task of the cartographer of our contemporary horizon is confronted
with an impossible task; the topography is un-navigable. One could at
most employ adjectives such as “plural”, “complex”, “contradictory”, and
“ambiguous”. One lives in a polyonymous time – it has many names; one
cannot capture its character in one word. Naming the present is difficult. One could at most refer to the many labels employed as currency to trade in
a bewildering market. The aim of the following is merely to convey a sense
of the direction of contemporary time. It is fragmentary, stylistically crude,
and even disjunctive. Each one of the cyphers represents a configuration
of immense dynamics, constituent dimensions, and implications. Thinking the divine obviously happens in a contingent horizon. Since
the mid-twentieth century, theology has become increasingly aware
of its own contextuality, and has also started to give more explicitly an
account thereof, although it had always been situated in concrete material
conditions. Nowadays, there is a heightened attentiveness, a finely tuned
sense of discernment of its own time in all its dimensions.92 Strictly, one
could or should have addressed a mapping of the landscape earlier in this
reflection. A decision was made to keep it back intentionally and relate it
to one important element of a theo-episteme, namely reconstruction. The
rationale of the project has not been to construe a creative doctrine of
God as such; such a venture would arguably assume a different shape. The task of the cartographer of our contemporary horizon is confronted • Globalisation:
Human
society
has
shrunk
through
modern
communication, transport, and trade, and simultaneously expanded
by a modernist disposition. ,
2010. The Trinity: Insights from thN: Liturgical Press. McGinn, B. 2015. Mysticism and the reformation: A brief survey. Acta Theologica 35(2):50‑65. https://doi.org/10.4314/actat.v35i2.4 Van Kuiken, E.J. 2017.“Ye worship ye know not what”? The apophatic turn and the Trinity. International Journal of Systematic Theology 19(4):401-420. https://doi. org/10. 1111/ijst.12227 Venter, R. 2015. A trinitarian approach to spirituality: Exploring the possibilities. HTS
Teologiese Studies/Theological Studies 71(1), Art. #2952, 7 pages. http://dx.doi. org/10.4102/hts.v71i1.2952 94 94 2022 Acta Theologica Supplementum 34 92
In a fine article, McGrath (2012:108, 113) argues for the cultivation of a “habit of theological
attentiveness”, referring to the church as a “community of discernment”. It is important to note
that he then develops the notion of a “theology of place” (pp. 116-123). 5.1
A horizon with many names The consequences are immense: much
greater networking and interdependence, but also disturbing growth
in inequality. • Anthropocene: The dawn of this new geological era highlights the
relation between the human being and nature and the alarming
destructive impact of societies’ consumer ethos. • Anthropocene: The dawn of this new geological era highlights the
relation between the human being and nature and the alarming
destructive impact of societies’ consumer ethos. • Identity: A human rights culture has moved issues of gender, sexual
orientation, race, dis/ability, and culture to centre stage. Identity
politics are advocating a re-centring of society and the full humanity of
all the subaltern persons. 95 Acta Theologica Supplementum 34 2022 This truncated account is admittedly subjective, one-sided, and
incomplete. Basically, every bullet can be substantiated with extensive
literature references. The purpose of this was to convey the impression of
a society in transition, a society confronted by overwhelming challenges. Often, the neologism “glocal” is used to convey the interplay between local
conditions and the global world. In every context, the configurations of the
cyphers mentioned should be worked out. The South African horizon will
be different from the one in Singapore, for example. And the doctrines of
God would differ. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter • Postcolony: The intricate interwovenness of colonisation, modernism,
and the West is being resisted in new political alignments, new
epistemic regimes, and new social configurations. In this instance, the
key is the deconstruction of an entire new metaphysic with a plethora
of new philosophies. • Capital: The tyranny and failure of economic systems have become
too obvious in the astounding and unsettling inequality among
societies and human beings. Economic injustice pervasively scars
human societies. • Poly-centres: International geo-politics are marked by nationalist
ambitions, oppositional alignments, and escalating conflict, with a
multitude of instances of concentrated violence and global impact. • Fourth Industrial Revolution: Science and technology have made strides
with fantastic achievements. The dawn of a digital society, for one
example, has drastically changed human life. It remains uncertain what
the accomplishments may ultimately entail for the entire spectrum of
human society, for example the dawn of sentient artificial intelligence. • Postsecularity: This rubric represents a bewildering scope of
contradictory and ambiguous religious trends: from secularisation
to fundamentalism. A sociology of religion points to the diminishing
of institutionalised religion, a renewed interest in spirituality, a return
of religion as public force, a shift to the South in Christianity, a
retrieval of traditional religions, and the proliferation of esoteric new
religious movements. • Posttruth: The correlation between interpretation and factual empirical
reality has collapsed. The sheer fabrication of distortions for political
power and reactionary aims is increasing and fuelled by the support of
social media. • Violence: The many faces of Ares is an outstanding feature of the
contemporary world: military, ethnic, gender, racial, criminal, epistemic,
and so on. The deep disrespect for the Other and for life is devastating. The inflation of the use of “trauma” as indicator has become striking. Naming our time as “traumatic” is not without strong warrant. • Melancholia: “Mood” has become a central rubric to gauge the character
of the present horizon. One cannot miss the frequent occurrence of
adjectives such as “insecure”, “uncertain”, “vulnerable”, and “tragic”
in literature. One realises that one’s time is an age of “anger”, “doom”,
and “disaster” – an age of sheer cynicism. At present, human life has a
certain darkness to it. 96 Venter In light of what was discussed in the previous sections, and of the
focus on the Trinity, one obvious and fundamental question transpires,
especially in light of the brief account of the horizon mapped. Does
trinitarian theology have the resources and the potential to address the
multiple challenges heuristically? This is clearly a far-reaching question; it
intersects with the question of the vitality of the Christian vision as such to
make a meaningful contribution to human sense-making at this stage of
civilisation. If the God symbol and specifically the Trinity symbol is central
to the identity of the Christian faith, this question is understandably of
cardinal significance. What is at stake in this section – the issue of correlation between
horizon and construction – is amply demonstrated in a fascinating book,
published recently with the intriguing title: God after the church lost
control (2022). The two Scandinavian authors – theologian Henriksen
and sociologist Repstad – address the problem of God and change,
considering the contemporary landscape in northern Europe. Traditional
theology is clearly in a crisis when it comes to speaking about God and its
relevance. By exploring issues such as the “morally intolerable God”, the
“politically dangerous God”, God as a “she”, and the religious plurality,
they probe the possibility of thinking God as “vulnerable love” (2022:140). They focus on the interplay between changing society, crisis of religion,
problematic traditional ways of understanding God, and the imperative of
a fresh naming. A reading of the history of theology, say from the 1960s, reveals a
commitment and enthusiasm to engage with the myriad social dilemmas
and exigencies, especially from the perspective of the confession of
God as triune. It is important to acknowledge and point out significant
work done in this regard. The key occupation was to apply the social
implications of the relational turn in trinitarian doctrine. A vast number
of publications were published in this respect. The proposals by Gunton
and Volf were described in earlier sections of this work. To a great extent,
one finds a similar enthusiasm for spirituality; this was also discussed. In
addition to this, a large scope of questions and problems were treated from
a trinitarian perspective. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God 93
For the sake of brevity, only outstanding publications will be referred to. Recommended further reading The Oxford series of “Very Short Introductions” of over 600 little books
written by experts is an outstanding resource for orienting one of the
vast fields of knowledge of our time. Many of these volumes have direct
relevance for mapping the landscape of our time. The following five are
examples of what is available. Copson, A. 2019. Secularism: A very short introduction. Oxford: Oxford University Press. https://doi.org/10.1093/actrade/9780198747222.001.0001 Copson, A. 2019. Secularism: A very short introduction. Oxford: Oxford University Press. https://doi.org/10.1093/actrade/9780198747222.001.0001 Coulmas, F. 2019. Identity: A very short introduction. Oxford: Oxford University Press. https://doi.org/10.1093/actrade/9780198828549.001.0001 Coulmas, F. 2019. Identity: A very short introduction. Oxford: Oxford University Press. https://doi.org/10.1093/actrade/9780198828549.001.0001 Ellis, E.C. 2018. Anthropocene: A very short introduction. Oxford: Oxford University Press. https://doi.org/10.1093/actrade/9780198792987.001.0001 Steger, M.B. 2020. Globalization: A very short introduction. 5th edition. Oxford: Oxford
University Press. https://doi.org/10.1093/actrade/9780198849452.001.0001 Young, R.J.C. 2020. Postcolonialism: A very short introduction. 2nd edition. Oxford: Oxford
University Press. https://doi.org/10.1093/actrade/9780198856832.001.0001 5.2
Responsibility of trinitarian adventures
A reminder about the exact rationale or intention of this work may be
appropriate. It is a reflection on the construction of a doctrine of God. It
is not another proposal for a new construction itself. The very notion of
interplay between time and doctrine is at play and should be examined. Writing doctrines of God is a hermeneutical and constructive practice, and
that activity must be examined. 97 97 Considering the doctrine of God Acta Theologica Supplementum 34 2022 and queering (Quero & Goh 2018). These represent only a fraction of the
scholarship available, but amply prove the body of matters addressed. There is evidently a pervasive conviction that the Trinity matters to
contemporary life. Inevitably, one moves to a point where one should account for the
phenomenon of theo-constructions. In the long history of theological
reflection and development of the doctrine of God, what has been
happening in theology since the 1960s is a fairly novel trend and requires
some form of perspective. Five comments will suffice: • Reconstruction hardly needs a warrant. The immense intra-canonical
plurality of naming and of traditions evidence a certain theological
datum: believers think from their contingent conditions. The God of 1&2
Samuel is profiled differently than the one in the Book of Revelation. This plurality is a given in Scripture. One should arguably put this
perspective more strongly: reconstructions are not only warranted;
they are required. One should speak the reality and the experience of
the divine afresh. It is part of the intersubjective encounter. A living
relationship issues an imperative of probing the inexhaustible riches
of the triune God continually. New ways of speaking accompany the
wonder of the experience. • Reconstruction hardly needs a warrant. The immense intra-canonical
plurality of naming and of traditions evidence a certain theological
datum: believers think from their contingent conditions. The God of 1&2
Samuel is profiled differently than the one in the Book of Revelation. This plurality is a given in Scripture. One should arguably put this
perspective more strongly: reconstructions are not only warranted;
they are required. One should speak the reality and the experience of
the divine afresh. It is part of the intersubjective encounter. A living
relationship issues an imperative of probing the inexhaustible riches
of the triune God continually. New ways of speaking accompany the
wonder of the experience. • The very nature of the practice is inherently hermeneutical and
rhetorical. It is about interpretation within a large conversation with
the Bible, the tradition, the context, and the person of the theologian
as participants. It is listening to some voices more intently. But it is
also about speaking, about construing an argument, with performative
intent – to do something. • The fundamental issue is obviously about the relation between
revelation and construction. The fairly long discussion on a genealogy
also moves into focus, in this instance. Venter One can mention some truly significant work93
on gender (Johnson 1992), sexual difference (Tonstad (2016), economy
(Meeks 1989), science (Polkinghorne 2004), evolution (Edwards 1999),
world religions (Heim 2001), culture and Africa (Kombo 2007), as well as
trauma and horror (Harrower 2019). A wide range of concerns transpires
in these proposals. Work not of the same scope, but also important, can
be referred to on ecology (Gorringe 2014), violence (Papanikolaou 2018), 93
For the sake of brevity, only outstanding publications will be referred to. 98 Acta Theologica Supplementum 34 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter imagination tries to translate the divine in categories that maintain a
sense of identifiability, recognisability, enabling believers to worship
the same God – and that is utterly crucial. • Reconstructions have as primary intent performance in mind: to enable
people to make sense. Conventionally, scholars intend to establish
intelligibility in dialogue with secular disciplines or non-Western
cultures, to advocate liberation and agency among the marginalized,
to further comfort in conditions of trauma, or to contribute to different
behaviour patterns. The catalogue of motivations is basically
limitless. The pertinent question is whether these performative effects
correspond with the corpus of behaviour patterns relative to the matrix
of the Christian faith. Does it encourage greater commitment? Does it
lead to more obedient lives? Does it liberate people? In short, do these
constructions cultivate patterns of faith, hope, and love? • Reconstructions have as primary intent performance in mind: to enable
people to make sense. Conventionally, scholars intend to establish
intelligibility in dialogue with secular disciplines or non-Western
cultures, to advocate liberation and agency among the marginalized,
to further comfort in conditions of trauma, or to contribute to different
behaviour patterns. The catalogue of motivations is basically
limitless. The pertinent question is whether these performative effects
correspond with the corpus of behaviour patterns relative to the matrix
of the Christian faith. Does it encourage greater commitment? Does it
lead to more obedient lives? Does it liberate people? In short, do these
constructions cultivate patterns of faith, hope, and love? A typical question that may surface is the one about the how, and
this cannot be casuistically answered. There is no recipe, no template to
be followed with faithful imagination; it is free, creative, captivating, and
subversive. What one finds in contemporary trinitarian ones are most
often an excavation of the possibilities of the relational paradigm, some
form of analogical thinking, and an exploration of typical and prominent
biblical motifs. The vitality of the biblical vision cannot be missed; it
possesses an immense surplus of meaning. The plurality of text types –
narrative, prophetic sapiential, apocalyptic literature, the movement from
creation to salvation to perfection, the emphases on bodiliness, struggle,
vulnerability, hospitality – all establish a pool of rhetorical resources that
can be consulted for great imaginative construals. Acta Theologica Supplementum 34 Revelation is not mechanical,
and construction is not projection. The crystallisation of Yahwism
through experience, the encounter with Jesus of Nazareth and his
resurrection, the tortuous route of the formulation cannot be thought
without the category of revelation. But, at the same time, the active
role of reflective intellectual work cannot be dismissed. Images of God
have been born in this complex organic entanglement of revelation
and thinking. • Reconstruction is fundamentally faithful imagining. Speaking about the
divine is carried by extraordinary feats of human creative imagination. But it is also faithful – faithful to the wisdom of very long traditions in
the Bible and in history, and to the possibility of doxology. The faithful • Reconstruction is fundamentally faithful imagining. Speaking about the
divine is carried by extraordinary feats of human creative imagination. But it is also faithful – faithful to the wisdom of very long traditions in
the Bible and in history, and to the possibility of doxology. The faithful 99 Considering the doctrine of God To the initial fundamental question about the ultimate potential of the
triune symbol to interact with the landscape constructively, one should
give a nuanced answer. There are indications of significant work already
done, but it is also evident that much work lies ahead. Whether it is a
question about the vitality of the symbol or rather one of the capacity of
theologians to think imaginatively enough should be considered. A host of
questions and challenges remain insufficiently addressed. One may point
to the imperative of decolonisation; the expansion of technology and the
reality of artificial intelligence; the suffocating growth of fundamentalism
and the erosion of truth; the depressing collective mood of cynicism; the
inattention of theology to the affective turn; agency of women and gender
violence, and the increased valuation of everyday life – all of these are in
dire need of trinitarian engagement. 100 Acta Theologica Supplementum 34 2022 Johnson, E.A.
2007. Quest for the living God: Mapping frontiers in the theology of God. London:
Continuum. Johnson, E.A. 2007. Quest for the living God: Mapping frontiers in the theology of God. London:
Continuum. Johnson, E.A. 2007. Quest for the living God: Mapping frontiers in the theology of God. London:
Continuum. Jones, S. & Lakeland, P. (Eds)
2005. Constructive theology: A contemporary approach to classical themes. Minneapolis, MN: Fortress Press (specifically Chapter 1). https://doi.org/10. 2307/j.ctv1hqdhmw Sakupapa, T.C. 2019. The Trinity in African Christian theology: An overview of contemporary
approaches. HTS Teologiese Studies/Theological Studies 75(1), a5460. https://
doi.org/10.4102/hts.v75i1.5460 Venter, R. 2018. God in Systematic Theology after Barth: Trends and perspectives. Stellenbosch Theological Journal 4(2):303-333. DOI: http://dx.doi.org/10.17570/
stj.2018.v4n2.a15 Verhoef, A.H. 2021. Decolonising the concept of the Trinity to decolonise the religious
education curriculum. HTS Teologiese Studies/Theological Studies 77(4), a6313. https://doi.org/10.4102/hts.v77i4.6313 Recommended further reading The recommendations cover three descriptions of developments in the
doctrine of God, and the remaining two address prominent questions on
African culture and the imperative of decolonisation. Venter Reformed theology and a sensitivity to be confessionally sound were
characteristic of this era. Reformed theology and a sensitivity to be confessionally sound were
characteristic of this era. • The Roman Catholic theologian Gaybba (1994; 2004) should be credited
for innovation and for incorporating tenets of social trinitarianism in his
doctrinal presentations. • The Roman Catholic theologian Gaybba (1994; 2004) should be credited
for innovation and for incorporating tenets of social trinitarianism in his
doctrinal presentations. • How the doctrine functioned as resource in the fight against apartheid
warrants a study of its own. The impression is, and this could be
rebutted, that the full critical resources of the doctrine of the Trinity
were not employed. There were attempts to envision God in terms of
Black Theology (see Maimela 1993) and of liberation in general (see
Nolan 1988), but the full political potential of the confession was
not excavated. • The period after the dawn of democracy in 1994 inaugurated much
greater trinitarian awareness and more encouragement to do creative
constructive work. Apart from Nürnberger (2016) in his ambitious two-
volume Systematic Theology, most of the reflections have taken the
form of doctoral dissertations or journal articles. Nürnberger (2016:407)
truncated the significance to a theodicy application. Theologians
such as Vosloo (2004) explored the Trinity for an ethic of hospitality;
Smit (2009) was interested in Reformed approaches, and Conradie
(2013), no enthusiast of social trinitarianism, proposed ways to do
trinitarian theology. A theologian/philosopher such as Verhoef (2019),
who completed a doctorate on Jenson in several articles, explored
innovative avenues for interpreting the Trinity, for example in dialogue
with poststructuralism. A number of PhDs focused on the Trinity with
multiple interests; the ones by Van Wyk (2013) on ecclesiology and by
Deetlefs (2018) on politics may be mentioned. There has clearly been
an interest to “catch up” with wider, international discourses. • The period after the dawn of democracy in 1994 inaugurated much
greater trinitarian awareness and more encouragement to do creative
constructive work. Apart from Nürnberger (2016) in his ambitious two-
volume Systematic Theology, most of the reflections have taken the
form of doctoral dissertations or journal articles. Nürnberger (2016:407)
truncated the significance to a theodicy application. Theologians
such as Vosloo (2004) explored the Trinity for an ethic of hospitality;
Smit (2009) was interested in Reformed approaches, and Conradie
(2013), no enthusiast of social trinitarianism, proposed ways to do
trinitarian theology. Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter 5.3.
A God for South Africa? It is a valid expectation to enquire about a doctrine of God for South Africa. How would one approach an intentionally constructive view of God for
this context? Four perspectives will be raised in this regard: the history of
doctrinal approaches; the current landscape; a suggestion about the way
ahead, and a note about a possible shape. One cannot really think about constructing a creative proposal without
a sense of what has happened in Systematic Theology. Unfortunately,
no history of this discipline is available in South Africa; one can at most
mention some impressions: • The textbook era of the mid-1970s would enable one some form of
sure footing. The trailblazing work done by theologians such as König
(1975), Durand (1976), and Heyns (1978) was important in the overall
development of the discipline of Systematic Theology. These theologies
were Euro-centric and the Trinity, although expressly recognised as
important, was not employed in a creative sense. The dominant role of 101 Acta Theologica Supplementum 34 2022 No reconstructive is possible without some attempt at describing the
character of the South African reality. It would be a banal cliché to label
this context as “complex”, but in all fairness, the country has contracted
most of the challenges available at present. The horizon mapped in 5.1 fits
this country’s landscape, only a dense specificity distinguishes the South
African one. A few of the features may be mentioned: • South Africa cannot escape the global ecological crisis. It is part of this
planetary exigency. • South Africa cannot escape the global ecological crisis. It is part of this
planetary exigency. • The racial legacy of apartheid is not something of the past, despite
constitutional change. Relational alienation, despite some strides
made, continues. The discourse on reconciliation has sadly been
eclipsed by radicalised ones. • The Hydra face of violence has become one of the outstanding features
of the country and it appears as if the faces, whether gender, farmer, or
sheer criminality, keep on proliferating. • The excessive economic disparity between rich and poor continues
to scar the face of this country. The ugly face of poverty in all its
manifestations does not diminish. • Increasingly, despite efforts to counter the so-called “state-capture”,
corruption on various levels of government continues to be the mode
of operation. • Increasingly, despite efforts to counter the so-called “state-capture”,
corruption on various levels of government continues to be the mode
of operation. • From a religious perspective, South African society is a smorgasbord
of what seems available and possible. One finds instances of increased
secularisation, fundamentalism, retrieval of traditional religions, African
Pentecostalism, and African initiated religions. • The collective mood is arguably one of depression and one of cynicism. • The collective mood is arguably one of depression and one of cynicism. The South African horizon is an epitome of plurality, contradictions,
and ambiguity. Theologians should navigate a course within these waters. Despite the bleak picture sketched, it remains an immensely fertile space
for doing intellectual work, for thinking the divine. The way ahead might take surprising directions. The proposal of this
limited study is the following: • The South African community of systematic theologians is a relatively
small network of thinkers and theologians; all have some form
of interest. Venter A theologian/philosopher such as Verhoef (2019),
who completed a doctorate on Jenson in several articles, explored
innovative avenues for interpreting the Trinity, for example in dialogue
with poststructuralism. A number of PhDs focused on the Trinity with
multiple interests; the ones by Van Wyk (2013) on ecclesiology and by
Deetlefs (2018) on politics may be mentioned. There has clearly been
an interest to “catch up” with wider, international discourses. • One specific field of research should be highlighted: the connection
of the Trinity to Africa. This is a significant development. These often
took the form of journal articles and PhD dissertations. This turn to the
cultural reality of the continent is a significant shift in South African
theology. One may refer, in this instance, to joint projects by Manganyi
and Buitendag (2013; 2017). • One’s impressions are somewhat ambiguous when taking stock of
trinitarian thinking in South Africa. It has always been realised that the
Trinity is somewhat important, and there are clearly evidences of more
creative engagements and reconstructive. But the outputs remain
limited, sporadic, and even incidental. • One’s impressions are somewhat ambiguous when taking stock of
trinitarian thinking in South Africa. It has always been realised that the
Trinity is somewhat important, and there are clearly evidences of more
creative engagements and reconstructive. But the outputs remain
limited, sporadic, and even incidental. 102 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Venter Venter Furthermore, the symbol of the Trinity is the ultimate resource for
sense-making. This requires more attention in the myriad attempts at
doing contextual theology. • Doctrines of God should be approached intentionally in a
multidisciplinary manner. To refer to the Bible does not imply that the
theologian has engaged with the state of scholarship of the Old and
the New Testament. The substantial work done by biblical scholars
should be integrated by Systematic Theology; one may merely refer, in
this instance, to Old Testament scholars such as J. Claassens and J. Gericke, who published extensively on God. South African theologians
have never been great patristic scholars. Greater attention to the study
of Early Christianity remains a future task. Some interaction has taken
place with Continental Philosophy of Religion, but much work remains
to be done, especially on phenomenology. • The neglect of the relational paradigm should be redressed. Within
a South African context, this could be employment with much fruit. Reformed scholars’ intuition to restrict the functioning of the Trinity to
action in the economia, the anxiety about speculation, and the neglect of
mimetic thinking should be questioned. The confession of a triune God
can function discursively in a variety of ways. This acknowledgement is
sine qua non for a vibrant trinitarian theology. • All violence, poverty, homophobia, and corruption render the doctrine
of the Trinity a public doctrine. The confession of God as triune could
contribute to a public discourse. Sporadic work by Koopman (for
example, 2007) on Trinity and public theology is important and should
be developed further. The connection between God and morality
should be argued more strongly and more publicly. • More recently, the insistence on doing de-colonising theology has
gained much momentum. Limited work has been completed so far
on the doctrine of God and specifically the Trinity. It is not yet clear
what a postcolonial God may resemble. Would a radicalised version
entail a dismissal of the entire genealogical trajectories as described
in chapter 2? Would the trinitarian canon of the fourth century be set
aside? If decolonisation – at minimum – refers to pluriversality (Mbembe
2016:37), it may entail an incorporation of marginalised voices – persons,
cultures, and religious traditions. This would be possible. Some work
has been done, but much work would lie ahead. Acta Theologica Supplementum 34 A plea may be in place: the reality of the divine is the
distinctive reality in theology; theological work should take this more
seriously and relate this more explicitly to the multiple social questions. 103 Venter Hardly anything, if
anything at all, has been done on African women and the Trinity, and
the Trinity in African Pentecostalism and African Initiated Churches. The inter-religious dialogue would require much more attention and • More recently, the insistence on doing de-colonising theology has
gained much momentum. Limited work has been completed so far
on the doctrine of God and specifically the Trinity. It is not yet clear
what a postcolonial God may resemble. Would a radicalised version
entail a dismissal of the entire genealogical trajectories as described
in chapter 2? Would the trinitarian canon of the fourth century be set
aside? If decolonisation – at minimum – refers to pluriversality (Mbembe
2016:37), it may entail an incorporation of marginalised voices – persons,
cultures, and religious traditions. This would be possible. Some work
has been done, but much work would lie ahead. Hardly anything, if
anything at all, has been done on African women and the Trinity, and
the Trinity in African Pentecostalism and African Initiated Churches. The inter-religious dialogue would require much more attention and 104 Acta Theologica Supplementum 34 2022 describe what truly happened in the missionary encounter between
the trinitarian vision and traditional religions. In this de-colonising
reconstruction, the performative function of the Trinity would require
pronounced attention; for one, the agency of the marginalised would
have to receive definitive prominence. The task ahead for construing a contextual doctrine of God seems
virtually an impossible task. There are simply too many considerations to
integrate. Maybe the notion of an encompassing constructive doctrine of
God is not viable. There are DNA elements that cannot be abandoned,
such as the ones mentioned in the first three proposals of this work. Reconstructions would be perspectival. However, a trinitarian habitus,
an entire way of thinking, is required in theology. When doing theology, a
trinitarian optic should be present. But a doctrine of God, as reconstruction,
would arguably remain, to refer to the title of Pohier’s touching theological
autobiography of God-in Fragments (1986). The South African God would
arguably be a kind of God-in-fragments. This is why it requires a network
of thinkers to engage in trinitarian imagining. 6. CONCLUSION A doctrine of God is important in any account of the Christian faith; it should
receive adequate and thorough attention. It is not merely yet another
element. God is the central symbol. This exposition should be consistently
trinitarian. This is how the divine is identified in this religion and this is
the one stable element in a constellation of cognitive dimensions; the
remainder – creation, salvation, hope – are inferences from this one datum. The Trinity is the point of departure – God is Father, Son, and Spirit. How
that confession is interpreted and represented can take many shapes
and forms. This short study was interested in the contemporary doctrine
of God. One of the basic proposals was that greater attention should be paid to
a genealogical description, and various trajectories were identified. These
trajectories signal radical shifts that took place and these changes should
be expressly mapped. Such a move expresses the historical nature of the
religions and carries a great deal of heuristic potential. The trajectories
discussed all manifest something revolutionary, namely the emergence of
exclusive monotheism, metaphysical trinitarianism, social trinitarianism,
and anatheism. The treatment took the form of vignettes, and more
detailed exploration should obviously happen in future. What crystallised is
an intricate interaction among social conditions, philosophical categories,
and profiles of God. Many questions can obviously also be raised. Some basic insights, however, transpired from this approach. The
understanding and articulation of the divine are undergoing mutations, but
always within the context of vehement contestation. Shifts do not take
place smoothly and acceptance is never unanimous; the old proceeds with
a resilient tenacity. Resistances to change in the Christian faith can take
several positions – a preference for a one-ness theology in monotheistic
theistic form, or for substance metaphysical trinitarianism with rejection
of social models of thinking. Within each trajectory, for example within the
Old Testament, whole. This internal plurality should be clearly observed
and hermeneutically navigated. A genealogical approach to a doctrine
of God is unavoidably a multidisciplinary undertaking: a conversation
between Old Testament, New Testament, Patristics, Philosophy, and
Systematic Theology. It is not a case of merely referring to a few sources
in those fields, but rather a proper engagement with the various states of
scholarship in these disciplines. The current profile is fairly blurred. Recommended further reading The recommendations include two attempts at mapping the features of
trinitarian discourse in South Africa, one on the decolonisation of the
Trinity, one on the global inter-religious dialogue and a final one on the
challenges of speaking God at a public university. Conradie, E.M. 2019. South African discourse on the triune God: Some reflections. HTS
Teologiese Studies/Theological Studies 75(1), a5483. https://doi.org/10.4102/
hts.v75i1.5483. Conradie, E.M. 2019. South African discourse on the triune God: Some reflections. HTS
Teologiese Studies/Theological Studies 75(1), a5483. https://doi.org/10.4102/
hts.v75i1.5483. Conradie, E.M. & Sakupapa, T.C. 2018 “Decolonizing the doctrine of the Trinity” or “The decolonizing doctrine of
the Trinity”? Journal of Theology for Southern Africa 161:37-53. Conradie, E.M. & Sakupapa, T.C. 2018 “Decolonizing the doctrine of the Trinity” or “The decolonizing doctrine of
the Trinity”? Journal of Theology for Southern Africa 161:37-53. Venter, R. 2013. Speaking God at a public university in South Africa: The challenge
of
epistemological
transformation. Studia
Historiae
Ecclesiasticae
39
Supplement:231-245. 2016. The Triune God in South African Systematic Theology since 1976. In:
D.F. Tolmie and R. Venter (eds), Theology and the post(apartheid) condition:
Genealogies and future directions (Bloemfontein: SUN MeDIA), pp. 156-168. https://doi.org/10.18820/9781920382919 2018. Thinking God in a global multi-religious context: Trends, challenges, and
possibilities. Journal for the Study of Religion 31(1) (2018) 70-85. DOI: http://
dx.doi.org/10.17159/2413-3027/2018/v31n1a4 105 Considering the doctrine of God Considering the doctrine of God Considering the doctrine of God Acta Theologica Supplementum 34 2022 Whatever the critique about the relational paradigm, the heuristic
potential can be rebutted only with difficulty. There is a long tradition of
thinking the Trinity, inevitably in terms of relationality, and modern thought
forms have radicalised that. The relational mode of conceptualisation
opens fruitful avenues for thinking the nature of being, of the human,
and of socio-ethics. The key, however, would be how to interpret the
relational identity of God. One productive suggestion could be to attend
more to motifs such as hiddenness and pursue an apophatic, after much
kataphasis has taken place. The treatment of a possible genealogy in this study is incomplete. The
first two trajectories – the birth gods and the rise of Yahwism – should
also receive attention. This would give a doctrine of God a much greater
intellectual respectability and generate possibilities of dialogue with more
disciplines in the humanities. One central question should be addressed in
a more detailed treatment: the precise role of revelation. How should one
understand the traditional emphasis on revelation amidst a range of social
and intellectual interactions? In the second proposal set – that of a grammar for speaking – a number
of insights have been produced. Concerning the who question, the question
of divine identity, one could be impressed by the vibrant scholarship of
theologians such as Barth, Jenson, and LaCugna. There is a real danger
that a younger generation of theologians could succumb to the danger
of forgetting. It would be sad if the work of a person such as LaCugna
be relegated to the margins of memory. The twentieth century, especially
since the 1960s, experienced a dense period of theo-reflection. Interests
move on and changes have taken place, but the work of theologians
should be intentionally remembered. Three major insights surfaced in the discussion of divine identity. The
Rahner Rule remains utterly crucial. Although there is clearly no uniform
interpretation of this axiom, the trend to focus on the economic activity
and shrug the shoulders about the alleged mystery of the immanent life,
will not do. LaCugna’s position is problematic, but she correctly points
to the mystery already present in the oikonomia. It is logically inevitable
to think “back” from the economic Trinity to the identity of the divine life. Naming in this regard is obviously always analogical and metaphoric. The category of the event prioritised by Barth is another major
perspective. 6. CONCLUSION There
is an uneasiness with the relational paradigm; some regard it as a deviation
from the typical classical Augustinian one; others deem it too adventurous. Some thinkers want to “retrieve” a classical model, others embrace a more
apophatic and postmodern approach. 106 Venter Considering the doctrine of God Finally, Jenson’s courageous embrace of temporality in his trinitarian
theology is another critical avenue that cannot be ignored in future thinking
on God. Attention to the what question, the question traditionally about
attributes, may capture one of the fundamental issues in a doctrine. What
does one refer to when one refers to the divine? What is “god”? The critique
of onto-theology has immensely complicated that. If God is not the highest
form of being, what exactly is “goodness”? The one critical achievement of
the trinitarian turn and the work of Barth is the realisation that all attempts
at attributing descriptors to God should be determined in a trinitarian
manner. It is surprising that scholars could even ponder this without the
filter of the triune reality. In the proposal, the notions of “hiddenness” and
“justice” have been attended to as prime associations for speaking God
in our time. It expresses not only dominant biblical traditions, but also
something of the yearnings of our time. One should consider the suggestion
that the attribute approach could be complemented by an approach from
narratology. Viewing God as character in a drama may yield perspectives
more commensurable with the dynamic of intersubjective relations than a
static metaphysical approach. This insight may be one of the contributions
of this study. Divine action has arguably become the central dilemma in the
human quest for God: where is God? The contemporary horizon, with its
excessive traumatic experiences, has intensified this intellectual struggle. This research proposes that it should be addressed in the doctrine of
God itself. If a theologian may find a rationale for a separate doctrine
of providence, it could he explored there in more detail, but structural
treatment should be tied as closely as possible to the identity question in
the doctrine of God. This study emphasised one basic direction – divine
action should be interpreted in a consistent trinitarian manner. This would
generate a textured view on God’s presence and action in the world. Each
one of the divine Persons is associated with a unique constellation of
motifs: for example, faithfulness, vulnerability, but also sheer occurrences
of historical novelty. On a trinitarian basis, it would be possible to speak
about God’s action in a manner defeating cynicism, extending comfort,
and being credible amidst suffering. Acta Theologica Supplementum 34 This underlines the movement of categories from the
anthropomorphic to being to relationship. What this implies has not yet
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Оригинальная статья І Original article Оригинальная статья І Original article Оригинальная статья І Original article DOI: 10.33925/1683-3031-2023-607 АННОТАЦИЯ Актуальность. Стоматологическая медицинская помощь является одним из наиболее массовых и социаль-
но значимых видов медицинской помощи населению РФ. Различают следующие виды медицинской стома-
тологической помощи детям в амбулаторных условиях: первичная доврачебная медико-санитарная стома-
тологическая помощь детскому населению (оказывается зубными врачами), первичная специализированная
медико-санитарная стоматологическая помощь детям (оказывается врачами-стоматологами и врачами-сто-
матологами детскими). Одним из критериев эффективности оказания стоматологической помощи является
показатель доступности стоматологической помощи, характеризующийся посещаемостью населением, в том
числе детским населением, медицинских учреждений, оказывающих помощь по профилю «Стоматология». Целью исследования является анализ динамики изменений объемов и видов стоматологической помощи
детскому населению 84 регионов РФ в 2020 и 2021 годах. Материалы и методы. В рамках деятельности Национального медицинского исследовательского центра
ФГБОУ ВО МГМСУ им. А.И. Евдокимова Минздрава России по профилю «Стоматология» был проведен анализ
отчетной документации 84 регионов РФ по основным разделам деятельности стоматологической службы за
2020 и 2021 годы. В данной статье представлен анализ видов медицинской стоматологической помощи, ока-
занной детям, и количества посещений детского населения по поводу оказания стоматологической помощи
в регионах РФ в 2020 и 2021 годах и динамика изменений изучаемых показателей. Результаты. В 2021 году на 27,66% увеличилось число регионов, где на одного ребенка в год приходилось
менее одного посещения в медицинские организации, оказывающие медицинскую помощь по профилю «Сто-
матология». В 2021 году по сравнению с 2020 годом отмечалось уменьшение числа посещений детьми меди-
цинских организаций, оказывающих стоматологическую помощь на 15,78%. В целом по 84 регионам РФ в
2021 году отмечается уменьшение объемов первичной доврачебной медико-санитарной стоматологической
помощи, оказываемой зубными врачами, и первично-специализированной медико-санитарной стоматологи-
ческой помощи, оказываемой врачами-стоматологами детскими, врачами стоматологами-хирургами, и уве-
личение объемов первично-специализированной медико-санитарной стоматологической помощи детскому
населению, оказываемой врачами-стоматологами (врачами-стоматологами общей практики). Ключевые слова: стоматология, дети, объемы помощи, виды помощи. Для цитирования: Янушевич ОО, Кисельникова ЛП, Зуева ТЕ, Золотницкий ИВ, Огарева АА. Особенности ока-
зания стоматологической помощи детскому населению среди субъектов Российской Федерации в 2020 и 2021
годах. Стоматология детского возраста и профилактика. 2023;23(2):105-131. DOI: 10.33925/1683-3031-2023-607. АКТУАЛЬНОСТЬ ботниками со средним медицинским образованием),
первичная врачебная медико-санитарная помощь
(оказывается врачами общей практики), первичная
специализированная
медико-санитарная
помощь
(оказывается врачами-специалистами). В стомато-
логии согласно Приказу от 19 августа 2021 года 866н
«Об утверждении классификатора работ (услуг), со-
ставляющих медицинскую деятельность» первичная
доврачебная медико-санитарная стоматологическая
помощь детскому населению оказывается зубными
врачами, первичная специализированная стоматоло-
гическая помощь – врачами стоматологами (врача-
ми-стоматологами общей практики) и врачами-сто-
матологами детскими. Стоматологическая медицинская помощь является
одним из наиболее массовых и социально значимых
видов медицинской помощи населению РФ. Это свя-
зано с высоким уровнем распространенности и ин-
тенсивности стоматологических заболеваний у детей
разного возраста в различных регионах РФ [1-4]. По данным проводимого в РФ в 2019 году Третьего
национального эпидемиологического стоматологи-
ческого обследования населения Российской Феде-
рации в среднем у детей 12-летнего возраста распро-
страненность кариеса составила 71%, у 15-летних
детей – 82%. Патология тканей пародонта была выяв-
лена у 37% 12-летних детей и 45% 15-летних детей [5]. Одним из критериев эффективности оказания сто-
матологической помощи является показатель доступ-
ности стоматологической помощи, характеризующий-
ся посещаемостью населением, в том числе детским
населением, медицинских организаций, оказываю-
щих помощь по профилю «Стоматология» [6, 7]. По данным проводимого в РФ в 2019 году Третьего
национального эпидемиологического стоматологи-
ческого обследования населения Российской Феде-
рации в среднем у детей 12-летнего возраста распро-
страненность кариеса составила 71%, у 15-летних
детей – 82%. Патология тканей пародонта была выяв-
лена у 37% 12-летних детей и 45% 15-летних детей [5]. Количество посещений детьми медицинских орга-
низаций является одним из объективных критериев,
входящих в объемы оказанной стоматологической
помощи, и ежегодно анализируется для планирова-
ния деятельности стоматологической службы регио-
на в последующие годы [8, 9]. Одним из критериев эффективности оказания сто-
матологической помощи является показатель доступ-
ности стоматологической помощи, характеризующий-
ся посещаемостью населением, в том числе детским
населением, медицинских организаций, оказываю-
щих помощь по профилю «Стоматология» [6, 7]. Медицинская помощь населению, в том числе
детскому населению, по профилю «Стоматология» в
рамках Программы государственных гарантий бес-
платного оказания гражданам Российской Федера-
ции медицинской помощи и соответствующих тер-
риториальных программ оказывается медицинскими
организациями разного уровня. Стоматологическая
помощь населению в амбулаторных условиях ока-
зывается в рамках первичной медико-санитарной
помощи. Согласно Федеральному закону от 21 но-
ября 2011 г. ABSTRACT Relevance. Dental care is one of the most common and socially significant types of medical care to the population
of the Russian Federation. The types of outpatient pediatric dental care are pre-medical primary dental care for 105 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Оригинальная статья І Original article children provided by dental therapists; specialized primary pediatric dental care provided by dentists and pediatric
dentists. Dental care availability is a dental care effectiveness criterium involving the population presentation,
including the children's population, to the medical institutions providing dental assistance. including the children's population, to the medical institutions providing dental assistance. The study aimed to conduct an analysis of the changes in the volume and types of pediatric dental care in 84 regions
of the Russian Federation in 2020 and 2021. g
p p
,
p
g
The study aimed to conduct an analysis of the changes in the volume and types of pediatric dental care in 84 regions
of the Russian Federation in 2020 and 2021. Material and methods. Within the framework of the activities of the National Medical Research Center of the Mos-
cow State Medical University, named after A.I. Evdokimov of the Ministry of Health of the Russian Federation on the
profile "Dentistry", the study analyzed the reporting documentation of 84 regions of the Russian Federation on the
main sections of the dental service for 2020 and 2021. The article presents an analysis of the types of dental care
provided to children, the number of child population visits for dental care in the regions of the Russian Federation
in 2020 and 2021 and the pattern of changes in the studied indicators. Results. In 2021, the number of regions where a child had less than one annual visit to a dental care provider in-
creased by 27.66%. In 2021de, compared with 2020, there was a decrease in the number of child visits to dental care
facilities by 15.78%. In general, in 84 regions of the Russian Federation in 2021, there is a decrease in the volume
of pre-medical primary dental care provided by dental therapists and specialized primary dental care provided by
pediatric dentists and oral surgeons and an increase in the specialized primary dental care volume to the child
population by dentists (general practice dentists). s: dentistry, children, care volume, types of care. Key words: dentistry, children, care volume, types of care. For citation: Yanushevich OO, Kiselnikova LP, Zueva TE, Zolotnitsky IV, Ogareva AA. Features of dental care provi-
sion for the child population in subjects of the Russian Federation in 2020 and 2021. Pediatric dentistry and dental
prophylaxis. 2023;23(2):105-131 (In Russ.). DOI: 10.33925/1683-3031-2023-607. АКТУАЛЬНОСТЬ №323-ФЗ «Об основах охраны здоровья
граждан в Российской Федерации» различают три
вида первичной медико-санитарной медицинской
помощи: первичная доврачебная медико-санитарная
помощь населению (оказывается медицинскими ра- Целью исследования является анализ динамики
изменений объемов и видов стоматологической по-
мощи детскому населению 84 регионов РФ в 2020 и
2021 годы. Оригинальная статья І Original article исследовательским центром по профилю «Сто-
матология» определен ФГБОУ ВО МГМСУ имени
А. И. Евдокимова Минздрава России. НМИЦ по про-
филю «Стоматология» функционирует под руковод-
ством главного внештатного специалиста-стомато-
лога Министерства здравоохранения РФ, академика
РАН, профессора, доктора медицинских наук, ректо-
ра ФГБОУ ВО МГМСУ имени А. И. Евдокимова Минз-
драва России Олега Олеговича Янушевича. исследовательским центром по профилю «Сто-
матология» определен ФГБОУ ВО МГМСУ имени
А. И. Евдокимова Минздрава России. НМИЦ по про-
филю «Стоматология» функционирует под руковод-
ством главного внештатного специалиста-стомато-
лога Министерства здравоохранения РФ, академика
РАН, профессора, доктора медицинских наук, ректо-
ра ФГБОУ ВО МГМСУ имени А. И. Евдокимова Минз-
драва России Олега Олеговича Янушевича. одного посещения в год, в 35 регионах – одно-два по-
сещения, более двух посещений выявлено только в
двух регионах: в Чувашской Республике (2,03 посеще-
ния на одного ребенка год) и в Ненецком автономном
округе (2,3 посещения на одного ребенка год). В 2021 году картина несколько изменилась: на
27,66% увеличилось число регионов, где на одного ре-
бенка в год приходилось менее одного посещения в
медицинские организации, оказывающие медицин-
скую помощь по профилю «Стоматология» и, соот-
ветственно, уменьшилось количество регионов, где
на одного ребенка в год было одно-два посещения. В 2021 году картина несколько изменилась: на
27,66% увеличилось число регионов, где на одного ре-
бенка в год приходилось менее одного посещения в
медицинские организации, оказывающие медицин-
скую помощь по профилю «Стоматология» и, соот-
ветственно, уменьшилось количество регионов, где
на одного ребенка в год было одно-два посещения. В рамках деятельности Национального медицин-
ского исследовательского центра ФГБОУ ВО МГМСУ
имени А. И. Евдокимова Минздрава России по про-
филю «Стоматология» был проведен анализ отчет-
ной документации 84 регионов РФ по основным раз-
делам деятельности стоматологической службы за
2020 и 2021 годы (Сведения о медицинской органи-
зации за 2020 и 2021 годы, Форма 30, Приказ Росста-
та: Об утверждении формы от 03.08.2018 г. №483). В 2021 году в большинстве регионов РФ (60 ре-
гионов) в год на одного ребенка приходилось ме-
нее одного посещения, в 22 регионах РФ на одного
ребенка в год отмечено одно-два посещения, более
двух посещений в год выявлено также только в двух
регионах: Чувашской Республике (2,05 посещения на
одного ребенка год) и Ненецком автономном округе
(2,23 посещения на одного ребенка год) (рис. 1). Анализировались отчетные формы 84 регионов
Центрального, Сибирского, Уральского, Северо-За-
падного, Приволжского, Южного, Северо-Кавказского
и Дальневосточного федеральных округов. Получен-
ные данные подвергались статистической обработке
с использованием метода дескриптивной статистики. Оригинальная статья І Original article Количество посещений на одного ребенка в год в
медицинских организациях, оказывающих помощь
по профилю «Стоматология» в 84 субъектах РФ в
2020 и 2021 годах, и динамика изменений представ-
лены в таблице 1. Одним из показателей, по которым оценивается
деятельность стоматологической службы регионов
РФ, является объем оказанной стоматологической
помощи населению. При оценке объемов помощи
одним из критериев является количество посещений
детского населения в медицинские организации,
оказывающие помощь по профилю «Стоматология». Поскольку в различных регионах численность дет-
ского населения значительно отличается, объектив-
ным показателем при оценке объемов стоматологи-
ческой помощи является количество посещений на
одного ребенка в год. Среднее число посещений на одного ребенка по
субъектам РФ в 2020 году составило 1,00 посещение,
в 2021 году – 0,84 посещения. В среднем отмечалось
уменьшение числа посещений детей в медицинские
организации, оказывающие медицинскую помощь
по профилю «Стоматология» на 15,78%. При сравнительном анализе данных за 2020 и
2021 годы по отдельным регионам РФ была выявлена
аналогичная тенденция. В большинстве регионов РФ
(61 регион) отмечается снижение объемов оказанной
стоматологической помощи детскому населению,
уменьшение количества посещений на одного ребен-
ка в год. При этом в ряде регионов снижение числа
посещений детского населения в медицинские орга-
низации, оказывающие стоматологическую помощь
в 2021 году, было более 50%, при сравнении с 2020 го-
дом. В Амурской области количество посещений в год
на одного ребенка уменьшилось на 61,73%, в Белго-
родской области – на 61,02%, во Владимирской обла-
сти – на 53,85%, в Костромской области – на 67,90%, в
Республике Мордовия – на 56,63%, в Чеченской Респу-
блике – на 55,42%, в Ярославской области – на 74,24%. В данной статье представлен анализ объемов и ви-
дов медицинской стоматологической помощи, ока-
занной детям, и количества посещений детского насе-
ления по поводу оказания стоматологической помощи
в 84 регионах РФ в 2020 и 2021 годах, представлена ди-
намика изменений изучаемых показателей. МАТЕРИАЛЫ И МЕТОДЫ В рамках реализации федерального проекта «Раз-
витие сети национальных медицинских исследо-
вательских центров» и внедрения инновационных
медицинских технологий национального проекта
«Здравоохранение» в 2019 году был создан Наци-
ональный медицинский исследовательский центр
по профилю «Стоматология». Приказом МЗ РФ от
20.01.2020 г. №30 Национальным медицинским 106 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis РЕЗУЛЬТАТЫ В рамах проведенного исследования было выявлено,
что имеются определенные различия в обеспеченно-
сти детей стоматологической помощью по количеству
посещений на одного ребенка в год между субъектами
РФ как в 2020-м, так и в 2021 году. Такая картина, веро-
ятно, во многом связана с различием географических
и социально-экономических условий, разным уров-
нем стоматологической заболеваемости, состояния
материально-технической базы службы и доступности
стоматологической помощи в разных субъектах РФ. Следует отметить ряд регионов (23 региона), в ко-
торых в 2021 году отмечается увеличение количества
посещений на одного ребенка: Архангельская об-
ласть, Волгоградская область, Вологодская область,
город Санкт-Петербург, Забайкальский край, Иркут-
ская область, Калининградская область, Калужская
область, Камчатский край, Карачаево-Черкесская Ре-
спублика, Краснодарский край, Красноярский край,
Курская область, Орловская область, Республика Бу-
рятия, Республика Дагестан, Республика Крым, Респу-
блика Саха (Якутия), Самарская область, Ставрополь- При анализе количества посещений детского на-
селения регионов РФ в 2020 году было выявлено, что
в 47 регионах на одного ребенка приходилось менее 107 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Оригинальная статья І Original article
Субъект РФ
Subject of the RF
2020 г. Year 2020
2021 г. РЕЗУЛЬТАТЫ Year 2021
Отличие
Difference
Отличие, %
Difference, %
Ярославская область / Yaroslavl Region
1.32
0.34
–0.98
–74.24
Костромская область / Kostroma Region
0.81
0.26
–0.55
–67.90
Амурская область / Amur Region
0.81
0.31
–0.50
–61.73
Белгородская область / Belgorod Region
1.18
0.46
–0.72
–61.02
Республика Мордовия / Republic of Mordovia
0.83
0.36
–0.47
–56.63
Чеченская Республика/ Chechen Republic
0.83
0.37
–0.46
–55.42
Владимирская область / Vladimir Region
0.78
0.36
–0.42
–53.85
Курганская область / Kurgan Region
0.98
0.50
–0.48
–48.98
Ивановская область / Ivanovo Region
1.18
0.61
–0.57
–48.31
Магаданская область / Magadan Region
1.59
0.83
–0.76
–47.80
Республика Алтай / Republic of Altai
1.97
1.06
–0.91
–46.19
Ростовская область / Rostov Region
0.64
0.37
–0.27
–42.19
Тамбовская область / Tombov Region
1.12
0.68
–0.44
–39.29
Тульская область / Tula Region
1.38
0.86
–0.52
–37.68
Новгородская область / Novgorod Region
0.59
0.38
–0.21
–35.59
Псковская область / Pskov Region
0.85
0.55
–0.30
–35.29
Липецкая область / Lipetsk Region
1.05
0.68
–0.37
–35.24
Сахалинская область / Sakhalin Region
1.43
0.95
–0.48
–33.57
Оренбургская область / Orenburg Region
1.24
0.83
–0.41
–33.06
Хабаровский край / Khabarovsk Territory
1.01
0.69
–0.32
–31.68
Республика Адыгея / Republic of Adygea
0.58
0.41
–0.17
–29.31
Кировская область / Kirov Region
1.23
0.87
–0.36
–29.27
Астраханская область / Astrakhan Region
0.61
0.44
–0.17
–27.87
Ленинградская область / Leningrad Region
0.84
0.61
–0.23
–27.38
Приморский край / Primorye Territory
1.42
1.04
–0.38
–26.76
Город Севастополь / City of Sevastopol
1.05
0.78
–0.27
–25.71
Омская область / Omsk Region
1.62
1.22
–0.40
–24.69
Республика Карелия / Republic of Karelia
0.77
0.58
–0.19
–24.68
Ульяновская область / Ulyanovsk Region
1.5
1.13
–0.37
–24.67
Мурманская область / Murmansk Region
1.07
0.81
–0.26
–24.30
Томская область / Tomsk Region
0.75
0.58
–0.17
–22.67
Республика Калмыкия / Republic of Kalmykia
0.75
0.58
–0.17
–22.67
Нижегородская область / Nizhny Novgorod Region
0.8
0.62
–0.18
–22.50
Республика Ингушетия / Republic of Ingushetia
0.90
0.71
–0.19
–21.11
Челябинская область / Chelyabinsk Region
0.90
0.71
–0.19
–21.11
Ханты-Мансийский автономный округ – Югра /
Khanty-Mansi Autonomous Area – Yugra
1.31
1.04
–0.27
–20.61
Алтайский край / Altai Territory
0.88
0.70
–0.18
–20.45
Республика Башкортостан / Republic of Bashkortostan
1.06
0.87
–0.19
–17.92
Тюменская область / Tyumen Region
0.72
0.60
–0.12
–16.67
Новосибирская область / Novosibirsk Region
0.8
0.67
–0.13
–16.25
Свердловская область / Sverdlovsk Region
1.00
0.84
–0.16
–16.00
Республика Марий Эл / Republic of Mari El
1.08
0.91
–0.17
–15.74
Рязанская область / Ryazan Region
0.64
0.54
–0.10
–15.63
Таблица 1. Продолжение / Сontinuation РЕЗУЛЬТАТЫ Количество посещений на 1 ребенка в субъектах РФ в 2020 и 2021 годах
Table 1. Number of visits per child in the RF subjects in 2020 and 2021 Оригинальная статья І Original article 108 Оригинальная статья І Original article Субъект РФ
Subject of the RF
2020 г. Year 2020
2021 г. 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis РЕЗУЛЬТАТЫ Year 2021
Отличие
Difference
Отличие, %
Difference, %
Саратовская область / Saratov Region
0.69
0.59
–0.10
–14.49
Удмуртская Республика / Republic of Udmurtia
1.16
1.03
–0.13
–11.21
Республика Тыва / Republic of Tyva
0.64
0.57
–0.07
–10.94
Еврейская автономная область / Jewish Autonomous Region
0.72
0.65
–0.07
–9.72
Республика Коми / Komi Republic
1.16
1.05
–0.11
–9.48
Республика Татарстан / Republic of Tatarstan
0.97
0.88
–0.09
–9.28
Московская область / Moscow Region
0.78
0.73
–0.05
–6.41
Республика Хакасия / Republic of Khakassia
0.79
0.74
–0.05
–6.33
Смоленская область / Smolensk Region
1.11
1.04
–0.07
–6.31
Воронежская область / Voronezh Region
0.98
0.94
–0.04
–4.08
Брянская область / Bryansk Region
1.23
1.18
–0.05
–4.07
Пензенская область / Penza Region
1.51
1.45
–0.06
–3.97
Республика Северная Осетия – Алания /
Republic of North Ossetia - Alania
0.26
0.25
–0.01
–3.85
Ненецкий автономный округ / Nenets Autonomous Area
2.3
2.23
–0.07
–3.04
Чукотский автономный округ / Chukotka Autonomous Area
1.22
1.19
–0.03
–2.46
Кабардино-Балкарская Республика / Kabardino-Balkarian Republic
0.94
0.93
–0.01
–1.06
Пермский край / Perm Territory
0.96
0.95
–0.01
–1.04
Кемеровская область – Кузбасс / Kemerovo Region - Kuzbass
1.03
1.02
–0.01
–0.97
Чувашская Республика / Chuvash Republic
2.03
2.05
0.02
+0.99
Волгоградская область / Volgograd Region
1.28
1.32
0.04
+3.13
Город Санкт-Петербург / City of St Petersburg
0.96
0.99
0.03
+3.13
Калужская область / Kaluga Region
1.05
1.09
0.04
+3.81
Ставропольский край / Stavropol Territory
0.86
0.91
0.05
+5.81
Архангельская область / Arkhangelsk Region
1.33
1.41
0.08
+6.02
Красноярский край / Krasnoyarsk Territory
1.03
1.10
0.07
+6.80
Курская область / Kursk Region
0.70
0.75
0.05
+7.14
Краснодарский край / Krasnodar Territory
0.67
0.72
0.05
+7.46
Ямало-Ненецкий автономный округ /
Yamal-Nenets Autonomous Area
1.21
1.31
0.10
+8.26
Самарская область / Samara Region
1.18
1.30
0.12
+10.17
Республика Бурятия / Republic of Buryatia
0.86
0.95
0.09
+10.47
Забайкальский край / Trans-Baikal Territory
0.90
1.01
0.11
+12.22
Камчатский край / Kamchatka Territory
0.93
1.05
0.12
+12.90
Республика Крым / Republic of Crimea
0.86
0.99
0.13
+15.12
Республика Саха (Якутия) / Republic of Sakha (Yakutia)
0.84
0.97
0.13
+15.48
Калининградская область / Kaliningrad Region
0.58
0.67
0.09
+15.52
Карачаево-Черкесская Республика /
Karachayevo-Circassian Republic
0.76
0.90
0.14
+18.42
Республика Дагестан / Republic of Dagestan
0.52
0.62
0.10
+19.23
Иркутская область / Irkutsk Region
1.07
1.29
0.22
+20.56
Тверская область / Tver Region
0.78
0.96
0.18
+23.08
Вологодская область / Vologda Region
0.82
1.22
0.40
+48.78
Орловская область / Orel Region
0.36
0.95
0.59
+163.89
Среднее / Mean
1.00
0.84
–0.16
–15.78
Уменьшение количества посещений на 1 ребенка в год / Decrease in the number of visits per child/ year 109 Оригинальная статья І Original article
Таблица 2. РЕЗУЛЬТАТЫ Динамика изменений распределения видов и объемов оказываемой стоматологической помощи
детскому населению по 84 субъектам РФ в 2020 и 2021 годах
Table 2. Pattern of changes in pediatric dental care type and volume distribution in 84 subjects of the RF in years 2020 and 2021
Рис. 1. Распределение регионов
по количеству посещений на одного ребенка в год
в медицинские организации,
оказывающие стоматологическую помощь
детскому населению субъектов РФ,
в 2020 и в 2021 годах
Fig. 1. Region distribution by dental visits
to pediatric dental care providers
in the RF per child a year in 2020 and 2021
Вид стоматологической помощи
Type of dental care
Объем в 2020 г., %
Volume in 2020, %
Объем в 2021 г., %
Volume in 2021, %
Отличия
Differences
Первичная доврачебная
стоматологическая помощь (зубные врачи)
Pre-medical primary dental care (dental therapists)
25.6
25.37
–0.23
Первичная специализированная
стоматологическая помощь (врачи-стоматологи)
Specialized primary dental care (dentists)
18.09
19.24
1.15
Первичная специализированная детская
стоматологическая помощь (врачи-стоматологи детские)
Specialized pediatric primary dental care (pediatric dentists)
45.16
44.75
–0.41
Первичная специализированная терапевтическая
стоматологическая помощь (врачи стоматологи-терапевты)
Specialized primary operative dentistry (operative dentists)
2.60
2.51
–0.09
Первичная специализированная хирургическая
стоматологическая помощь (врачи стоматологи-хирурги)
specialized primary oral surgery (oral surgeons)
8.53
8.10
–0.43
Уменьшение объемов помощи / Decrease in care volume
Увеличение объемов помощи / Increase in care volume Оригинальная статья І Original article Оригинальная статья І Original article ский край, Тверская область, Чувашская Республика,
Ямало-Ненецкий автономный округ. Среди данных
регионов наиболее выраженные изменения выявле-
ны в Вологодской области (увеличение на 48,78%) и
Орловской области (увеличение на 163,89%). принимали участие врачи стоматологи-терапевты. Первичная специализированная терапевтическая
стоматологическая помощь детям составила 2,6% в
2020 году и 2,51% в 2021 году (рис. 2, 3). принимали участие врачи стоматологи-терапевты. Первичная специализированная терапевтическая
стоматологическая помощь детям составила 2,6% в
2020 году и 2,51% в 2021 году (рис. 2, 3). Анализ динамики изменений распределения объе-
мов и видов помощи детскому населению в субъектах
РФ в 2020 и 2021 годах показал следующие измене-
ния. В среднем в структуре оказываемой стоматоло-
гической помощи произошло уменьшение объемов
первичной доврачебной стоматологической помощи
(на 0,23%), первичной специализированной детской
стоматологической помощи (на 0,41%), первичной
специализированной хирургической стоматологиче-
ской помощи (на 0,43%) и первичной специализиро-
ванной терапевтической стоматологической помощи
(на 0,09%). При этом отмечается увеличение объемов
первичной специализированной стоматологической
помощи, оказываемой врачами-стоматологами об-
щей практики (на 1,15%). Полученные данные свидетельствуют о тенден-
ции к уменьшению объемов оказанной медицин-
ской стоматологической помощи детскому населе-
нию большинства регионов РФ и, опосредованно, об
уменьшении ее доступности. Для оценки видов и объемов стоматологической
медицинской помощи населению РФ проведен ана-
лиз данных из отчетных форм за 2020 и 2021 годы
по посещениям к врачам разных стоматологических
специальностей (Сведения о медицинской органи-
зации за 2021 год, Форма 30, Приказ Росстата: Об ут-
верждении формы от 03.08.2018 г. №483). В связи с
тем, что корректно рассчитать объемы специализи-
рованной ортодонтической и ортопедической сто-
матологической помощи невозможно, данные виды
помощи из анализа были исключены. Таким образом, анализ видов и объемов стоматоло-
гической медицинской помощи детскому населению
РФ показал, что в 2021 году отмечается определенная
тенденция к уменьшению объемов оказанной помощи
детям по сравнению с 2020 годом. Отмечается незначи-
тельное уменьшение объемов первичной доврачебной
медико-санитарной стоматологической помощи, ока-
зываемой зубными врачами, и первично-специали-
зированной медико-санитарной стоматологической
помощи населению, оказываемой врачами-стомато-
логами детскими, врачами стоматологами-терапевта-
ми и врачами стоматологами-хирургами. Отмечается
увеличение объемов первично-специализированной
медико-санитарной стоматологической помощи, ока-
зываемой врачами-стоматологами (врачами-стомато-
логами общей практики). Данный факт опосредован-
но свидетельствует об уменьшении доступности для
детского населения РФ специализированной детской
и хирургической стоматологической помощи. Данные по структуре видов и объемов оказыва-
емой стоматологической помощи детскому населе-
нию по 84 субъектам РФ в 2020 и 2021 годах, а также
динамика изменений представлены в таблице 2. Оригинальная статья І Original article При оценке видов и объемов стоматологиче-
ской помощи населению РФ было выявлено, что и в
2020-м, и в 2021 году наибольший объем занимает
первичная специализированная детская стоматоло-
гическая помощь, оказываемая врачами-стоматоло-
гами детскими. В 2020 году объем первичной специ-
ализированной детской стоматологической помощи
составил 45,16%, в 2021 году – 44,75%. Объем первичной доврачебной стоматологиче-
ской помощи, оказываемой зубными врачами, был
достаточно значительным, составил 25,6% в 2020
году и 25,37% в 2021 году. Следующим по объему
видом помощи была первичная специализирован-
ная стоматологическая помощь, которую оказыва-
ют врачи-стоматологи (врачи-стоматологи общей
практики). В 2020 году данный вид помощи составил
18,09%, в 2021 году – 19,24%. Первичная специализи-
рованная хирургическая стоматологическая помощь
детскому населению, оказываемая врачами стома-
тологами-хирургами, в 2020 году составила 8,53% от
общего числа посещений, в 2021 году – 8,1%. Оригинальная статья І Original article Таблица 2. Динамика изменений распределения видов и объемов оказываемой стоматологической помощи
детскому населению по 84 субъектам РФ в 2020 и 2021 годах
Table 2. Pattern of changes in pediatric dental care type and volume distribution in 84 subjects of the RF in years 2020 and 2021
Вид стоматологической помощи
Type of dental care
Объем в 2020 г., %
Volume in 2020, %
Объем в 2021 г., %
Volume in 2021, %
Отличия
Differences
Первичная доврачебная
стоматологическая помощь (зубные врачи)
Pre-medical primary dental care (dental therapists)
25.6
25.37
–0.23
Первичная специализированная
стоматологическая помощь (врачи-стоматологи)
Specialized primary dental care (dentists)
18.09
19.24
1.15
Первичная специализированная детская
стоматологическая помощь (врачи-стоматологи детские)
Specialized pediatric primary dental care (pediatric dentists)
45.16
44.75
–0.41
Первичная специализированная терапевтическая
стоматологическая помощь (врачи стоматологи-терапевты)
Specialized primary operative dentistry (operative dentists)
2.60
2.51
–0.09
Первичная специализированная хирургическая
стоматологическая помощь (врачи стоматологи-хирурги)
specialized primary oral surgery (oral surgeons)
8.53
8.10
–0.43
Уменьшение объемов помощи / Decrease in care volume
Увеличение объемов помощи / Increase in care volume Рис. 2. Распределение видов и объемов стоматологической
помощи детскому населению РФ в 2020 году, %
Fig. 2. Distribution of dental care types and volume
to the child population in the RF in 2020, % Рис. 3. Распределение видов и объемов стоматологической
помощи детскому населению РФ в 2021 году, %
Fig. 3. Distribution of dental care types and volume
to the child population in the RF in 2021, % Рис. 2. Распределение видов и объемов стоматологической
помощи детскому населению РФ в 2020 году, %
Fig. 2. Distribution of dental care types and volume
to the child population in the RF in 2020, % Рис. 2. Распределение видов и объемов стоматологической
помощи детскому населению РФ в 2020 году, %
Fig. 2. Distribution of dental care types and volume
to the child population in the RF in 2020, % 110 ВЫВОДЫ 1. В 2021 году на 27,66% увеличилось число регионов,
где на одного ребенка в год приходилось менее одного
посещения медицинских организаций, оказывающих
медицинскую помощь по профилю «Стоматология». 2. В 2021 году по сравнению с 2020 годом отме-
чалось уменьшение числа посещений детьми меди-
цинских организаций, оказывающих стоматологи-
ческую помощь, на 15,78%. Согласно Приказу Минздрава России от 13.11.2012
№910н (ред. от 21.02.2020) «Об утверждении По-
рядка оказания медицинской помощи детям со
стоматологическими заболеваниями» амбулатор-
ная стоматологическая помощь детям оказывается
врачами-стоматологами детскими, врачами-стома-
тологами (врачами стоматологами общей практи-
ки), врачами стоматологами-хирургами и зубными
врачами [12]. Проведенный анализ отчетной доку-
ментации за 2020 и 2021 годы показал, что, поми-
мо вышеперечисленных специалистов, в оказании
стоматологической помощи детскому населению 3. В целом по 84 регионам РФ в 2021 году отмеча-
ется уменьшение объемов первичной доврачебной
медико-санитарной
стоматологической
помощи,
первичной специализированной детской стомато-
логической помощи, первичной специализирован-
ной хирургической стоматологической помощи и
увеличение объемов первичной специализирован-
ной стоматологической помощи, оказываемой вра-
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служенный врач Российской Федерации, доктор
медицинских наук, профессор, ректор Московско-
го
государственного
медико-стоматологическо-
го университета имени А. И. Евдокимова, главный
внештатный специалист-стоматолог Министерства
здравоохранения Российской Федерации, Москва,
Российская Федерация
Для переписки: olegyanushevich@mail.ru
ORCID: https://orcid.org/0000-0003-0059-4980 Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова,
Москва, Российская Федерация Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова,
Москва, Российская Федерация
Для переписки: tatyana_zueva@mail.ru
ORCID: https://orcid.org/0000-0002-5489-5888 Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова,
Москва, Российская Федерация Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова,
Москва, Российская Федерация
Для переписки: tatyana_zueva@mail.ru
ORCID: https://orcid.org/0000-0002-5489-5888 Для переписки: tatyana_zueva@mail.ru
ORCID: https://orcid.org/0000-0002-5489-5888 Золотницкий Игорь Валерьевич, доктор меди-
цинских наук, профессор, проректор, исполняющий
обязанности заведующего кафедрой пропедевтики
ортопедической стоматологии Московского госу-
дарственного медико-стоматологического универ-
ситета имени А. И. Евдокимова, главный внештат-
ный специалист по стоматологии департамента
здравоохранения города Москвы, Москва, Россий-
ская Федерация Кисельникова Лариса Петровна, доктор ме-
дицинских наук, профессор, заведующая кафедрой
детской стоматологии Московского государственно-
го медико-стоматологического университета имени
А. И. Евдокимова, главный внештатный специалист
стоматолог-детский
Центрального
федерального
округа Министерства здравоохранения Российской
Федерации, Москва, Российская Федерация
Для переписки: lpkiselnikova@mail.ru
ORCID: https://orcid.org/0000-0003-2095-9473 Для переписки: igorzolot@mail.ru
ORCID: https://orcid.org/0000-0001-7717-0540 Огарева Анна Алексеевна, кандидат медицин-
ских наук, ассистент кафедры детской стоматологии
Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова,
Москва, Российская Федерация
Для переписки: ann.ogareva@gmail.com
ORCID: https://orcid.org/0000-0003-0538-8107 Огарева Анна Алексеевна, кандидат медицин-
ских наук, ассистент кафедры детской стоматологии
Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова, Огарева Анна Алексеевна, кандидат медицин-
ских наук, ассистент кафедры детской стоматологии
Московского государственного медико-стоматоло-
гического университета имени А. И. Евдокимова, Автор, ответственный за связь с редакцией:
Зуева Татьяна Евгеньевна, кандидат медицин-
ских наук, доцент кафедры детской стоматологии Автор, ответственный за связь с редакцией: Москва, Российская Федерация
Для переписки: ann.ogareva@gmail.com
ORCID: https://orcid.org/0000-0003-0538-8107 Москва, Российская Федерация
Для переписки: ann.ogareva@gmail.com
ORCID: https://orcid.org/0000-0003-0538-8107 Зуева Татьяна Евгеньевна, кандидат медицин-
ских наук, доцент кафедры детской стоматологии REFERENCES The prevalence of den- 5. Peterson PE, Kuzmina EM. The prevalence of den- 112 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis INFORMATION ABOUT THE AUTHORS Oleg O. Yanushevich, DMD, PhD, DSc, Professor, Ac-
ademician of the Russian Academy of Sciences, Honored
Doctor of the Russian Federation, Rector of A. I. Yevdo-
kimov Moscow State University of Medicine and Den-
tistry, Chief freelance dentist of the Ministry of Health
of the Russian Federation, Moscow, Russian Federation
For correspondence: olegyanushevich@mail.ru
ORCID: https://orcid.org/0000-0003-0059-4980 Igor V. Zolotnitskii, DMD, PhD, DSc, Prorector, Act-
ing Head of the Department of Introduction to Prosth-
odontics, A. I. Yevdokimov Moscow State University
of Medicine and Dentistry, Chief Freelance Dentist of
the Moscow Healthcare Department, Moscow, Russian
Federation For correspondence: igorzolot@mail.ru
ORCID h
//
id
/0000 0001 7717 0540 For correspondence: igorzolot@mail.ru
ORCID: https://orcid.org/0000-0001-7717-0540 Larisa P. Kiselnikova, DMD, PhD, DSc, Professor,
Head of the Department of Pediatric Dentistry, A. I. Yev-
dokimov Moscow State University of Medicine and Den-
tistry, Chief freelance pediatric dentist of the Central
Federal District of the Ministry of Health of the Russian
d
i
i
d
i Anna A. Ogareva, DMD, PhD, Assistant Professor,
Department of Pediatric Dentistry, A. I. Yevdokimov
Moscow State University of Medicine and Dentistry,
Moscow, Russian Federation
For correspondence: ann.ogareva@gmail.com
ORCID: https://orcid.org/0000-0003-0538-8107 Federation, Moscow, Russian Federation
For correspondence: lpkiselnikova@mail.ru
ORCID Id: https://orcid.org/0000-0003-2095-9473 Конфликт интересов:
Авторы декларируют отсутствие
конфликта интересов/
Сonflict of interests:
The authors declare no conflict of interests
Поступила / Article received 15.03.2023
оступила после рецензирования / Revised 04.04.2023
Принята к публикации / Accepted 11.04.2023 Corresponding author: Corresponding author:
Tatyana E. Zueva, DMD, PhD, Associate Professor,
Department of Pediatric Dentistry, A. I. Yevdokimov
Moscow State University of Medicine and Dentistry,
Moscow, Russian Federation. For correspondence: tatyana_zueva@mail.ru
ORCID: https://orcid.org/0000-0002-5489-5888 113 2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
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https://dr.ntu.edu.sg/bitstream/10356/89512/1/The%20tumour%20microenvironment%20creates%20a%20niche%20for%20the%20self-renewal%20of%20tumour-promoting%20macrophages%20in%20colon%20adenoma.pdf
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English
| null |
The tumour microenvironment creates a niche for the self-renewal of tumour-promoting macrophages in colon adenoma
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Nature communications
| 2,018
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cc-by
| 16,452
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2018 Soncin, I., Sheng, J., Chen, Q., Foo, S., Duan, K., Lum, J., et al. (2018). The tumour
microenvironment creates a niche for the self‑renewal of tumour‑promoting macrophages
in colon adenoma. Nature Communications, 9, 582‑. Soncin, I., Sheng, J., Chen, Q., Foo, S., Duan, K., Lum, J., et al. (2018). The tumour
microenvironment creates a niche for the self‑renewal of tumour‑promoting macrophages
in colon adenoma. Nature Communications, 9, 582‑. The tumour microenvironment creates a niche for
the self‑renewal of tumour‑promoting
macrophages in colon adenoma Soncin, Irene; Sheng, Jianpeng; Chen, Qi; Foo, Shihui; Duan, Kaibo; Lum, Josephine;
Poidinger, Michael; Zolezzi, Francesca; Karjalainen, Klaus; Ruedl, Christiane 2018 https://hdl.handle.net/10356/89512 https://doi.org/10.1038/s41467‑018‑02834‑8 © 2018 The Author(s) (Nature Publishing Group).This article is licensed under a Creative
Commons Attribution 4.0 International License, which permits use, sharing, adaptation,
distribution and reproduction in any medium or format, as long as you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise
in a credit line to the material. If material is not included in the article’s Creative
Commons license and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Downloaded on 24 Oct 2024 11:57:28 SGT ARTICLE | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications The tumour microenvironment creates a niche for
the self-renewal of tumour-promoting
macrophages in colon adenoma 1,
although with a clear increase in the proportion of the tumoural
CD11b+F4/80−neutrophil fraction (up to 70% of total CD11b+
cells) (Supplementary Fig. 1). Tissue-resident macrophages are also naturally present in
various tumours, including lung and mammary tumours where
macrophages are monocyte-derived22,23 or even self-renewing24. The developmental origins and the maintenance kinetics of
resident macrophages in intestinal tumours, however, have not
been studied thus far. MHCIIlow macrophages accumulate during tumour progres-
sion. A previous study reported that F4/80hi tissue-resident
macrophages in the colon LP progressively disappear with
increasing age17. We therefore analysed the myeloid cell subsets
present in the fetal colon (embryonic day 19.5) and compared
them to those present in the colon LP of young and old mice
(from 1 week to 12 months). In contrast to Bain et al.,17 our
analysis clearly found that all three macrophage fractions (I–III)
were maintained across all age groups with only minor fluctua-
tions detected in their frequency (Fig. 2a). The predominant
tissue-resident macrophage subset in the fetal colon was the F4/
80hiMHCIIlow fraction. However, as a function of age this mac-
rophage subset almost disappeared to leave mainly F4/80hiMH-
CIIhi cells to represent the colon tissue-resident macrophages
(fraction I) (Fig. 2b). This decrease in MHCIIlow cell representa-
tion suggests that the LP environment gradually changes with age,
perhaps due to the establishment of a mature microbiota and/or a
tonic increase in inflammatory mediators17. h
l Here we show that the colon LP of normal adult mice contains
a major population of F4/80hiMHCIIhi macrophages and a
population of gradually disappearing F4/80hiMHCIIlow cells. Both
F4/80hi macrophage populations are relatively slowly replenished
by BM-derived cells, particularly MHCIIlow cells, compared to
F4/80int monocyte/macrophage cells that have a fast turnover. The tumour microenvironment, however, is enriched with F4/
80hiMHCIIlow macrophages. Furthermore, while F4/80hiMH-
CIIlow macrophages do not require CCR2+ monocytes for their
maintenance in the healthy gut LP or in tumours, we found that
F4/80hiMHCIIhi macrophages become independent from CCR2+
monocytes in tumours only, potentially due to tumour-derived
colony stimulating factor-1 (CSF1) that supports macrophage
self-renewal. Moreover, we demonstrate that CCR2-independent
intratumoural
F4/80hi
macrophages,
and
not
the
CCR2-
dependent monocytes or monocyte-derived macrophages, sup-
port cancer progression, suggesting this particular F4/80hi cell
fraction as an attractive therapeutic target. y
Given the high frequency of F4/80hiMHCIIlow cells in colon
polyps (Fig. The tumour microenvironment creates a niche for
the self-renewal of tumour-promoting
macrophages in colon adenoma The same LP populations were also identified in colon
polyps but with markedly different frequencies (Fig. 1b). In
these tumours, neutrophils (fraction III) were the most abun-
dant myeloid cell type (>40%) followed by monocyte-derived
cells (fraction II) and tumour-resident macrophages (fraction
I). Interestingly, eosinophils in tumours represented only a
minor fraction (~3%) of the total CD11b+ cells. Furthermore,
tumour-resident macrophages (fraction I) were clearly enriched
in MHCIIlow cells (Fig. 1a,b), which were almost neglectable in
normal adult colon LP. M
acrophages comprise a heterogeneous population of
tissue-resident immune cells that contribute to tissue
homeostasis, support the host defence system and can
impact on the initiation and propagation of several diseases,
including cancer (reviewed in refs.1,2). The majority of tissue-
resident macrophages are established prenatally3–5 and are mainly
independent from any further haematopoietic input due to their
capacity to self-renew in situ to maintain population size6–10. For
example, microglia, the resident macrophage population of the
central nervous system, develop early during embryonic devel-
opment from yolk sac precursors and effectively self-maintain
in situ throughout adulthood5,6,11. Although the majority of
the tissue-resident macrophages are maintained independently
from bone marrow (BM)-derived monocytes, numerous studies
have demonstrated that macrophages of certain tissues, including
the dermis5,12, mammary gland13, heart14, pancreas15 and
intestine5,16,17, require the input of monocytes to retain their pool
during adulthood. In particular, the intestinal macrophages
depend on the constant and fast replenishment of circulating
blood Ly6Chi monocytes, not only during inflammation but also
under normal healthy conditions16–18. In a healthy unperturbed
colon, monocytes attracted to the intestine gradually differentiate
into tissue-resident macrophages by losing Ly6C expression, up-
regulating the expression of macrophage markers, such as
CX3CR1,
F4/80,
CD64
and
CD11c16,19,
and
secreting
or
responding to the anti-inflammatory cytokine interleukin-10 20,21. Mice lacking either the chemokine receptor CCR2 or its ligand
CCL2 have reduced numbers of intestinal macrophages17, thus
implying that the homeostatic recruitment of macrophage pre-
cursors to the gut lamina propria (LP) may be dependent on a
CCR2–CCL2 axis. Upon intestinal inflammation, tissue-resident
macrophages are still derived from circulating monocytes, but
convert from being anti-inflammatory macrophages to highly
Toll-like receptor-responsive inflammatory cells16. Characterization of myeloid subpopulations in the colon LP of
untreated C57BL/6J mice and in “spontaneously” formed tumours
obtained
from
5-month-old ApcMin/+
mice confirmed the
presence of all myeloid subpopulations described in Fig. The tumour microenvironment creates a niche for
the self-renewal of tumour-promoting
macrophages in colon adenoma Irene Soncin1, Jianpeng Sheng1, Qi Chen1, Shihui Foo2, Kaibo Duan2, Josephine Lum2,
Francesca Zolezzi2,3, Klaus Karjalainen1 & Christiane Ruedl1 Irene Soncin1, Jianpeng Sheng1, Qi Chen1, Shihui Foo2, Kaibo Duan2, Josephine Lum2, Michael Poidinger
2,
Francesca Zolezzi2,3, Klaus Karjalainen1 & Christiane Ruedl1 Circulating CCR2+ monocytes are crucial for maintaining the adult tissue-resident
F4/80hiMHCIIhi macrophage pool in the intestinal lamina propria. Here we show that
a subpopulation of CCR2-independent F4/80hiMHCIIlow macrophages, which are the
most abundant F4/80hi cells in neonates, gradually decline in number in adulthood; these
macrophages likely represent the fetal contribution to F4/80hi cells. In colon adenomas of
ApcMin/+ mice, F4/80hiMHCIIlow macrophages are not only preserved, but become the
dominant subpopulation among tumour-resident macrophages during tumour progression. Furthermore, these pro-tumoural F4/80hiMHCIIlow and F4/80hiMHCIIhi macrophages can
self-renew in the tumour and maintain their numbers mostly independent from bone marrow
contribution. Analyses of colon adenomas indicate that CSF1 may be a key facilitator of
macrophage self-renewal. In summary, the tumour microenvironment creates an isolated
niche for tissue-resident macrophages that favours macrophage survival and self-renewal. 1 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. 2 Singapore Immunology Network,
Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Singapore 138648, Singapore. 3Present address: GALDERMA R&D, 06902
Sophia Antipolis Cedex France. Klaus Karjalainen and Christiane Ruedl jointly supervised this work. Correspondence and requests for materials should be
addressed to K.K. (email: Klaus@ntu.edu.sg) or to C.R. (email: Ruedl@ntu.edu.sg) | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS| (2018) 9:582 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 M treated ApcMin/+ mice (Fig. 1). Similar as previously reported5,
colon LP CD45+ cells comprised three major myeloid cell
fractions (I–III) after excluding CD11chiMHCIIhi dendritic
cells:
F4/80hiCD11b+
tissue-resident
(tumour-resident
in
polyps) macrophages (fraction I), F4/80intCD11b+ (fraction II)
and F4/80lowCD11b+ neutrophils (fraction III) (Fig. 1a). The
majority (>85%) of fraction I cells expressed high levels of
major histocompatibility complex (MHC) class II and only a
small proportion (~5%) of cells were low for MHC class II
expression. Fraction II could be further subdivided into four
distinct subpopulations in a waterfall-shaped distribution on a
Ly6C vs. MHCII dot plot consisting of three distinct monocyte-
differentiation stages (P1: Ly6ChiMHCII−; P2: Ly6ChiMHCII+;
P3: Ly6C−MHCII+) and one eosinophil fraction (Ly6C−MHCII
−) (Fig. 1a). We then analysed the presence of these sub-
populations in DSS-accelerated colon tumours from ApcMin/+
mice. The tumour microenvironment creates a niche for
the self-renewal of tumour-promoting
macrophages in colon adenoma 1b), we assessed whether there was an association
between tumour progression and the frequency of MHCIIlow
macrophages. Therefore, for this analysis we opted for the
spontaneous tumour model of ApcMin/+ mice, which generate
progressively adenomas up to 5–6 mm in size that is rarely
achieved in the DSS-accelerated tumour model. Indeed, we
observed an increasing abundance of this cell fraction in
conjunction with tumour progression until MHCIIlow macro-
phages became a dominant cell type among tumour-resident
macrophages (Fig. 2c). This observation clearly suggests that the
tumour microenvironment favours the differentiation or main-
tenance of MHCIIlow macrophages. | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 b Bar charts of the distinct myeloid cell subpopulations obtained
from the colon LP (n = 8) (upper bar chart) and 2–3 mm colon tumours (n = 20) (lower bar chart). White bars: fraction I; light blue bars: fraction II and
black bars: fraction III. Error bars represent the s.e.m. c Pie charts show the proportions of F4/80hi tissue-resident macrophages (MHCIIhi and MHCIIlow),
monocytes (P1), monocyte-derived macrophages (P2–P3), neutrophils and eosinophils across colon LP and tumours Taken together, these data assert that the tumour microenvir-
onment can modify the properties of MHCIIhi tissue-resident
macrophages such that they also become CCR2 independent like
MHCIIlow cells in the LP and hence are presumably able to
maintain themselves without monocyte replenishment. Intratumoural macrophages are independent from CCR2+
monocytes. Given that circulating Ly6Chi monocytes express
high levels of CCR2 receptor25, we decided to examine the pre-
sence of this receptor on tissue-resident and monocyte-derived
macrophages in colon LP and adenoma polyps. The aim of this
experiment was to determine the potential origins of these cells. In the LP, consistent with being descendants of classical mono-
cytes, Ly6ChiMHCII−(P1), Ly6ChiMHCIIhi (P2) and Ly6C
−MHCII+ (P3) cells in fraction II subset had the highest and most
homogeneous expression of CCR2 (Fig. 3a)17, whereas tissue-
resident F4/80hiMHCIIhi and F4/80hiMHCIIlow macrophages
were highly heterogeneous in their CCR2 expression levels, from
very high to very low levels (Fig. 3a and Supplementary Fig. 2). As
expected, numbers of monocytes and monocyte-derived cells (P1,
P2 and P3) in fraction II were severely diminished in Ccr2−/−
mice (Fig. 3b). In addition, MHCIIhi tissue-resident macrophages
were also mostly CCR2 dependent, whereas MHCIIlow cells were
not, which is likely a reflection of their markedly lower CCR2
expression level (Fig. 3b). hi Turnover of colon lamina propria myeloid cells. We utilized the
KitMerCreMer/R26 fate mapping mouse5—where yellow florescence
protein (YFP) expression can be induced in early BM progenitors
—to monitor the cell population turnover rates driven by the BM
input in colon LP myeloid cell populations. After injecting adult
mice with tamoxifen, the labelling index of tissue-resident mac-
rophages (F4/80hiMHCIIlow and MHCIIhi), monocytes (P1),
monocyte-derived macrophages (P2–P3 fractions), eosinophils
and neutrophils was analysed at different time points over a
period of 5 months. As anticipated, all tested myeloid popula-
tions, with the exception of tissue-resident macrophages, were
rapidly labelled with YFP and reached a plateau by 1–2 weeks
after the final tamoxifen injection. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 c Pie charts show the proportions of F4/80hi tissue-resident macrophages (MHCIIhi and MHCIIlow
monocytes (P1), monocyte-derived macrophages (P2–P3), neutrophils and eosinophils across colon LP and tumours 40
Fraction
I
Fraction
II
Fraction
III
30
15
10
% Of CD11b+ myeloid cells
5
0
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
Neutrophils
Colon LP
MHCIIlow
P1
MHCIIhigh
b
c Colon LP
Fr I
Fr II
Fr III
F4/80
CD11b
MHCII
Ly6C
105
6
85
15
18
3
56
P2
P1
eos
P3
104
104
105
103
103
0
0
104
105
103
0
105
104
104
105
103
103
0
0
105
104
103
102
0
a MHCII
Ly6C
105
6
85
15
18
3
56
P2
P1
eos
P3
104
104
105
103
103
0
0
104
105
103
0
105
104
103
102
0 Colon LP
Fr I
Fr II
Fr III
F4/80
CD11b
Ly6C
105
104
104
105
103
103
0
0
a b a Fraction
I
Fraction
II
Fraction
III
% Of CD11b+ myeloid cells
50
40
15
10
5
0
E
N
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
Neutrophils
Colon tumour
P2
P3
Eosinophils
Neutrophils 36
57
104
105
103
0
105
104
103
102
0 36
57
P2
P3
eos
P1
104
105
103
0
104
105
103
0
105
104
103
102
0
105
104
103
102
0
30
26
19
9
MHCII
Ly6C Colon tumour
Fr I
Fr II
Fr III
F4/80
CD11b
105
104
104
105
103
103
102
0
0 P2
P3
eos
P1
104
105
103
0
105
104
103
102
0
30
26
19
9
MHCII
Ly6C MHCII Fig. 1 Myeloid cell heterogeneity in colon lamina propria and colon tumours. WT C57BL/6J and ApcMin/+ mice aged 6 weeks old were administered
drinking water supplemented with dextran sodium sulphate for 7 days, and analysed 4 weeks later. a Flow cytometry representative dot plots of colon
lamina propria (LP) (upper panel) and tumour cell subpopulations (lower panel). Three different myeloid fractions (I–III) are defined by the differential
expression of F4/80. Fraction I represents F4/80hi tissue-resident macrophages, which can be further subdivided into MHCIIhi and MHCIIlow. Fraction II
contains monocytes (P1), two monocyte-derived macrophage subpopulations (P2 and P3) and eosinophils (eos), based on differential expression of MHCII
and Ly6C. Fr III consists of neutrophils. Gating strategy is shown in Supplementary Fig. 4. Results
Sk
d Skewed representation of myeloid cells in colon adenomas. We
first profiled colon LP myeloid subpopulations from wild-type
(WT) C57BL/6J mice, previously treated with dextran sodium
sulphate (DSS), and compared them with their tumoural
counterparts obtained from the polyps collected from DSS- NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 40
Fraction
I
Fraction
II
Fraction
III
Fraction
I
Fraction
II
Fraction
III
30
15
10
% Of CD11b+ myeloid cells
% Of CD11b+ myeloid cells
5
0
50
40
15
10
5
0
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
Neutrophils
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
Neutrophils
Colon LP
Colon LP
MHCIIlow
Colon tumour
Colon tumour
P1
P2
P3
Eosinophils
Neutrophils
MHCIIhigh
Fr I
Fr II
Fr III
Fr I
Fr II
Fr III
F4/80
CD11b
F4/80
CD11b
MHCII
Ly6C
105
6
85
15
18
36
57
3
56
P2
P2
P3
eos
P1
P1
eos
P3
104
104
105
103
103
0
0
104
105
103
0
104
105
103
0
104
105
103
0
105
104
104
105
103
103
0
0
105
104
104
105
103
103
102
0
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
30
26
19
9
MHCII
Ly6C
a
b
c
Fig. 1 Myeloid cell heterogeneity in colon lamina propria and colon tumours. WT C57BL/6J and ApcMin/+ mice aged 6 weeks old were administered
drinking water supplemented with dextran sodium sulphate for 7 days, and analysed 4 weeks later. a Flow cytometry representative dot plots of colo
lamina propria (LP) (upper panel) and tumour cell subpopulations (lower panel). Three different myeloid fractions (I–III) are defined by the differentia
expression of F4/80. Fraction I represents F4/80hi tissue-resident macrophages, which can be further subdivided into MHCIIhi and MHCIIlow. Fraction
contains monocytes (P1), two monocyte-derived macrophage subpopulations (P2 and P3) and eosinophils (eos), based on differential expression of MHC
and Ly6C. Fr III consists of neutrophils. Gating strategy is shown in Supplementary Fig. 4. b Bar charts of the distinct myeloid cell subpopulations obtaine
from the colon LP (n = 8) (upper bar chart) and 2–3 mm colon tumours (n = 20) (lower bar chart). White bars: fraction I; light blue bars: fraction II an
black bars: fraction III. Error bars represent the s.e.m. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Representative dot plots of F4/80hiMHCIIhi and F4/80hiMHCIIlow
subpopulations obtained from fetal colon (E19.5) and from colons of mice aged 2 days, 1, 3, 4, 6 and 8 weeks and 12 months. The bar chart represents the
age-dependent ratio between F4/80hiMHCIIhi (blue) and F4/80hiMHCIIlow (red) subpopulations in the colon LP. E19.5: n = 1 (pool of 15 embryos obtained
from 3 different pregnant mice); day 2: n = 2 (each group pool of 5 mice); 1 week: n = 2 (each group pool of 5 mice); 3 weeks: n = 6 mice; 4 weeks: n = 5
mice; 6 weeks: n = 4; 8 weeks: n = 7 and 12 months: n = 4 mice. Error bars represent the s.e.m. c Representative flow cytometry analysis (left panel) and
mean percentage of F4/80hiMHCIIhi and F4/80hiMHCIIlow subpopulations (right panel) in tumours of different sizes (0.5–6.0 mm in diameter) obtained
from ApcMin/+ mice. 0.5 mm: n = 3; 1 mm: n = 3; 2 mm: n = 4; 3–4 mm: n = 3; 5–6 mm: n = 4. Error bars represent the s.e.m. Scale bars: 2.5 mm. Gating
strategy is shown in Supplementary Fig. 4 ression influence the ratio between F4/80hiMHCIIhi and F4/80hiMHCIIlow subpopulations. a F4/80hi tissue-resident “frozen” to the level corresponding to the time for their entrap-
ment in this niche that became independent from new inputs
(Fig. 4 and Fig. 5a). replaced by BM-derived monocytes only very slowly, if at all. Our
fate mapping data indicated that it took several months to fully
replace F4/80hiMHCIIhi cells, whereas MHCIIlow macrophages
exhibited minimal labelling (Fig. 4), suggesting that these tissue-
resident macrophages retained their original fetal seed population
for a long period. Of note, when tamoxifen was administered during embryogen-
esis (Fig. 5b, upper panel), all myeloid cell subpopulations—
including tissue-resident macrophages in the healthy LP and
colon tumours—exhibited similar YFP tagging patterns (Fig. 5b,
lower panel). This result was expected, as all of these cells are the
progeny of classical haematopoietic stem cells, as previously
described5. Intratumoural resident macrophages expand by self-renewal. We noted that F4/80hiMHCIIlow tumour-resident macrophages
in particular seemed to expand in polyps and thus wanted to
determine if this effect was due to increased self-renewal or the
recruitment of new cells. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 3 Weeks
3 Weeks
1 Week
E19.5
2 Days
Ly6C
I
II
III
4 Weeks
6 Weeks
8 Weeks
12 months
Fraction I
% Of CD11b+ myeloid cells
Subset (%)
25
20
15
10
5
0
100
80
60
40
20
0
3 Weeks
3 Weeks
4 Weeks
6 Weeks
8 Weeks
12 Months
4 Weeks
0.5 mm
1 mm
2 mm
3–4 mm
5–6 mm
6 Weeks
8 Weeks
1 Week
2 Days
E19.5
12 Months
4 Weeks
6 Weeks
8 Weeks
12 Months
F4/80
105
2
11
0.5
0.5
4
2
1.5
5
7
2
3
12
0.3
0.3
0.3
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
105
104
104
103
103
0
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
MHCII
0.5 mm
19
18
79
13
83
2
2
96
95
75
22
70
21
73
32
63
45
49
62
36
1 mm
2 mm
3–4 mm
5–6 mm
Ly6C
MHCII
47
49
50
93
6
51
CD11b
Age
Subset (%)
100
MHCIIlow
MHCIIhigh
MHCIIlow
MHCIIhigh
80
60
40
20
0
Tumour diameter
a
b
c
Fig. 2 Ageing and tumour progression influence the ratio between F4/80hiMHCIIhi and F4/80hiMHCIIlow subpopulations. a F4/80hi tissue-resident
macrophages persist in the colon LP after birth into adulthood, as shown by the representative flow cytometry dot plots and bar chart. Error bars represen
the s.e.m. b The frequency of colon F4/80hiMHCIIlow cells rapidly declines after birth. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Gating
strategy is shown in Supplementary Fig 4 3 Weeks
I
II
III
4 Weeks
6 Weeks
8 Weeks
12 months
Fraction I
% Of CD11b+ myeloid cells
25
20
15
10
5
0
3 Weeks
4 Weeks
6 Weeks
8 Weeks
12 Months
F4/80
105
2
11
0.5
0.5
4
2
1.5
5
7
2
3
12
0.3
0.3
0.3
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
0
0
CD11b
a a 3 Weeks
1 Week
E19.5
2 Days
Ly6C
4 Weeks
6 Weeks
8 Weeks
12 Months
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
MHCII
18
79
13
83
2
2
96
95
75
22
47
49
50
93
6
51
b b Subset (%)
100
80
60
40
20
0
3 Weeks
4 Weeks
6 Weeks
8 Weeks
1 Week
2 Days
E19.5
12 Months
Age
MHCIIlow
MHCIIhigh Age 0.5 mm
1 mm
2 mm
3–4 mm
5–6 mm
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
105
104
103
102
0
MHCII
0.5 mm
19
70
21
73
32
63
45
49
62
36
1 mm
2 mm
3–4 mm
5–6 mm
Ly6C
MHCII
Subset (%)
100
MHCIIlow
MHCIIhigh
80
60
40
20
0
Tumour diameter
c c Tumour diameter Tumour diameter Fig. 2 Ageing and tumour progression influence the ratio between F4/80hiMHCIIhi and F4/80hiMHCIIlow subpopulations. a F4/80hi tissue-resident
macrophages persist in the colon LP after birth into adulthood, as shown by the representative flow cytometry dot plots and bar chart. Error bars represent
the s.e.m. b The frequency of colon F4/80hiMHCIIlow cells rapidly declines after birth. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Representative dot plots of F4/80hiMHCIIhi and F4/80hiMHCIIlow
subpopulations obtained from fetal colon (E19.5) and from colons of mice aged 2 days, 1, 3, 4, 6 and 8 weeks and 12 months. The bar chart represents th
age-dependent ratio between F4/80hiMHCIIhi (blue) and F4/80hiMHCIIlow (red) subpopulations in the colon LP. E19.5: n = 1 (pool of 15 embryos obtaine
from 3 different pregnant mice); day 2: n = 2 (each group pool of 5 mice); 1 week: n = 2 (each group pool of 5 mice); 3 weeks: n = 6 mice; 4 weeks: n = 5
mice; 6 weeks: n = 4; 8 weeks: n = 7 and 12 months: n = 4 mice. Error bars represent the s.e.m. c Representative flow cytometry analysis (left panel) an
mean percentage of F4/80hiMHCIIhi and F4/80hiMHCIIlow subpopulations (right panel) in tumours of different sizes (0.5–6.0 mm in diameter) obtaine
from ApcMin/+ mice. 0.5 mm: n = 3; 1 mm: n = 3; 2 mm: n = 4; 3–4 mm: n = 3; 5–6 mm: n = 4. Error bars represent the s.e.m. Scale bars: 2.5 mm. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 These data suggest rapid
replacement of myeloid cell populations by BM-derived cells
(Fig. 4). hi
hi Interestingly, we found that tumour-resident MHCIIhi and
MHCIIlow macrophages both expressed relatively low levels of
CCR2 compared to their LP counterparts and, consistently, their
numbers did not change in Ccr2−/−mice. Conversely, fraction II
cells (P1, P2 and P3) in polyps behaved exactly like their WT
counterparts (Fig. 3a, c). g
Although it is widely considered that gut F4/80hiMHCIIhi cells
lose their prenatal origin and are replaced by CCR2+ monocytes
in adulthood16, our data clearly demonstrated that they are in fact NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 3 ARTICLE | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Again, our adult fate mapping analyses
in KitMerCreMer/R26ApcMin/+ mice showed that the recruitment of
new cells to DSS-accelerated adenomas was not increased and
that the population turnover rate of F4/80hiMHCIIlow cells was
comparable to that of the LP (Fig. 5a). A decrease in YFP labelling
was observed in tumour-resident F4/80hiMHCIIhi cells when
compared to their LP counterparts. This is probably due to the
fact that the labelling index of macrophages in polyps was We found clear evidence, however, for increased self-renewal
of F4/80hi macrophages that could explain their capacity to
expand in polyps. Firstly, tumour-resident macrophages were
clearly in an active phase of the cell cycle unlike those in the LP,
as indicated by the expression of the Ki-67 cellproliferation
marker
(Fig. 6a). Secondly,
RNA-sequencing
(RNA-seq)
analyses of cell-sorted, purified tumour-resident F4/80hi (both
MHCIIhi and MHCIIlow) cells showed a clear upregulation of
several cell cycle regulators, including cyclin-dependent kinase
Cdk1, cyclin-A2 and -B2, E2f2, NEK family of serine/threonine NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 4 4 kinases and various cell division cycle and mini chromosome
because F4/80hiMHCIIlow macrophages gradually disappear in
Lamina propria
Tumour
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
0.6
3
0.5
3
93
20
9
61
1.8
8
28
43
22
0.2
0.8
0.4
75
3
II
I
II
I
II
I
II
I
–103
103
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
104
105
0
–103
103
104
105
0
Fraction I
Fraction II
Fraction I
Fraction II
Eosinophils
ns
ns
***
***
***
***
***
**
ns
ns
***
***
10,000
300
6000
4000
2000
0
6000
6000
4000
2000
2000
3000
3000
6000
3000
0
3000
9000
9000
4000
1000
2000
1000
0
0
3000
2000
1000
0
0
WT
Ccr2–/–
WT
Ccr2–/–
WT
Ccr2–/–
WT
Ccr2–/–
0
200
100
0
5000
0
2500
2000
1500
1000
500
0
25,000
20,000
15,000
10,000
5000
0
P1
P2
P3
P1
P2
P3
Absolute no. Absolute no. Absolute no. Absolute no. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 MHCIIlow
MHCIIhigh
Eosinophils
MHCIIlow
MHCIIhigh
8000
6000
4000
2000
0
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
F4/80
CD11b
Ly6C
MHCII
F4/80
CD11b
MHCII
105
104
103
0
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
102
102
102
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
6
11
42
18
80
42
17
16
9
7
1
76
23
73
5
5
48
0.5
a
b
c
Ly6C
CCR2
Fig. 3 Lamina propria and intratumoural colon F4/80hiCD11b+ macrophage subsets show differential CCR2-driven monocyte dependence. a Fluorescence-
activated cell sorting histograms showing CCR2 expression profiles on distinct colon LP and intratumoural myeloid cell subpopulations: fraction I consists
of MHCIIlow and MHCIIhi cells, and fraction II consists of monocytes (P1) and monocyte-derived macrophages (P2 and P3). b Myeloid cell profiling in WT
and Ccr2−/−colon LP. Representative flow cytometry analysis with F4/80 and CD11b-expressing myeloid subpopulations, obtained from the colon LP of
WT and Ccr2−/−mice. Fractions I and II were further dissected for MHCII and Ly6C expression (left panel). The absolute numbers of eosinophils, F4/
80hiMHCIIhi and F4/80hiMHCIIlow tissue-resident macrophages and P1–P3 subpopulations are shown (right panel). The bar chart represents the mean
number of mice in each group and the error bars represent the s.e.m. (WT; n = 7 and Ccr2−/−; n = 14). c Myeloid cell profiling in WT and Ccr2−/−colon
tumours as described above for LP cells. Bar charts show the mean±s.e.m. of absolute numbers of eosinophils, F4/80hiMHCIIhi and F4/80hiMHCIIlow
tissue-resident macrophages and P1–P3 subpopulations obtained from ApcMin/+ (n = 8) and ApcMin/+Ccr2−/−(n = 9) mice. Statistical significance was
determined using an unpaired Student's t-test. **P<0.001; ***P < 0.0001; ns, not significant. Gating strategy is shown in Supplementary Fig. 4
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8
ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Eosinophils
MHCIIlow
MHCIIhigh
8000
6000
4000
2000
0
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
F4/80
CD11b
Ly6C
MHCII
C
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
102
102
102
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
6
11
42
18
80
42
17
16
9
7
1
76
23
73
5
5
48
0.5
c II
I
II
I
Ccr2–/–ApcMin/+
ApcMin/+
F4/80
CD11b
L
C
105
105
104
104
103
103
0
0
102
102
105
104
103
0
105
104
103
0
6
11
42
5
5
48
c 105
104
103
0
105
104
103
102
0
18
80 c ***
ns
ns
6000
3000
0
9000
4000
3000
2000
1000
0
Absolute no. Eosinophils
MHCIIlow
MHCIIhigh
8000
6000
4000
2000
0 105
104
103
0
105
104
103
102
0
42
17
16
9 ***
***
**
6000
3000
3000
9000
2000
1000
0
0
25,000
20,000
15,000
10,000
5000
0
P1
P2
P3
Absolute no. Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+ Ly6C
MHCII
105
104
103
0
102
105
104
103
0
23
73 105
104
103
0
102
105
104
103
0
7
1
76
0.5 Fig. 3 Lamina propria and intratumoural colon F4/80hiCD11b+ macrophage subsets show differential CCR2-driven monocyte dependence. a Fluorescence-
activated cell sorting histograms showing CCR2 expression profiles on distinct colon LP and intratumoural myeloid cell subpopulations: fraction I consists
of MHCIIlow and MHCIIhi cells, and fraction II consists of monocytes (P1) and monocyte-derived macrophages (P2 and P3). b Myeloid cell profiling in WT
and Ccr2−/−colon LP. Representative flow cytometry analysis with F4/80 and CD11b-expressing myeloid subpopulations, obtained from the colon LP of
WT and Ccr2−/−mice. Fractions I and II were further dissected for MHCII and Ly6C expression (left panel). The absolute numbers of eosinophils, F4/
80hiMHCIIhi and F4/80hiMHCIIlow tissue-resident macrophages and P1–P3 subpopulations are shown (right panel). The bar chart represents the mean
number of mice in each group and the error bars represent the s.e.m. (WT; n = 7 and Ccr2−/−; n = 14). c Myeloid cell profiling in WT and Ccr2−/−colon
tumours as described above for LP cells. Bar charts show the mean±s.e.m. | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Lamina propria
Tumour
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
–103
103
104
105
0
–103
103
104
105
0
Fraction I
Fraction II
a
CCR2 a 0.6
3
0.5
3
93
20
9
61
1.8
8
28
43
22
0.2
0.8
0.4
75
3
II
I
II
I
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
Fraction I
Fraction II
Eosinophils
ns
ns
***
***
***
***
10,000
300
6000
4000
2000
0
6000
4000
2000
2000
3000
1000
0
WT
Ccr2–/–
WT
Ccr2–/–
WT
Ccr2–/–
WT
Ccr2–/–
0
200
100
0
5000
0
2500
2000
1500
1000
500
0
P1
P2
P3
Absolute no. Absolute no. MHCIIlow
MHCIIhigh
F4/80
CD11b
MHCII
105
104
103
0
b
Ly6C 0.6
3
0.5
3
93
20
9
61
1.8
8
28
43
22
0.2
0.8
0.4
75
3
II
I
II
I
105
105
104
104
103
103
0
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
0
105
104
103
102
0
105
104
103
102
0
105
104
103
0
105
104
103
0
105
104
103
0
Fraction I
Fraction II
Eosino
ns
***
10,000
WT
Ccr2
WT
Ccr2–/–
5000
0
2500
2000
1500
1000
500
0
P1
Absolute no. Absolute no. F4/80
CD11b
MHCII
105
104
103
0
b
Ly6C b Eosinophils
ns
ns
***
10,000
300
6000
4000
2000
0
200
100
0
5000
0
Absolute no. MHCIIlow
MHCIIhigh b ***
***
***
6000
4000
2000
2000
3000
1000
0
WT
Ccr2–/–
WT
Ccr2–/–
WT
Ccr2–/–
0
2500
2000
1500
1000
500
0
P1
P2
P3
Absolute no. II
I
II
I
***
***
***
**
ns
ns
6000
3000
6000
3000
0
3000
9000
9000
4000
2000
1000
0
3000
2000
1000
0
0
Cc
Cc
Cc
25,000
20,000
15,000
10,000
5000
0
P1
P2
P3
Absolute no. Absolute no. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 of absolute numbers of eosinophils, F4/80hiMHCIIhi and F4/80hiMHCIIlow
tissue-resident macrophages and P1–P3 subpopulations obtained from ApcMin/+ (n = 8) and ApcMin/+Ccr2−/−(n = 9) mice. Statistical significance was
determined using an unpaired Student's t-test. **P<0.001; ***P < 0.0001; ns, not significant. Gating strategy is shown in Supplementary Fig. 4 because F4/80hiMHCIIlow macrophages gradually disappear in
the adult LP and probably do not vigorously self-renew, and
that F4/80hiMHCIIhi cells are predominantly maintained in the
LP by recruited BM-derived cells and, therefore, do not depend
on in situ self-renewal. kinases and various cell division cycle and mini-chromosome
maintenance proteins family members, in comparison to their
LP counterparts (Fig. 6b). Interestingly, we could not find any
evidence for active cell cycling of F4/80hiMHCIIhi and F4/
80hiMHCIIlow cells in the LP (Fig. 6a, b). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 This effect could be | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 5 NATURE COMMUNICATIONS| (2018) 9:582 ARTICLE Tumour microenvironment alters resident macrophage phe-
tumour-associated macrophages (TAMs)26,27, with MHCIIlow
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
20 Weeks
Analysis of YFP+ cells
Neutrophils
102
102
103
103 104 105
–103
104
105
0
0
103
103 104 105
–103
104
105
0
0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
103 104 105
–103
104
105
0
0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
103 104 105
–103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
0
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103 104 105
–103 0
103
104
105
0
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103 104 105
–103 0
103
104
105
0
–103
103
103 104 105
–103
104
105
0
0
102
103
103 104 105
–103
104
105
0
103
104
105
0
0
102
103
103
104 105
–103
104
105
0
0
103 104 105
–103 0
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
5 Months
YFP+ after normalization (%)
CD11b
YFP
100
80
60
40
20
0
Neutrophils
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
20 Weeks
YFP
F4/80
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
KitMerCreMer/R26
Tamoxifen
13.8
12.3
1.9
7.8
11.3
10.9
10.7
11.8
5.8
4.8
5.2
5.3
4.1
1.3
5.7
7.6
14.3
22.9
3.3
6.5
6.5
7.1
8.4
14.9
12.8
13.5
13
5.5
0.8
0.4
0.3
4.5
24.9
21.9
12.6
11.7
2.5
12.9
13.6
13.7
13
14.1
a
b
c
Tracing of BM-precursors
Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834- NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
20 Weeks
Analysis of YFP+ cells
Neutrophils
102
102
103
103 104 105
–103
104
105
0
0
103
103 104 105
–103
104
105
0
0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
103 104 105
–103
104
105
0
0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
103 104 105
–103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
0
0
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
103 104 105
–1030
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103 104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103
104 105
–103 0
103 104 105
–103 0
103
104
105
0
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103
104
105
0
–103
103 104 105
–103 0
103
104
105
0
–103
103
103 104 105
–103
104
105
0
0
102
103
103 104 105
–103
104
105
0
103
104
105
0
0
102
103
103
104 105
–103
104
105
0
0
103 104 105
–103 0
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
5 Months
CD11b
YFP
YFP
F4/80
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
KitMerCreMer/R26
Tamoxifen
13.8
12.3
1.9
7.8
11.3
10.9
10.7
11.8
5.8
4.8
5.2
5.3
4.1
1.3
5.7
7.6
14.3
22.9
3.3
6.5
6.5
7.1
8.4
14.9
12.8
13.5
13
5.5
0.8
0.4
0.3
4.5
24.9
21.9
12.6
11.7
2.5
12.9
13.6
13.7
13
14.1
a
b
Tracing of BM-precursors a Neutrophils
102
102
103
103 104 105
–103
104
105
0
0
103 104 105
–103 0
103
104
105
0
–103
103
103 104 105
–103
104
105
0
0
102
103
103 104 105
–103
104
105
0
103
104
105
0
0
102
103
103
104 105
–103
104
105
0
0
103 104 105
–103 0
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
5 Months
CD11b
YFP
F4/80
13.8
12.3
5.7
7.6
14.3
22.9
b b c YFP+ after normalization (%)
100
80
60
40
20
0
Neutrophils
MHCIIlow
MHCIIhigh
P1
P2
P3
Eosinophils
1 Week
2 Weeks
3 Weeks
4 Weeks
7 Weeks
20 Weeks
c Neutrophils Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 4 Intestinal tissue-resident macrophages exhibit a slower, gradual replacement by bone marrow-derived cells compared to other gut myeloid cells. Schematic representation of the adult fate mapping protocol using KitMerCreMer/R26 mice. Mice aged 6 weeks were injected with tamoxifen five times an
groups of 4–8 animals were sacrificed 1, 2, 3, 4, 7 and 20 weeks later. b Representative flow cytometry analysis indicating the labelling efficiency of distinc
colon myeloid cell populations as defined in Fig. 1: MHCIIlow-expressing and MHCIIhi-expressing tissue-resident macrophages, monocytes and monocytes
derived macrophages (P1–P3), eosinophils and neutrophils. Neutrophils acted as internal controls for labelling efficiency and the tracings are from the sam
mouse. Gating strategy is shown in Supplementary Fig. 4. c The bar chart represents the mean percentage of yellow florescence protein-positive (YFP+
cells after normalization to the percentage of YFP+ neutrophils. The error bars represent the s.e.m. | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 5 Identification of self-renewing colon macrophage subsets in healthy and tumour tissue. a Dextran sodium sulphate (DSS)-induced intestinal
adenoma formation was combined with adult fate mapping using the KitMerCreMer/R26 and KitMerCreMer/R26ApcMin/+ mice (schematic representation
shown in upper panel). Representative flow cytometry analysis illustrates the colon myeloid cell subset labelling efficiency. Gating strategy is shown in
Supplementary Fig. 4. The bar chart represents the mean percentage of yellow fluorescence protein-positive (YFP+) cells after normalization to the
percentage of YFP+ neutrophils±s.e.m. A total of 15 KitMerCreMer/R26ApcMin/+ and 20 KitMerCreMer/R26 6–8-week-old mice were subjected to DSS
treatment. Statistical significance was determined by two-way ANOVA followed by Bonferroni test; ***P < 0.001. For reasons of clarity, the non-significant
differences between groups were not indicated. b Embryonic fate mapping confirms the prenatal origins of intestinal LP and tumoural tissue-resident F4/
80hiMHCIIhi and F4/80hiMHCIIlow macrophages. Pregnant KitMerCreMer/R26 and KitMerCreMer/R26ApcMin/+ mice (E10.5) were administered tamoxifen by
intraperitoneal injection, as schematically illustrated in the upper panel. The offspring were then subjected to DSS treatment at 8 weeks old and
sacrificed 4 weeks later for tissue and cell isolation. Representative flow cytometry analysis shows the labelling efficiency of each myeloid cell subset
analyzed. Gating strategy is shown in Supplementary Fig. 4. The bar chart represents the mean percentage of YFP+ cells after normalization to the
percentage of YFP+ neutrophils. The error bars represent the s.e.m. A total of 4 KitMerCreMer/R26ApcMin/+ and 6 KitMerCreMer/R26 mice were analysed. E,
embryonic day; P1, monocytes; P2 and P3, monocyte-derived macrophages. Statistical significance was determined by two-way ANOVA followed by
Bonferroni test. For reasons of clarity, the non-significant differences between groups were not indicated Of note, although hierarchical clustering between distinct colon
myeloid cells clearly identified F4/80hiMHCIIhi and F4/80hiMH-
CIIlow as demarcated populations, “high/low” pairs always formed
more closely related couples in tumours or in the LP than “low/
low” or “high/high” comparisons between samples. Although
these data suggested a close relationship between F4/80hiMHCIIhi
and F4/80hiMHCIIlow cells (Fig. 6g), ingenuity pathway analysis
(IPA®) revealed that multiple pathways involved in glycolysis,
gluconeogenesis, urea cycle, hypoxia-inducible factor-1 and
colorectal cancer metastasis signalling were significantly upregu-
lated in F4/80hiMHCIIlow cells (P < 0.05 by right-tailed Fisher’s
exact test, Supplementary Fig. 3, red arrows). Clearly, tumour-
resident F4/80hiMHCIIlow cells not only accumulate, but also undergo a robust metabolic reprogramming during tumour
progression. CSF1 maintains and expands pro-tumoural resident macro-
phages. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 DSS
DSS
Tumour formation
Tracing of BM-precursors
Tamoxifen
Tamoxifen
KitMerCreMer/R26
ApcMin/+
1 Week
4 Weeks
10 Weeks
1 week
4 Weeks
8 Weeks old
6–7 Weeks
Analysis of
YFP+ cells
Neutrophils
P1
CD45
Lamina propria
YFP
P2
P3
Eosinophils
2
7
10
10
10
10
11
MHCIIhigh
MHCIIlow
Neutrophils
P1
CD45
100
***
***
***
80
60
YFP+ after normalization (%)
40
20
0
Tumour
YFP
P2
P3
Eosinophils
4
7
15
14
15
14
15
MHCIIhigh
MHCIIlow
Neutrophils
P1
P2
P3
Eosinophils
MHCIIhigh
MHCIIlow
120
LP
Tumour
100
80
60
YFP+ after normalization (%)
40
20
0
Neutrophils
P1
P2
P3
Eosinophils
MHCIIhigh
MHCIIlow
Neutrophils
P1
CD45
YFP
P2
P3
Eosinophils
65
69
65
63
69
64
64
MHCIIhigh
MHCIIlow
Neutrophils
P1
CD45
YFP
P2
P3
Eosinophils
60
62
61
62
62
61
62
MHCIIhigh
MHCIIlow
Analysis of
YFP+ cells
E10.5
KitMerCreMer/R26
ApcMin/+
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103 0
105
105
104
104
103
103
–103
0
0
–103
105
105
104
104
103
103
–103
0
0
–103
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
–103
–103
105
105
104
104
103
103
0
0
–103
105
105
104
104
103
103
0
0
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
a
b
Tumour formation
Tracing of BM-precursors
g. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 For reasons of clarity, the non-significant differences between groups were not indicated DSS
Tumour formation
Tamoxifen
KitMerCreMer/R26
ApcMin/+
1 Week
4 Weeks
6–7 Weeks
Analysis of
YFP+ cells
Neutrophils
P1
CD45
Lamina propria
YFP
P2
P3
Eosinophils
2
7
10
10
10
10
11
MHCIIhigh
MHCIIlow
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103 0
a
Tracing of BM-precursors DSS
Tracing of BM-precursors
Tamoxifen
10 Weeks
1 week
4 Weeks
8 Weeks old
Neutrophils
P1
CD45
YFP
P2
P3
Eosinophils
65
69
65
63
69
64
64
MHCIIhigh
MHCIIlow
Analysis of
YFP+ cells
E10.5
KitMerCreMer/R26
ApcMin/+
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
b
Tumour formation b a Neutrophils
P1
CD45
Tumour
YFP
P2
P3
Eosinophils
4
7
15
14
15
14
15
MHCIIhigh
MHCIIlow
–103
105
105
104
104
103
103
–103
0
0
–103
105
105
104
104
103
103
–103
0
0
–103
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0
105
105
104
104
103
103
–103
–103
0
0 Neutrophils
P1
CD45
YFP
P2
P3
Eosinophils
60
62
61
62
62
61
62
MHCIIhigh
MHCIIlow
105
105
104
104
103
103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
105
105
104
104
103
103
–103
0
0
–103
105
105
104
104
103
103
0
0
–103
105
105
104
104
103
103
0
0
–103 100
***
***
***
80
60
YFP+ after normalization (%)
40
20
0
YFP
Neutrophils
P1
P2
P3
Eosinophils
MHCIIhigh
MHCIIlow
120
LP
Tumour
100
80
60
YFP+ after normalization (%)
40
20
0
Neutrophils
P1
P2
P3
Eosinophils
MHCIIhigh
MHCIIlow
YFP 100
***
***
***
80
60
YFP+ after normalization (%)
40
20
0
Neutrophils
P1
P2
P3
Eosinophils
MHCIIhigh
MHCIIlow
LP
Tumour Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 5 Identification of self-renewing colon macrophage subsets in healthy and tumour tissue. a Dextran sodium sulphate (DSS)-induced intestinal
enoma formation was combined with adult fate mapping using the KitMerCreMer/R26 and KitMerCreMer/R26ApcMin/+ mice (schematic representation
own in upper panel). Representative flow cytometry analysis illustrates the colon myeloid cell subset labelling efficiency. Gating strategy is shown
pplementary Fig. 4. The bar chart represents the mean percentage of yellow fluorescence protein-positive (YFP+) cells after normalization to the
rcentage of YFP+ neutrophils±s.e.m. A total of 15 KitMerCreMer/R26ApcMin/+ and 20 KitMerCreMer/R26 6–8-week-old mice were subjected to DSS
atment. Statistical significance was determined by two-way ANOVA followed by Bonferroni test; ***P < 0.001. For reasons of clarity, the non-signific
ferences between groups were not indicated. b Embryonic fate mapping confirms the prenatal origins of intestinal LP and tumoural tissue-resident F
hiMHCIIhi and F4/80hiMHCIIlow macrophages. Pregnant KitMerCreMer/R26 and KitMerCreMer/R26ApcMin/+ mice (E10.5) were administered tamoxifen
raperitoneal injection, as schematically illustrated in the upper panel. The offspring were then subjected to DSS treatment at 8 weeks old and
crificed 4 weeks later for tissue and cell isolation. Representative flow cytometry analysis shows the labelling efficiency of each myeloid cell subse
alyzed. Gating strategy is shown in Supplementary Fig. 4. The bar chart represents the mean percentage of YFP+ cells after normalization to the
rcentage of YFP+ neutrophils. The error bars represent the s.e.m. A total of 4 KitMerCreMer/R26ApcMin/+ and 6 KitMerCreMer/R26 mice were analysed
bryonic day; P1, monocytes; P2 and P3, monocyte-derived macrophages. Statistical significance was determined by two-way ANOVA followed by
nferroni test. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 4 Intestinal tissue-resident macrophages exhibit a slower, gradual replacement by bone marrow-derived cells compared to other gut myeloid cells. a
Schematic representation of the adult fate mapping protocol using KitMerCreMer/R26 mice. Mice aged 6 weeks were injected with tamoxifen five times and
groups of 4–8 animals were sacrificed 1, 2, 3, 4, 7 and 20 weeks later. b Representative flow cytometry analysis indicating the labelling efficiency of distinct
colon myeloid cell populations as defined in Fig. 1: MHCIIlow-expressing and MHCIIhi-expressing tissue-resident macrophages, monocytes and monocytes-
derived macrophages (P1–P3), eosinophils and neutrophils. Neutrophils acted as internal controls for labelling efficiency and the tracings are from the same
mouse. Gating strategy is shown in Supplementary Fig. 4. c The bar chart represents the mean percentage of yellow florescence protein-positive (YFP+)
cells after normalization to the percentage of YFP+ neutrophils. The error bars represent the s.e.m. Tumour microenvironment alters resident macrophage phe-
notype. Besides the upregulation of genes involved in cell pro-
liferation, macrophages in polyps clearly adopted a distinct
metabolic signature. Similar to cancer cells, which can alter their
metabolism in aerobic glycolysis and production of lactate (so-
called Warburg effect) macrophages in polyps increased the
transcript levels of key glycolytic genes, such as ENO1, GAPDH,
TPI1, PGAM and LDHA, with the F4/80hiMHCIIlow macro-
phages expressing the highest levels (Fig. 6c). In addition, our
RNA-seq found that both tissue-resident subsets displayed
enhanced transcripts of ARG1, typical for M2-polarized and tumour-associated macrophages (TAMs)26,27, with MHCIIlow
macrophages being the major producers of ARG1 within tumours
(Fig. 6c–e). The expression of other urea cell cycle genes, such as
Arginase-2 (ARG2) and Arginosuccinate synthetase 1 (AAS1),
was increased in tumour-resident macrophages as well (Fig. 6c). Moreover, several transcripts of matrix metalloproteases (MMPs),
such as MMP2, MMP9 and MMP12, involved in the degradation
of extracellular matrix and promotion of metastasis were upre-
gulated in both subpopulations of tissue-resident macrophages
when compared to their LP counterparts (Fig. 6f), suggesting a
possible contribution of these cells in tumour progression. NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 6 6 ARTICLE | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications Discussion
h The macrophages found resident in the majority of tissues are
established during fetal development and are able to locally self-
maintain in adulthood without any further input from BM hae-
matopoietic progenitors4. In tissues exposed to a low-grade
inflammation, however, such as the intestine and skin, or to
mechanical stress, such as the heart, the maintenance of these
cells relies not on self-renewal, but on a continuous replenish-
ment by infiltrating BM-derived CCR2-dependent Ly6Chi circu-
lating monocytes12,14,17. To evaluate the pro-tumoural potential of different types of
tumour-resident macrophages, polyp numbers and their sizes
were assessed in Ccr2−/−ApcMin/+ and anti-CSF1R injected
ApcMin/+ mice. Clearly, CCR2 deficiency, although strongly
reduced monocytes and monocyte-derived macrophage P2 and
P3 populations, had no effect on colonic polyp numbers in Ccr2
−/−ApcMin/+ mice compared to their ApcMin/+ littermates in the
DSS-accelerated tumour model (Fig. 7c, upper panel). In contrast,
depletion of tumour-resident F4/80hi macrophages lowered
significantly the polyp counts in the colon (P < 0.05 by unpaired g
y
Here we confirmed that in adult mice the vast majority of
colon LP macrophages are derived from CCR2-dependent BM
progenies. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 CSF1 is a crucial growth factor for macrophage pro-
liferation and survival, and supports the self-maintenance of
tissue-resident macrophages26–29. Here, we compared the levels
of CSF1 in the colon LP and colon adenomas and detected a
progressive increase in the amounts of CSF1 as a function of
polyp size (Fig. 7a). In addition, when the effects of CSF1 were
neutralized upon exposure to an anti-CSF1 receptor neutralizing
antibody in tumour-bearing ApcMin/+ mice, we observed a strong | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:582 7 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 reduction in the number of both F4/80hiMHCIIlow and F4/
80hiMHCIIhi tumour-resident macrophages, whereas the num-
bers of monocytes and monocyte-derived macrophages were
unperturbed (Fig. 7b). All together, these data strongly suggest
that F4/80hi-resident macrophages exposed to high levels of CSF1
(and likely other tumour niche factors) in the tumour micro-
environment gain the ability to self-renew within the tumour. Student's t-test) and the tumours formed were of smaller size
(P < 0.001 by unpaired Student's t-test) suggesting their pro-
tumoural role in established tumours (Fig. 7c, lower panel). Discussion
h resident macrophages (fraction I) in addition to a classical
monocyte-derived series of F4/80intCD11b+ cells (fraction II). We
found one exception to this rule, whereby LP-resident F4/
80hiMHCIIlow macrophages that could be detected in high
numbers only in young mice were also clearly found in adult Ccr2
−/−mice. Interestingly, our fate mapping experiments showed
that LP-resident F4/80hi macrophages exhibited a very slow
turnover rate compared to other myeloid populations in the LP. In particular, F4/80hiMHCIIlow macrophages showed minimal
labelling after adult mouse tamoxifen administration, which
suggests that they are a fetal-derived population that gradually
disappear with age. This phenomenon is perhaps expected due to
increased competition to occupy the niche by monocyte-derived
cells, which can then efficiently differentiate into F4/80hiMHCIIhi
macrophages17. p
g
The lymphoid and myeloid composition of colon adenomas of
ApcMin/+ mice differed from that of the colon LP. In the LP, we
found that lymphoid cells (mainly B cells) were the predominant
cell type, whereas in adenomas the majority consisted of myeloid
cells and were almost devoid of lymphocytes and, notably, eosi-
nophils that were the most common LP myeloid cells. Neutrophils
and monocytes were strongly increased in adenomas, but interest-
ingly also F4/80hiMHCIIlow macrophages. These MHCIIlow cells are
usually a minor population in LP, but clearly “flourished” in the
tumour environment in our model to such an extent that in large
polyps they became the dominant population among F4/80hi cells. Our fate mapping studies and analyses of Ccr2−/−mice showed that
MHCIIlow macrophages expanded in situ in adenomas by self-
renewal, without any recruitment of new cells. This finding was also
supported by our RNA-seq and Ki-67 analyses showing that
MHCIIlow macrophages in polyps were actively cycling, unlike
macrophages found in the colon LP. Similarly, the tumour micro-
environment was also conducive for the self-renewal of F4/
80hiMHCIIhi macrophages. This subpopulation of macrophages
was also actively cycling and had become independent from CCR2-
dependent inputs from the BM. The tumour milieu, however, did
not favour monocyte differentiation to macrophages. In adenomas,
monocytes (P1) got “stuck” at the beginning of the “waterfall”
maturation pathway to macrophages (P3), which resulted in the F4/
80hi cells becoming the dominant macrophage population. This
effect has also been reported in the context of mammary cancers13. Our data clearly suggest that unique characteristics of the tumour
niche can trigger self-renewal of F4/80hi macrophages. Discussion
h CSF1 is
certainly a key component of the tumour niche to manipulate
macrophage self-renewal, but other cues can also contribute to this
process and, therefore, reconstruction of the tumour niche would be
very illuminating. −/−mice. Interestingly, our fate mapping experiments showed
that LP-resident F4/80hi macrophages exhibited a very slow
turnover rate compared to other myeloid populations in the LP. In particular, F4/80hiMHCIIlow macrophages showed minimal
labelling after adult mouse tamoxifen administration, which
suggests that they are a fetal-derived population that gradually
disappear with age. This phenomenon is perhaps expected due to
increased competition to occupy the niche by monocyte-derived
cells, which can then efficiently differentiate into F4/80hiMHCIIhi
macrophages17. Similarly to the gut, an analogous tissue-resident macrophage
subpopulation that expresses low levels of MHCII was recently
characterized in the skin dermis—another tissue exposed to
commensal microbes and under tonic, low-level inflammation30. Consistent with the colon tissue-resident macrophages, the der-
mal F4/80hiMHCIIhi fraction is clearly BM derived, whereas the
recruitment of the F4/80hiMHCIIlow subset in the dermis occurs
mainly during embryogenesis and rapidly declines
during
adulthood5,12. To date, the developmental origins of intestinal TAMs and
their maintenance in colon adenomas have not been extensively
studied. As macrophages have become potential targets for cancer
therapy31, we therefore decided to study the dynamics of myeloid
populations in colon adenomas. In breast cancer13, glioma32 and
lung metastases33 TAMs can arise from Ly6Chi circulating
monocytes. Their influx and positioning in the tumour is medi-
ated by chemokines (e.g., CCL2), growth factors (e.g., CSF1) and
complement
components31,34. In
addition,
a
recent
study
demonstrated that local proliferation of TAMs contributes to
their accumulation in spontaneous mammary tumours24. Many
have speculated that in cancers exposed to microbes, such as
colon cancer, the recruitment of macrophages and their main-
tenance might differ from cancers located in “sterile” sites, such as
breast cancers31. According to the “M1/M2 paradigm”, mainly based on in vitro
polarization
experiments,
pro-inflammatory
M1macrophages
display anti-tumoural characteristics, whereas alternatively acti-
vated M2-macrophages are generally considered pro-tumoural. Both populations can co-exist in the tumour, although M2-like
MHCIIlow TAMs are claimed to enrich in hypoxic and M1-like
MHCIIhi TAMs in normoxic areas22,36. Due to the complex
environment of the gut, intestinal tissue-resident macrophages do Here, we have analysed in detail the origins and maintenance
of macrophage subpopulations in colon tumours obtained from
ApcMin/+ mice, which is a widely used animal model for intestinal Fig. Discussion
h These macrophages included F4/80hiMHCIIhi LP- p
g
p
g
LP neutrophils 1
LP neutrophils 2
LP neutrophils 3
Tumour neutrophils 3
LP monocytes 1
LP monocytes 2
LP monocytes 3
Tumour monocytes 3
Tumour F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIhi 2
Tumour F4/80hiMHCIIhi 3
LP F4/80hiMHCIIhi 3
LP F4/80hiMHCIIlo 3
LP F4/80hiMHCIIlo 2
LP F4/80hiMHCIIlo 1
LP F4/80hiMHCIIhi 2
LP F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIlo 1
Tumour F4/80hiMHCIIlo 2
Tumour F4/80hiMHCIIlo 3
Tumour monocytes 2
Tumour monocytes 1
Tumour neutrophils 2
Tumour neutrophils 1
LP
Tumour
LP
Tumour
Sample
MHCIIlow
MHCIIlow
F4/80
MHCIIhigh
MHCIIhi MHCIIlo MHCIIhi MHCIIlo
MHCIIlo
MHCIIhi
MHCIIhi
MHCIIlo
Pdk3
Ganc
Pdha1
Dlat
Pdk2
Arg2
Aldoc
Ass1
Ldhb
H6pd
Arg1
Pdk1
Dcxr
Pgam1
Pgm2
Eno1b
Eno1
Tpi1
Ldha
Pkm
Mdh1
Pgk1
Gapdh
200
200
150
150
100
100
50
50
PC3
0
0
–50
–50
–100
–100
–150
–150
–200 –100
0
100
200
300
PC1
PC2
Nek3
Nek2
Cdc25c
Cdc20
Cks1b
Ccnb2
Cdca3
Cdca71
Prc1
Nek4
Cdk1
E2f2
Rrm1
Mcm5
Mcm2
Mcm3
Mcm7
Mki67
Mcm4
Cdca8
Ccna2
MHCIIhigh
MHCIIlow
MHCIIhigh
Ki67
6
1
27
23
30
20
10
ns
Low High Low
25
5000
Arg1
ns
ns
Low High
Low
LP
Tumour
High
Low High
Low
LP
0.05
0.15
0.20
0.25
0.30
0.35
0.10
Tumour
High
***
**
4000
Mean fluorescence
intensity (MFI) of Arg1
3000
Tumour neutrophils
Tumour monocytes
Tumour F4/80hiMHCIIlo
Tumour F4/80hiMHCIIhi
LP F4/80hiMHCIIhi
LP F4/80hiMHCIIlo
LP monocytes
LP neutrophils
2000
1000
0
20
15
10
Relative expression
(fold change)
5
0
High
ns
***
**
% Of Ki67+
0
LP
Tumour
LP
0.0
2.0
–2.0
Tumour
Arg1
***
***
***
***
Low High
Low
LP
LP
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
0
104
105
0
104
105
0
104
105
0
0
104
104
105
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
Tumour
Tumour
4
***
***
***
***
*
***
*
ns
ns
ns
ns
ns
MMP2
MMP9
MMP12
3
2
1
0
Relative expression (fold-change)
20
15
10
5
0
8
6
4
2
0
High
a
b
c
d
e
f
g
NATURE COMMUNICATIONS| (2018) 9:582
| DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommu MHCIIhi MHCIIlo MHCIIhi MHCIIlo
Nek3
Nek2
Cdc25c
Cdc20
Cks1b
Ccnb2
Cdca3
Cdca71
Prc1
Nek4
Cdk1
E2f2
Rrm1
Mcm5
Mcm2
Mcm3
Mcm7
Mki67
Mcm4
Cdca8
Ccna2
LP
0.0
2.0
–2.0
Tumour
b LP
Tumour
MHCIIlow
MHCIIlow
F4/80
MHCIIhigh
MHCIIhigh
Ki67
6
1
27
23
30
20
10
ns
Low High Low High
ns
***
**
% Of Ki67+
0
LP
Tumour
0
104
105
0
104
105
0
104
105
0
0
104
104
105
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
a b 30
20
10
ns
Low High Low High
ns
***
**
% Of Ki67+
0
LP
Tumour LP
Tumour
MHCIIlow
MHCIIlow
F4/80
MHCIIhigh
MHCIIhigh
Ki67
6
1
27
23
0
104
105
0
104
105
0
104
105
0
0
104
104
105
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
0
104
105
103
–103
a a Nek4
Cdk1
E2f2
Rrm1
Mcm5
Mcm2
Mcm3
Mcm7
Mki67
Mcm4
Cdca8
MHCIIlow
MHCIIhigh
25
5000
Arg1
ns
ns
Low High
Low
LP
Tumour
High
Low High
Low
LP
Tumour
High
***
**
4000
Mean fluorescence
intensity (MFI) of Arg1
3000
2000
1000
0
20
15
10
Relative expression
(fold change)
5
0
Arg1
***
***
***
***
Low High
Low
LP
LP
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
Tumour
Tumour
4
***
***
***
***
*
***
*
ns
ns
ns
ns
ns
MMP2
MMP9
MMP12
3
2
1
0
Relative expression (fold-change)
20
15
10
5
0
8
6
4
2
0
High
d
e
f LP
Tumour
Sample
MHCIIlo
MHCIIhi
MHCIIhi
MHCIIlo
Pdk3
Ganc
Pdha1
Dlat
Pdk2
Arg2
Aldoc
Ass1
Ldhb
H6pd
Arg1
Pdk1
Dcxr
Pgam1
Pgm2
Eno1b
Eno1
Tpi1
Ldha
Pkm
Mdh1
Pgk1
Gapdh
c
g MHCIIlow
MHCIIhigh
25
5000
Arg1
ns
ns
Low High
Low
LP
Tumour
High
***
**
4000
Mean fluorescence
intensity (MFI) of Arg1
3000
2000
1000
0
20
15
10
Relative expression
(fold change)
5
0
Arg1
***
***
***
***
Low High
Low
LP
LP
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
102
0
103
104
105
Tumour
Tumour
High
d
e c c d f f Low High
Low
LP
Tumour
High
4
***
***
***
***
*
***
*
ns
ns
ns
ns
ns
MMP2
MMP9
MMP12
3
2
1
0
Relative expression (fold-change)
20
15
10
5
0
8
6
4
2
0
f e Relative expression (fold-change) g LP neutrophils 1
LP neutrophils 2
LP neutrophils 3
Tumour neutrophils 3
LP monocytes 1
LP monocytes 2
LP monocytes 3
Tumour monocytes 3
Tumour F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIhi 2
Tumour F4/80hiMHCIIhi 3
LP F4/80hiMHCIIhi 3
LP F4/80hiMHCIIlo 2
LP F4/80hiMHCIIlo 1
LP F4/80hiMHCIIhi 2
LP F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIlo 1
Tumour F4/80hiMHCIIlo 2
Tumour F4/80hiMHCIIlo 3
Tumour monocytes 2
Tumour monocytes 1
Tumour neutrophils 2
Tumour neutrophils 1
200
200
150
150
100
100
50
50
PC3
0
0
–50
–50
–100
–100
–150
–150
–200 –100
0
100
200
300
PC1
PC2
0.05
0.15
0.20
0.25
0.30
0.35
0.10
Tumour neutrophils
Tumour monocytes
Tumour F4/80hiMHCIIlo
Tumour F4/80hiMHCIIhi
LP F4/80hiMHCIIhi
LP F4/80hiMHCIIlo
LP monocytes
LP neutrophils
g 0.05
0.15
0.20
0.25
0.30
0.35
0.10 LP neutrophils 1
LP neutrophils 2
LP neutrophils 3
Tumour neutrophils 3
LP monocytes 1
LP monocytes 2
LP monocytes 3
Tumour monocytes 3
Tumour F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIhi 2
Tumour F4/80hiMHCIIhi 3
LP F4/80hiMHCIIhi 3
LP F4/80hiMHCIIlo 3
LP F4/80hiMHCIIlo 2
LP F4/80hiMHCIIlo 1
LP F4/80hiMHCIIhi 2
LP F4/80hiMHCIIhi 1
Tumour F4/80hiMHCIIlo 1
Tumour F4/80hiMHCIIlo 2
Tumour F4/80hiMHCIIlo 3
Tumour monocytes 2
Tumour monocytes 1
Tumour neutrophils 2
Tumour neutrophils 1
0.05
0.15
0.20
0.25
0.30
0.35
0.10 200
200
150
150
100
100
50
50
PC3
0
0
–50
–50
–100
–100
–150
–150
–200 –100
0
100
200
300
PC1
PC2 NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 tumourigenesis35, and focused on both subsets of tissue-resident
F4/80hi macrophages in the colon LP. | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications Discussion
h 6 Tissue-resident macrophages in polyps expand by self-renewal and undergo metabolic reprogramming. a Proliferative activity of the LP and tumoural
tissue-resident macrophage subpopulations. Intracellular expression levels of Ki-67 in adult F4/80hiMHCIIhi and F4/80hiMHCIIlow cells, obtained from the
colon LP of wild-type mice and colon tumours from dextran sodium sulphate-treated ApcMin/+ mice. Representative dot plot and bar chart representing
data obtain for colon LP (n = 5 mice) and tumours (n = 10 mice). Error bars represent the s.e.m. Statistical significance was determined by two-way
ANOVA followed by Bonferroni test; **P < 0.01; ***P < 0.001; ns, not significant. b, c Myeloid subpopulations were sorted from pooled colon LP and
tumours of 13-15 mice in three independent experiments in an RNA-seq study: Ly6ChiMHCII−monocytes, Ly6GhiF4/80−CD11b+ neutrophils, F4/
80hiMHCIIhi and F4/80hiMHCIIlow tissue-resident macrophage subpopulations. Gating strategy is shown in Supplementary Fig. 4. b Heat map and
clustering of cell cycle-associated transcripts enriched in intratumoural F4/80hiMHCIIhi and F4/80hiMHCIIlow subsets. The heat map was generated with
log2 transformed RPKM values and with the row/gene median subtracted. c Heat map and clustering of glycolysis- and urea cycle-associated transcripts
enriched in intratumoural F4/80hiMHCIIhi and F4/80hiMHCIIlow subsets. The heat map was generated with log2 transformed FPKM values and with the
row/gene median subtracted. d Quantitative PCR of Arg-1 expression levels in the LP and intratumoural F4/80hiMHCIIhi and F4/80hiMHCIIlow
subpopulations. The qPCR results shown are representative of sorted macrophage subpopulations obtained from pooled LP and tumours of 13-15 mice. Error bars represent the s.e.m. Statistical significance was determined by two-way ANOVA followed by Bonferroni test; ***P<0.001 e Representative flow
cytometry histograms (upper panels) and bar charts (lower panel) showing the intracellular mean of fluorescence intensity (MFI) of ARG1 expression in
colon LP and intratumoural F4/80hiMHCIIhi and F4/80hiMHCIIlow cells. Error bars represent the s.e.m. Statistical significance was determined by two-way
ANOVA followed by Bonferroni test; **P<0.01; ***P < 0.001; ns, not significant. f Differential expression of distinct metalloproteinases (Mmp2, Mmp9 and
Mmp12) in LP and intratumoural F4/80hiMHCIIhi and F4/80hiMHCIIlow subsets measured by qPCR. Error bars represent the s.e.m. Statistical significance
was determined by two-way ANOVA followed by Bonferroni test; *P<0.05; ***P < 0.001; ns, not significant. g Transcriptome analysis of distinct colon LP
and tumoural myeloid cell populations. Discussion
h Hierarchical clustering of the LP and intratumoural monocytes (P1), neutrophils and tissue-resident macrophage
(F4/80hiMHCIIhi and MHCIIlow) subsets | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:582 9 CSF1
8
Relative expression
(fold change)
6
4
2
0
LP
0.5 mm
1–1.5 mm
2 mm
3–4 mm
5–6 mm
d–4
DSS
ApcMin/+
Tumour formation
Anti-CSF1R
Ab or
isotype Ab
4 Weeks
Analysis of colon tumours
1 Week
d–1
ApcMin/+
+ isotype Ab
F4/80
105
105
104
104
103
103
0
0
105
104
103
0
105
105
104
104
103
102
103
0
105
104
103
102
0
0
105
104
103
0
105
105
104
104
103
102
103
0
105
104
103
102
0
0
105
104
103
0
105
104
103
0
ApcMin/+
+ anti-CSF1R Ab
CD11b
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
MHCII
Ly6C
Fraction I
Fraction II
Eosinophils
9
I
II
I
II
18
50
26
72
46
20
5
19
31
14
84
18
0.5
46
22
28
8
P1
P2
P3
***
***
***
12,000
9000
6000
3000
3000
2000
1000
0
3000
2000
1000
0
0
9000
6000
3000
0
9000
20,000
ns
ns
ns
15,000
10,000
5000
40
Polyps count
Polyps count
4 Weeks
1 Week
DSS
ApcMin/+
ApcMin/+
Ccr2–/–ApcMin/+
Tumour formation
4 Weeks
2 weeks
Anti-CSF1R Ab
or isotype Ab
1 Week
DSS
Tumour formation
30
20
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
10
0
40
30
20
10
0
40
12
9
6
3
0
30
20
10
0
30
15
10
5
0
20
10
0
0
6000
3000
0
Absolute no. Absolute no. MHCIIlow
MHCIIhigh
Total polyps
Total polyps
*
Polyps < 2 mm
Polyps < 2 mm
Polyps > 2 mm
Polyps > 2 mm
**
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ + isotype Ab
ApcMin/+ + anti-CSF1R Ab
a
b
c
Fig. 7 Pro-tumoural F4/80hi resident macrophages depend on CSF1. a Intra-tumoural CSF1 levels gradually increase with tumour progression. Colon
polyps of different sizes (ranging from 0.5 mm to 6 mm in diameter) were processed for quantitative PCR analysis of Csf1 expression. Data were norm
to β-actin and are expressed as fold change in Csf1 expression from 0.5 mm tumours. Discussion
h MHCIIlow
MHCIIhigh
ApcMin/+ + isotype Ab
ApcMin/+ + anti-CSF1R Ab
b b Eosinophils
P1
P2
P3
***
***
***
12,000
9000
6000
3000
3000
2000
1000
0
3000
2000
1000
0
0
9000
6000
3000
0
9000
20,000
ns
ns
ns
15,000
10,000
5000
0
6000
3000
0
Absolute no. Absolute no. MHCIIlow
MHCIIhigh
ApcMin/+ + isotype Ab
ApcMin/+ + anti-CSF1R Ab P1
P2
P3
3000
2000
1000
0
9000
20,000
ns
ns
ns
15,000
10,000
5000
0
6000
3000
0
Absolute no. ApcMin/+ + isotype Ab
ApcMin/+ + anti-CSF1R Ab CD11b
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
40
Polyps count
Polyps count
4 Weeks
1 Week
DSS
ApcMin/+
ApcMin/+
Ccr2–/–ApcMin/+
Tumour formation
4 Weeks
2 weeks
Anti-CSF1R Ab
or isotype Ab
1 Week
DSS
Tumour formation
30
20
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
Polyps count/colon
10
0
40
30
20
10
0
40
12
9
6
3
0
30
20
10
0
30
15
10
5
0
20
10
0
Total polyps
Total polyps
*
Polyps < 2 mm
Polyps < 2 mm
Polyps > 2 mm
Polyps > 2 mm
**
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
c ApcMin/+
Ccr2–/–
ApcMin/+
ApcMin/+
Ccr2–/–
ApcMin/+
40
yps count
30
20
Polyps count/colon
Polyps count/colon
10
0
30
20
10
0
Total polyps
Polyps < 2 mm ApcMin/+
Ccr2–/–
ApcMin/+
Polyps count/colon
30
20
10
0
Polyps < 2 mm ApcMin/+
Ccr2–/–
ApcMin/+
Polyps count/colon
15
10
5
0
Polyps > 2 mm c c Apc
Apc
Polyps count/colon
Polyps count/colon
40
30
20
10
0
40
30
20
10
0
Total polyps
*
Polyps < 2 mm
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab p
Polyps count/colon
40
30
20
10
0
Polyps < 2 mm
b
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab Polyps count/colon
12
9
6
3
0
Polyps > 2 mm
**
ApcMin/+ +
isotype Ab
ApcMin/+ +
anti-CSF1R Ab Polyps > 2 mm Fig. 7 Pro-tumoural F4/80hi resident macrophages depend on CSF1. a Intra-tumoural CSF1 levels gradually increase with tumour progression. Colon LP and
polyps of different sizes (ranging from 0.5 mm to 6 mm in diameter) were processed for quantitative PCR analysis of Csf1 expression. Discussion
h b Anti-CSF1 receptor (CSF1R) antibody depletes intratumou
80hiMHCIIlow and F4/80hiMHCIIhi macrophages. The 12-week-old dextran sodium sulphate (DSS)-treated ApcMin/+ mice were injected intraperito
with rat IGg2a or anti-CSF1R blocking antibody (400 μg/mouse) at days −4 and −1 before collection of the colons, as shown in the schematic
representation (upper panel). Polyps were processed and cells were analysed by flow cytometry for the presence of different myeloid cell popula
defined in Fig. 1. Gating strategy is shown in Supplementary Fig. 4. The bar charts show the means±s.e.m. of absolute numbers of F4/80hiMHCIIhi a
80hiMHCIIlow tissue-resident macrophage subpopulations, monocytes (P1), monocyte-derived macrophages (P2–P3) and eosinophils (control mice
and anti-CSF1R Ab injected mice: n = 5). Statistical significance was determined using an unpaired Student’s t-test. ***P < 0.0001; ns, not signific
CSF1R blockade but not CCR2 deficiency reduces tumour burden in ApcMin/+ mice. Polyp counts in the colon of ApcMin/+ (n = 9) and Ccr2−/−ApcMin/
(n = 9) (upper panel) and anti-rat IgG2a injected ApcMin/+ (n = 11) and anti-CSF1R Ab injected ApcMin/+ mice (n = 9) (lower panel). Statistical signifi
was determined using an unpaired Student’s t-test. *P < 0.05 and **P < 0.001
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 CSF1
8
Relative expression
(fold change)
6
4
2
0
LP
0.5 mm
1–1.5 mm
2 mm
3–4 mm
5–6 mm
d–4 d–1
a a Relative exp
(fold chan
4
2
0
LP
0.5 mm
1–1.5 mm
2 mm
3–4 mm
5–6 mm
d–4
DSS
ApcMin/+
Tumour formation
Anti-CSF1R
Ab or
isotype Ab
4 Weeks
Analysis of colon tumours
1 Week
d–1
ApcMin/+
+ isotype Ab
F4/80
105
105
104
104
103
103
0
0
105
104
103
0
105
105
104
104
103
102
103
0
105
104
103
102
0
0
105
104
103
0
105
105
104
104
103
102
103
0
105
104
103
102
0
0
105
104
103
0
105
104
103
0
ApcMin/+
+ anti-CSF1R Ab
CD11b
MHCII
Ly6C
Fraction I
Fraction II
Eosinophils
9
I
II
I
II
18
50
26
72
46
20
5
19
31
14
84
18
0.5
46
22
28
8
P1
P2
P3
***
***
***
12,000
9000
6000
3000
3000
2000
1000
0
3000
2000
1000
0
0
9000
6000
3000
0
9000
20,000
ns
ns
ns
15,000
10,000
5000
0
6000
3000
0
Absolute no. Absolute no. | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications Methods
M Mice. KitMerCreMer/R26 mice were generated in our laboratory as previously
described5. Ccr2−/−(B6.129S4-Ccr2tm1lfc/J)45, C57BL/6J and ApcMin/+ (C57BL/6J-
ApcMin/J)46 mice were obtained from The Jackson Laboratory (USA). ApcMin/+
mice were backcrossed with KitMerCreMer/R26 mice or Ccr2−/−mice to obtain
ApcMin/+KitMerCreMer/R26 and Ccr2−/−ApcMin/+ mice, respectively. Mice were
specific-pathogen-free (SPF) and maintained under a 12 h light/dark cycle with
standard chow diet provided ad libitum. Both male and female mice (6–8 weeks
old) were used for all experiments and were equally distributed within experi-
mental and control groups. The paired-end reads were mapped to the Mouse GRCm38/mm10 reference
genome using the STAR alignment tool49. Mapped reads were summarized to the
gene level using featureCounts (V1.5.0-P1) software50 and with GENCODE gene
annotation51. Genes with an average number of reads per sample <10 in all cell
subpopulations were filtered out from subsequent analyses. For differentially
expressed gene (DEG) analysis, the limma/voom pipeline was used as
recommended by the MicroArray Quality Control (MAQC) project52 as one of the
best performing RNA-seq data analysis pipelines. Comparisons between different
cell populations were performed using limma and DEGs were selected with
Benjamini–Hochberg adjusted P-values < 0.05. Hierarchical clustering and
principal component analysis were performed with Log2 transformed value of
RPKM (Reads Per Kilobase of transcript per Million mapped reads). All analyses
were carried out in R version 3.1.2 (URL http://www.R-project.org/). All transgenic mice were bred and housed under SPF conditions in the Nanyang
Technological University animal facility. This study was carried out in strict
accordance with the recommendations of the NACLAR (National Advisory
Committee for Laboratory Animal Research) guidelines under the Animal & Birds
(Care and Use of Animals for Scientific Purposes) Rules of Singapore. The protocol
was approved by the Institutional Animal Care and Use Committee of the Nanyang
Technological University of Singapore. Spontaneous and chemical-induced intestinal tumourigenesis. ApcMin/+ mice
spontaneously develop macroscopically detectable adenomas mainly in the small
intestine and only few in the colon in aged mice35. Therefore, for all our experi-
ments (with exception of the experiments shown in Fig. 2 and Supplementary
Fig. 1) we opted for a chemically induced colitis model which selectively enhances
the development of adenomas in the colon in a relatively short time of 5 weeks. Mice were terminated when showing symptoms of anaemia in combination with
weight loss and/or other signs of physical discomfort. h “
”
d l
l
d
h
f Flow cytometry. Methods
M Single-cell suspensions were then stained and subsequently
analysed by a BD Fortessa 5 laser flow cytometer (BD Bioscience). Data were
analysed using a FlowJo software (TreeStar, Ashland, OR, USA). The following
antibodies were used: APC/Cy7-labelled anti-CD11b (clone: M1/70, 1:600) and
BV605-labelled anti-Ly6C (HK1.4, 1:600) were purchased from Biolegend (San
Diego, CA, USA). PE-labelled anti-EMR1 (also known as F4/80) (clone: BM8,
1:600), PE/Cy7-labelled anti-CD45.2 (clone: 104, 1:600), APC-labelled anti-CD11c
(clone: N418, 1:600) and eFluor®450-labelled anti-MHCII (clone:M5/114.15.2,
1:800) were obtained from eBioscience (San Diego, CA, USA). FITC-labelled
polyclonal sheep anti-human/mouse ARG1 (1:20) and APC-labelled anti-CCR2
(Cat. No. FAB5538A) (1:20) were obtained from R&D System (Minneapolis, MN,
USA), FITC-labelled anti-Mouse/Rat Ki-67 (clone SolA15, 1:40) from Miltenyi
Biotech (Bergisch-Gladbach, Germany) and BUV395-labelled anti-Ly6G (clone:
1A8, 1:600) from BD (San Diego, CA, USA) In the “spontaneous” tumour model, mice were analysed at the age of
6–7 months, whereas in case of chemically induced tumourigenesis, 6–8-week-old
female and male ApcMin/+ mice (and correspondent control C57BL/6J mice) were
treated with 1.5 or 1.25% (w/v) DSS (50,000 Da; MP Biomedicals, Santa Ana, CA,
USA), respectively, in the drinking water for 1 week and sacrificed 4 weeks later at
an age of 3–4 months. Colorectal polyp counts. Polyps in each colon were macroscopically counted and
categorized as >2 mm (large) and <2 mm (small). Colon tumours were measured
ex vivo with the help of a sliding caliper. Tamoxifen-inducible fate mapping mouse model. KitMerCreMer/R26 and ApcMin/
+KitMerCreMer/R26 mice were used for cell fate mapping to delineate the ontogeny of
intratumoural macrophages. Then, 80 mg tamoxifen (Sigma, T5648; Sigma-
Aldrich, St. Louis, MO, USA) was dissolved in 6 ml corn oil (C8267; Sigma) and a
total of 4 mg tamoxifen per mouse was administered for five consecutive days by
gavage for adult labelling or by intraperitoneal injection in pregnant mothers
(embryonic day 10.5) for embryonic labelling. After tamoxifen administration, c-
kit+ BM-derived haematopoietic stem cells (HSCs) in ApcMin/+KitMerCreMer/R26
mice were labelled with yellow fluorescent protein (YFP+) and all cells deriving
from YFP+ BM-HSCs would maintain YFP expression. This effect enables to
distinguish whether adult intratumoural macrophages are derived from adult BM-
definitive haematopoiesis or from embryonic haematopoiesis. Adult and fetal
labelling experimental protocols are shown in Fig. 5. Anti-CSF1R antibody treatment. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 Tumours were manually cut, minced and digested in 2% IMDM containing 1
mg/ml Collagenase D and 20 U/ml DNase I (Life Technologies, Carlsbad, CA,
USA) under shaking conditions at 37 °C for 1 h. Digested tissue was subsequently
mashed through a cell strainer to obtain a cell suspension. The leukocyte
population was enriched by 35% Percoll and further processed for flow cytometry
and, if required, cell sorting using a FACSAria cell sorter (BD Biosciences, San Jose,
CA, USA). Gating strategy used for sorting is shown in Supplementary Fig. 4. Macrophages are not only crucial mediators of many pathol-
ogies (ranging from infectious to metabolic diseases and cancers)
but also influence the outcome of anticancer therapeutics, such as
chemotherapy, radiotherapy and even immunotherapy40–42. As
such, these cells are becoming attractive candidates for ther-
apeutic intervention. Manipulation of macrophage numbers43
and/or reprogramming their phenotype44 are techniques cur-
rently used to target macrophages in a therapeutic context. A
better understanding of their ontogeny in different tumours, such
as the data presented here for colon cancer, will facilitate the
development of successful innovative macrophage-based ther-
apeutic approaches. Transcriptomics analysis by RNA-sequencing. Total RNA was extracted using
the ArcturusTM PicoPureTM RNA Isolation Kit (Cat. No. KIT0214, Thermo Fisher
Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. All
mouse RNAs were analysed using an Agilent Bioanalyser (Agilent, Santa Clara, CA,
USA) for quality assessment; the RNA Integrity Number range was 6.5–10, with a
median of 9.2. Complementary DNA (cDNA) libraries were prepared from 2 ng
total RNA starting material and 1 μl of a 1:50,000 dilution of ERCC RNA Spike in
Controls (Cat. No. 4456740, Ambion® Thermo Fisher Scientific) using the
SMARTSeq v2 protocol48 with the following modifications: (1) addition of 20 µM
TSO; and (2) use of 250 pg cDNA with 1/5 reaction of Illumina Nextera XT kit
(Cat. No. FC-131–1024, Illumina, San Diego, CA, USA). The length distribution of
the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on
the Perkin Elmer Labchip (Cat. No. CLS760672, Perkin Elmer, Waltham, MA,
USA). All eight samples were subjected to an indexed paired-end sequencing run of
2 × 51 cycles on an Illumina HiSeq 2500 system (Illumina) under rapid run mode
(17 samples/lane). Discussion
h Data were normalized
to β-actin and are expressed as fold change in Csf1 expression from 0.5 mm tumours. b Anti-CSF1 receptor (CSF1R) antibody depletes intratumoural F4/
80hiMHCIIlow and F4/80hiMHCIIhi macrophages. The 12-week-old dextran sodium sulphate (DSS)-treated ApcMin/+ mice were injected intraperitoneally
with rat IGg2a or anti-CSF1R blocking antibody (400 μg/mouse) at days −4 and −1 before collection of the colons, as shown in the schematic
representation (upper panel). Polyps were processed and cells were analysed by flow cytometry for the presence of different myeloid cell populations
defined in Fig. 1. Gating strategy is shown in Supplementary Fig. 4. The bar charts show the means±s.e.m. of absolute numbers of F4/80hiMHCIIhi and F4/
80hiMHCIIlow tissue-resident macrophage subpopulations, monocytes (P1), monocyte-derived macrophages (P2–P3) and eosinophils (control mice: n = 6
and anti-CSF1R Ab injected mice: n = 5). Statistical significance was determined using an unpaired Student’s t-test. ***P < 0.0001; ns, not significant. c
CSF1R blockade but not CCR2 deficiency reduces tumour burden in ApcMin/+ mice. Polyp counts in the colon of ApcMin/+ (n = 9) and Ccr2−/−ApcMin/+ mice
(n = 9) (upper panel) and anti-rat IgG2a injected ApcMin/+ (n = 11) and anti-CSF1R Ab injected ApcMin/+ mice (n = 9) (lower panel). Statistical significance
was determined using an unpaired Student’s t-test. *P < 0.05 and **P < 0.001 not follow the rigid M1–M2 classification37 and “classic” M1 and
M2 features are often shared by the same cells20,38. Similar mixed
M1/M2 phenotype is observed in macrophages of colon tumours,
since both F4/80hi MHCIIhi and MHCIIlow populations express
the classical M2 marker ARG1, but also many glycolytic genes
typical for M1 macrophages, although the F4/80hiMHCIIlow- not follow the rigid M1–M2 classification37 and “classic” M1 and
M2 features are often shared by the same cells20,38. Similar mixed
M1/M2 phenotype is observed in macrophages of colon tumours,
since both F4/80hi MHCIIhi and MHCIIlow populations express
the classical M2 marker ARG1, but also many glycolytic genes
typical for M1 macrophages, although the F4/80hiMHCIIlow- resident macrophage subset shows always the highest transcript
levels. As observed in other tumour models22,36,39, F4/80hiMH-
CIIlow cells, with their augmented metabolic switch together with
their progressive predominance during tumour development,
represent a pro-tumoural macrophage subpopulation also in
intestinal adenomas. 10 NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications 10 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications
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the manuscript prior to submission. This work was supported by MOE2014-T2-1-011
and MOE2016-T2-1-012 Ministry of Education Tier2 grants to C.R. The authors would like to thank Monika Tetlak for providing excellent mouse man-
agement. The authors would also like to thank Insight Editing London for proofreading
the manuscript prior to submission. This work was supported by MOE2014-T2-1-011
and MOE2016-T2-1-012 Ministry of Education Tier2 grants to C.R. agement. The authors would also like to thank Insight Editing London for proofreading
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therapy. Immunity 41, 49–61 (2014). Methods
M Twelve-week-old DSS-treated ApcMin/+ mice
were injected intraperitoneally with rat Ig2a isotype control (Biolegend) or anti-
CSF1R blocking antibody (Clone AFS98, BioXCell, West Lebanon, NH, USA) (400
μg/mouse) at days −4 and −1 before collection of the colons. Quantitative PCR (qPCR) analysis. cDNA was generated using SuperScript III
Reverse Transcriptase (Cat. No. 18080093, Invitrogen, Carlsbad, CA, USA)
according to the manufacturer’s instructions. Quantitative real-time PCR was then
performed using the FAST 2× qPCR Master mix (PrecisionFAST-SY, Primerdesign
Ltd, Cambridge, UK). Reactions were run on a real-time qPCR system (Illumina,
San Diego, CA, USA). Samples were normalized to β-actin, and data represent the
mean of triplicate analyses and are displayed as a fold change from F4/80hiMHCIIhi
LP macrophages unless otherwise stated. The primer sequences were as follows:
Arg1; Fwd: gaatctgcatgggcaacc, Rev: gaatcctggtacatctgggaac; Mmp2; Fwd: taacctg-
gatgccgtcgt, Rev: ttcaggtaataagcacccttgaa; Mmp9; Fwd: catccagtatctgtatggtcgtg, Rev:
gctgtggttcagttgtggtg; Mmp12; Fwd: gctgctcccatgaatgaca, Rev: aagcattgcacacggttgt;
Csf1; Fwd: ggtggaactgccagtatagaaag, Rev: tcccatatgtctccttccataaa; β-actin Fwd:
aaggccaaccgtgaaaagat, Rev: gtggtacgaccagaggcatac. Myeloid cell isolation. LP myeloid cells were isolated as previously described47. Briefly, after CO2 killing of the mice, the colon was opened and rinsed with
phosphate-buffered saline (PBS) to remove the luminal contents. To remove the
epithelium, the colon was incubated in 25 ml PBS with 1.3 mM EDTA under
shaking conditions at 37 °C for 1 h. After incubation, the colon was washed in 2%
Iscove’s modified Dulbecco’s medium (IMDM) to remove the EDTA solution, and
then minced. The colon pieces were digested in 2% IMDM containing 1 mg/ml
Collagenase D (Roche, Switzerland) under shaking conditions at 37 °C for 1.5 h. The digested tissue was then gently mashed through a 150 μm cell strainer. The
leukocyte population was enriched using a 70/40% Percoll gradient (GE Healthcare
Life Science, Chicago, IL, USA). Low-density cells at the interface were harvested
and processed further for flow cytometry. Statistical analysis. Statistical analysis was performed using GraphPad Prism
6 software (GraphPad Software, La Jolla, CA, USA). All values are expressed as the
mean±s.e.m as indicated in the legend. Samples were analysed by unpaired Stu-
dent’s t-test (two-tailed) or Bonferroni two-way analysis of variance (ANOVA). A
P-value of <0.05 was considered to be statistically significant. 11 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:582 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-02834-8 27. Tagliani, E. et al. Coordinate regulation of tissue macrophage and dendritic
cell population dynamics by CSF-1. J. Exp. Med. 208, 1901–1916 (2011). Data availability. The accession number for the RNA-seq data reported in this
paper is GEO: GSE90153. Original flow cytometry data are deposited in the NTU
Open Access Data Repository DR-NTU (https://researchdata.ntu.edu.sg/dataset. xhtml?persistentId=doi:10.21979/N9/EQXCRF). All other data are available from
the authors upon reasonable request. 28. Hume, D. A., Pavli, P., Donahue, R. E. & Fidler, I. J. The effect of human
recombinant macrophage colony-stimulating factor (CSF-1) on the murine
mononuclear phagocyte system in vivo. J. Immunol. 141, 3405–3409 (1988). p
p
y
p
g
xhtml?persistentId=doi:10.21979/N9/EQXCRF). All other data are available from
the authors upon reasonable request. p
the authors upon reasonable request. 29. Stanley, E. R. & Chitu, V. CSF-1 receptor signaling in myeloid cells. Cold
Spring Harb. Perspect. Biol. 6, a021857 (2014). Received: 11 July 2017 Accepted: 2 January 2018 Received: 11 July 2017 Accepted: 2 January 2018 © The Author(s) 2018 Author contributions 25. Shi, C. & Pamer, E. G. Monocyte recruitment during infection and
inflammation. Nat. Rev. Immunol. 11, 762–774 (2011). Conceptualization: C.R.; methodology: I.S., J.S., S.F., J.L. and F.Z.; investigation: I.S., J.S. and Q.C.; formal analysis: I.S.; bioinformatic analysis: K.D. and M.P.; writing (original
draft): C.R.; writing (review and editing): C.R. and K.K.; visualization: I.S.; funding
acquisition: C.R.; supervision: J.S., K.K. and C.R. 26. Tushinski, R. J. et al. Survival of mononuclear phagocytes depends on a
lineage-specific growth factor that the differentiated cells selectively destroy. Cell 28, 71–81 (1982). 12 NATURE COMMUNICATIONS| (2018) 9:582 | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications | DOI: 10.1038/s41467-018-02834-8| www.nature.com/naturecommunications Additional information Open Access This article is licensed under a Creative Commons
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Сведения об авторах
Ад Tugareva A. V., Head of the Laboratory of Geology of
Jurassic and Pre-Jurassic Deposits, V. I. Shpilman Research
and Analytical Centre for the Rational Use of the Subsoil,
Tyumen, phone: 8(3452)621896, e-mail: tugoreva@crru.ru Tugareva A. V., Head of the Laboratory of Geology of
Jurassic and Pre-Jurassic Deposits, V. I. Shpilman Research
and Analytical Centre for the Rational Use of the Subsoil,
Tyumen, phone: 8(3452)621896, e-mail: tugoreva@crru.ru Тугарева Аделина Вольдемаровна, заведующий ла-
бораторией геологии юрских и доюрских отложений,
Научно-аналитический центр рационального недрополь-
зования им. В. И. Шпильмана, г. Тюмень, тел. 8(3452)621896, e-mail: tugoreva@crru.ru Chernova G. A., Senior Researcher of the Laboratory of
Geology
of
Jurassic
and
Pre-Jurassic
Deposits,
V. I. Shpilman Research and Analytical Centre for the Ra-
tional Use of the Subsoil, Tyumen, phone: 8(3452)621896 Чернова Галина Александровна, старший научный
сотрудник лаборатории геологии юрских и доюрских
отложений, Научно-аналитический центр рационально-
го недропользования им. В. И. Шпильмана, г. Тюмень,
тел. 8(3452)621896 Yakovleva N. P., Senior Researcher of the Laboratory
of Geology of Jurassic and Pre-Jurassic Deposits,
V. I. Shpilman Research and Analytical Centre for the Ra-
tional Use of the Subsoil, Tyumen, phone: 8(3452)621896 Яковлева Наталья Павловна, старший научный
сотрудник лаборатории геологии юрских и доюрских
отложений, Научно-аналитический центр рационально-
го недропользования им. В. И. Шпильмана, г. Тюмень,
тел. 8(3452)621896 Moroz M. L., Researcher of the Laboratory of Geology
of Jurassic and Pre-Jurassic Deposits, V. I. Shpilman Re-
search and Analytical Centre for the Rational Use of the
Subsoil, Tyumen, phone: 8(3452)621896 Мороз Мария Леонидовна, научный сотрудник ла-
боратории геологии юрских и доюрских отложений,
Научно-аналитический центр рационального недрополь-
зования им. В. И. Шпильмана, г. Тюмень, тел. 8(3452)621896 Мороз Мария Леонидовна, научный сотрудник ла-
боратории геологии юрских и доюрских отложений,
Научно-аналитический центр рационального недрополь-
зования им. В. И. Шпильмана, г. Тюмень, тел. 8(3452)621896 5.
Перспективы нефтегазоносности отложений зоны контакта юры с триасом и палеозоем на территории
ХМАО-Югры / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: мате-
риалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 34–52. 5. Перспективы нефтегазоносности отложений зоны контакта юры с триасом и палеозоем на территории
ХМАО-Югры / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: мате-
риалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 34–52. р
у
р
ф
6. Закономерности геологического строения и перспективы нефтегазоносности триасового НГК территории
ХМАО — Югры / Н. П. Яковлева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 10–23. р
у
р
ф
7. Шпильман В. И. Проблемы освоения нефтегазового комплекса и возможные пути их решения // Пути реали-
зации нефтегазового потенциала ХМАО. – Ханты-Мансийск, – 1998. – С. 7–13. 8. Геологическое строение и перспективы нефтегазоносностидоюрских отложений Карабашской зоны
А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: материалы XIX науч.-
практ. конф. – Т. 1. – Ханты-Мансийск, 2016. – С. 71–88. 9. Вещественный состав и нефтегазоносность триасовых грабенообразных впадин западной части территории
ХМАО — Югры / Н. П. Яковлева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVIII науч.-практ. конф. – Т. 2. – Ханты-Мансийск, 2015. – С. 126–137. 10. Вещественный состав и перспективы нефтегазоносностидоюрских отложений Когалымской вершины Сур-
гутского свода / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVII науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2014. – С. 228–237. 11. Новые данные о вещественном составе доюрских отложений Карабашской зоны (в пределах территории
ХМАО — Югры) / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XIX науч.-практ. конф. – Т. 2. – Ханты-Мансийск, 2016. – С. 56–74. 5.
Перспективы нефтегазоносности отложений зоны контакта юры с триасом и палеозоем на территории
ХМАО-Югры / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: мате-
риалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 34–52. 5.
Перспективы нефтегазоносности отложений зоны контакта юры с триасом и палеозоем на территории
ХМАО-Югры / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: мате-
риалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 34–52.
6.
Закономерности геологического строения и перспективы нефтегазоносности триасового НГК территории
ХМАО — Югры / Н. П. Яковлева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVI науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2013. – С. 10–23.
7.
Шпильман В. И. Проблемы освоения нефтегазового комплекса и возможные пути их решения // Пути реали-
зации нефтегазового потенциала ХМАО. – Ханты-Мансийск, – 1998. – С. 7–13.
8.
Геологическое строение и перспективы нефтегазоносностидоюрских отложений Карабашской зоны
А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры: материалы XIX науч.-
практ. конф. – Т. 1. – Ханты-Мансийск, 2016. – С. 71–88.
9.
Вещественный состав и нефтегазоносность триасовых грабенообразных впадин западной части территории
ХМАО — Югры / Н. П. Яковлева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVIII науч.-практ. конф. – Т. 2. – Ханты-Мансийск, 2015. – С. 126–137.
10. Вещественный состав и перспективы нефтегазоносностидоюрских отложений Когалымской вершины Сур-
гутского свода / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XVII науч.-практ. конф. – Т. 1. – Ханты-Мансийск, 2014. – С. 228–237.
11. Новые данные о вещественном составе доюрских отложений Карабашской зоны (в пределах территории
ХМАО — Югры) / А. В. Тугарева [и др.] // Пути реализации нефтегазового и рудного потенциала ХМАО — Югры:
материалы XIX науч.-практ. конф. – Т. 2. – Ханты-Мансийск, 2016. – С. 56–74. УДК 622.621.317
ПРОБЛЕМЫ ИЗМЕРЕНИЯ ПАРАМЕТРОВ
ДВИЖЕНИЯ БУРИЛЬНОЙ КОЛОННЫ
PROBLEMS OF MEASURING OF MOTION PARAMETERS
OF THE DRILL STRING В. А. Кузнецов, С. А. Михеев
V. A. Kuznetsov, S. A. Mikheew Самарский государственный технический университет, г. Самара 66 Нефть и газ Нефть и газ Для управления процессом бурения и спускоподъемными операциями на буро-
вых установках глубокого бурения необходима информация о параметрах движе-
ния колонны бурильных труб и талевого блока. На основе этой информации опре-
деляются такие технологические параметры и показатели процесса бурения, как
механическая скорость бурения проходка, скорость спуска и подъема бурильной
колонны, положение талевого блока, длина спущенной в скважину колонны бу-
рильных труб, глубина скважины. Эта информация используется также для авто-
матизации процесса бурения и спускоподъемных операций. С помощью кинематиче-
ской схемы спускоподъемного
механизма буровой установки
(рис. 1) показаны возможные
методы измерения параметров
движения бурильной колонны. С помощью кинематиче-
ской схемы спускоподъемного
механизма буровой установки
(рис. 1) показаны возможные
методы измерения параметров
движения бурильной колонны. Рис. 1. Кинематическая схема спускоподъемного
механизма буровой установки Несущей
конструкцией
спускоподъемного
механизма
служат вышка 1, а сам он пред-
ставляет собой полиспастную
систему, которая включает в
себя буровую лебедку 2, при-
водимую в движение двигате-
лем 3, кронблок 4 (неподвиж-
ные блоки), талевый блок 5
(подвижные блоки), талевый
канат 6 и крюк 7. Неподвиж-
ный конец талевого каната 10
крепится к основанию буровой
установки, а подвижный конец Несущей
конструкцией
спускоподъемного
механизма
служат вышка 1, а сам он пред-
ставляет собой полиспастную
систему, которая включает в
себя буровую лебедку 2, при-
водимую в движение двигате-
лем 3, кронблок 4 (неподвиж-
ные блоки), талевый блок 5
(подвижные блоки), талевый
канат 6 и крюк 7. Неподвиж-
ный конец талевого каната 10
крепится к основанию буровой
установки, а подвижный конец Рис. 1. Кинематическая схема спускоподъемного
механизма буровой установки 11 навивается на барабане буровой лебедки. На крюк через промежуточное звено (вертлюг) пущена колонна бурильных
труб 9. Полиспастная система за счет применения нескольких подвижных и не-
подвижных блоков позволяет снизить нагрузку на лебедку и увеличить грузоподъ-
емность спускоподъемного механизма. у
Измерять перемещение бурильной колонны можно следующими способами [1]:
• с помощью дополнительного мерного тросика 12 и мерного барабана 13; у
Измерять перемещение бурильной колонны можно следующими способами [1]:
12
б
б
13 у
Измерять перемещение бурильной колонны можно следующими способами [1]:
• с помощью дополнительного мерного тросика 12 и мерного барабана 13; у
Измерять перемещение бурильной колонны можно следующими способами [1]: Измерять перемещение бурильной колонны можно следующими способами [1]
• с помощью дополнительного мерного тросика 12 и мерного барабана 13; • по углу поворота одного из шкивов талевого блока; • по углу поворота одного из шкивов кронблока; • по перемещению талевого каната; • по углу поворота барабана лебедки. Нефть и газ Каждый из этих способов имеет свои достоинства и недостатки, при этом в
большинстве случаев, кроме первого способа, в измерительную цепь входят эле-
менты спускоподъемного механизма, которые при их нестабильности становятся
источниками погрешности измерения. Недостатки первого способа обусловлены
сложностью конструкции и низкой надежностью работы, связанной с возможно-
стью обрыва мерного тросика при закручивании струн каната талевого блока. Способ измерения по углу поворота одного из шкивов талевого блока позволя-
ет сравнительно просто устанавливать и обслуживать первичный измерительный
преобразователь, так как талевый блок может быть опущен в удобное для этой
цели положение. Функция преобразования при этом способе измерения имеет вид , (1) , (1) № 5, 2017 67 Нефть и газ Нефть и газ где φ — угол поворота рабочего шкива талевого блока; n — коэффициент оснастки
талевой системы для выбранного шкива; h — перемещение бурильной колонны;
dш — диаметр шкива талевого блока; dк — диаметр талевого каната. Например, при параметрах талевой системы dш = 900 мм, dк = 28 мм, установке
преобразователя на самый быстрый шкив (при числе шкивов 5n = 10) угол поворо-
та рабочего шкива составит 0,215 рад (12,3 0) при перемещении бурильной колон-
ны на 1 см. Из выражения (1) видно, что источником погрешности измерения является не-
стабильность параметров dш и dк. Относительное значения этой погрешности оп-
ределяется выражением , где ∆ш — допустимый износ шкивов; ∆к — изменения диаметра каната при его
вытяжке. Обычно ∆ш = 1мм, а ∆к = 2 мм, при этом δ = 0,32 %. де ∆ш — допустимый износ шкивов; ∆к — изменения диаметра каната при его
вытяжке. Обычно ∆ш = 1мм, а ∆к = 2 мм, при этом δ = 0,32 %. Однако следует учесть, что эта погрешность является мультипликативной, то
есть носит интегральный характер, поэтому при спуске колонны на 25 м (на одну
свечу) она составит 8 см. Существенным недостатком привязки первичного преобразователя к одному из
шкивов талевого блока являются подвижность этого блока и отсутствие проводно-
го канала связи для передачи измерительной информации. Частичное решение
этой проблемы предложено в [2]. Способ измерения перемещения бурильной колонны по углу поворота одного
из шкивов кронблока имеет те же метрологические характеристики, что и преды-
дущий способ (по углу поворота одного из шкивов талевого блока), но позволяет
организовать стационарный проводной канал связи для обеспечения питания из-
мерительной аппаратуры и передачи измерительной информации. Недостатки это-
го способы связаны со сложностью монтажа и обслуживания измерительного пре-
образователя на большой высоте. Нефть и газ На кафедре информа-
ционно-измерительной
техники
Самарского
государственного тех-
нического университе-
та разработан ряд ме-
тодов автоматической
коррекции погрешно-
стей, связанных с из-
менением радиуса на-
вивки талевого каната
на барабане лебедки. При
разработке
этих методов стави-
лась задача коррекции
всех
перечисленных
погрешностей измере-
ния, для чего необхо-
дима
информация
о
значении рабочего ра-
диуса навивки последнего витка талевого каната [3, 4]. На рисунке 3 представлена кинематическая схема механического устройства, по-
зволяющего автоматически корректировать перечисленные погрешности измерения. Устройство содержит подпружиненный рычаг 1, по которому скользит скоба 2,
прижатая к ходовому концу талевого каната, редуктор 3, подсоединенный к валу
буровой лебедки 4, дисковый вариатр 5 и преобразователь частоты вращения
в число-импульсный код 6. Рис. 3. Кинематическая схема механического устройства
для автоматической коррекции погрешностей,
связанных с измерением радиуса навивки
талевого каната на барабане лебедки Рис. 3. Кинематическая схема механического устройства
для автоматической коррекции погрешностей,
связанных с измерением радиуса навивки
талевого каната на барабане лебедки На кафедре информа-
ционно-измерительной
техники
Самарского
государственного тех-
нического университе-
та разработан ряд ме-
тодов автоматической
коррекции погрешно-
стей, связанных с из-
менением радиуса на-
вивки талевого каната
на барабане лебедки. При
разработке
этих методов стави-
лась задача коррекции
всех
перечисленных
погрешностей измере-
ния, для чего необхо-
дима
информация
о
Рис. 3. Кинематическая схема механического устройства
для автоматической коррекции погрешностей,
связанных с измерением радиуса навивки
талевого каната на барабане лебедки На кафедре информа-
ционно-измерительной
техники
Самарского
государственного тех-
нического университе-
та разработан ряд ме-
тодов автоматической
коррекции погрешно-
стей, связанных с из-
менением радиуса на-
вивки талевого каната
на барабане лебедки. На кафедре информа-
ционно-измерительной
техники
Самарского
государственного тех-
нического университе-
та разработан ряд ме-
тодов автоматической
коррекции погрешно-
стей, связанных с из-
менением радиуса на-
вивки талевого каната
на барабане лебедки. При
разработке
этих методов стави-
лась задача коррекции
всех
перечисленных
погрешностей измере-
ния, для чего необхо-
дима
информация
о
значении рабочего ра- Рис. 3. Кинематическая схема механического устройства
для автоматической коррекции погрешностей,
связанных с измерением радиуса навивки
талевого каната на барабане лебедки Нефть и газ Так как талевый канат свит из отдельных, периодически чередующихся прядей
стальной проволоки, то способ измерения перемещения бурильной колонны по
перемещению талевого каната основан на возможности модуляции магнитного
потока этими прядями при движении талевого каната. Однако этот способ не на-
шел применения из-за сложности первичного измерительного преобразователя и
его низкой надежности работы. Наиболее удобным является метод измерения перемещения бурильной колон-
ны по углу поворота барабана буровой лебедки. В этом случае измерительная ап-
паратура находится в зоне прямого доступа обслуживающего персонала, что об-
легчает ее монтаж и обслуживание. Угол поворота барабана лебедки связан с пе-
ремещением бурильной колонны зависимостью , (2) (2) где φδ — угол порота барабана лебедки, рад; Ко — коэффициент оснастки талевой
системы; h — перемещение бурильной колонны; dδ — диаметр барабана лебедки;
dк — диаметр талевого каната; n — номер слоя навивки талевого каната на бараба-
не лебедки. Из выражения (2) видно, что коэффициент преобразования зависимо-
сти φδ(h) изменяется при переходе с одного слоя навивки талевого каната на бара-
бане лебедки на другой, что приводит к существенной погрешности измерения. ру
р
у
р
р
Относительное значение этой погрешности определяется выражением . . № 5, 2017 Нефть и газ
№ 5, 2017 68 Нефть и газ На рисунке 2 представлен график зависимости погрешности измерения при пе-
реходе с одного слоя навивки на другой при параметрах талевой системы dδ = 600
мм, dк = 28 мм, макс n = 5. Рис. 2. График зависимости
относительной погрешности
измерения перемещения бурильной
колонны по углу поворота барабана
лебедки при переходе одного
слоя навивки талевого каната
на барабане лебедки на другой Рис. 2. График зависимости
относительной погрешности
измерения перемещения бурильной
колонны по углу поворота барабана
лебедки при переходе одного
слоя навивки талевого каната
на барабане лебедки на другой Рис. 2. График зависимости
относительной погрешности
измерения перемещения бурильной
колонны по углу поворота барабана
лебедки при переходе одного
слоя навивки талевого каната
на барабане лебедки на другой Максимальная погрешность измерения при переходе на пятый слой навивки
составляет 15,1 %. К погрешности измерения, представленной на рисунке 2, до-
бавляется погрешность, связанная с изменением диаметра талевого каната при его
вытяжке, а также связанная с нерегулярностью навивки талевого каната на бара-
бане лебедки. Наличие большой погрешности измерения сдерживает применение этого спо-
соба измерения, несмотря на ряд его преимуществ. Для автоматической коррекции
погрешности измерения необходимо знать значение радиуса навивки последнего
витка талевого каната на барабане лебедки. диуса навивки последнего витка талевого каната [3, 4]. диуса навивки последнего витка талевого каната [3, 4]. диуса навивки последнего витка талевого каната [3, 4]. На рисунке 3 представлена кинематическая схема механического устройства, по-
воляющего автоматически корректировать перечисленные погрешности измерения. На рисунке 3 представлена кинематическая схема механического устройства, по-
зволяющего автоматически корректировать перечисленные погрешности измерения. Устройство содержит подпружиненный рычаг 1, по которому скользит скоба 2,
прижатая к ходовому концу талевого каната, редуктор 3, подсоединенный к валу
буровой лебедки 4, дисковый вариатр 5 и преобразователь частоты вращения
в число-импульсный код 6. Вариатор состоит из подвижного в осевом направлении ролика 7 и диска 8. Р
1
б
2 Вариатор состоит из подвижного в осевом направлении ролика 7 и диска 8. Рычаг 1 с помощью скобы 2 следит за последним витком навивки талевого ка-
ната и занимает положение соответствующее текущему значению радиуса навив Вариатор состоит из подвижного в осевом направлении ролика 7 и диска 8. Рычаг 1 с помощью скобы 2 следит за последним витком навивки талевого ка-
ната и занимает положение, соответствующее текущему значению радиуса навив- № 5, 2017 Нефть и газ № 5, 2017 Нефть и газ 69 ки. Этот рычаг определяет положение ролика 7 и изменяет передаточное отноше-
ние вариатора при изменении радиуса навивки талевого каната таким образом,
чтобы «цена» электрических импульсов на выходе электрического преобразовате-
ля 6 осталась неизменной ки. Этот рычаг определяет положение ролика 7 и изменяет передаточное отноше-
ние вариатора при изменении радиуса навивки талевого каната таким образом,
чтобы «цена» электрических импульсов на выходе электрического преобразовате-
ля 6 осталась неизменной Если периодическое отношение редуктора — Кр, передаточное отношение ва-
риатора — Кв, коэффициент преобразования преобразователя — 6-
П
K
, то с уче-
том (2) зависимость между числом выходных электрических импульсов устройст-
ва № 4 и перемещением бурильной колонны будет иметь вид )
nd
d
(
К
K
h
К
K
N
K
В
Р
О
П
+
⋅
=
δ
2
. (3) (3) Для преобразования частоты вращения в число-импульсный код удобно ис-
пользовать серийный оптоэлектронный растровый преобразователь типа ВЕ-178,
генерирующий 1 000 электрических импульсов за один оборот вала и имеющий
функцию определения направления вращения, при этом Кп = 159,236 ИМП/рад. фу
р
р
р
р
Если задать «цену» одного импульса 1 см, то из (3) определяется требуемое пе-
редаточное отношение редуктора и вариатора. . . При параметрах талевой системы К0 = 10; h = 1, dδ = 600 мм, d = 2,5 см, n = 1
получим Кр Кв = 30,71. Библиографический список
1.
Бражников В. А., Кузнецов В. А. Информационные устройства для определения эффективности управления
процессом бурения. – М.: Недра, 1978. – 108 с.
2.
А. С. 1416677 СССР, МКИ3Е21 В45/00. Устройство для передачи информации / В. А. Кузнецов, В. Н. Евдо-
кимов, М. В. Фалеева. – № 3925995/22-03; заявл. 11.07.85.
3.
А. С. 71399/СССР, МКИ3Е21В45/00. Устройство для измерения вертикальных перемещений бурового инст-
румента / В. А. Кузнецов, В. Г. Шуваев. – № 2587980/22-03; заявл. 06.03.78; опубл. 05.02.80, Бюл. № 5. – 2 с.
4.
А. С. 451836 СССР, МКИ3Е21В45/00. Устройство для измерения вертикальных перемещений бурового инстру-
мента / В. А. Кузнецов, С. С. Торопов, Е. Г. Иванов. – № 1910026/22-3; заявл. 17.04.73; опубл. 30.11.74, Бюл. № 44. – 3 с.
5.
А. С. 1596459 СССР, МКИ3НОЗМ 1/22. Преобразователь линейных перемещений в код / В. А. Кузнецов,
В. Н. Яшин. – № 4467213/24-24; заявл. 29.07.88; опубл. 30.09.90, Бюл. № 36. – 4 с.
6.
А. С. 1580553 СССР, МКИ3Н03М1/30. Преобразователь линейных перемещений в код / В. А. Кузнецов. –
№ 4431899/24-24; заявл. 30.05.88; опубл. 23.07.90, Бюл. № 27. – 2 с.
7.
А. С. 1363465 СССР, МКИ3Н03М1/26. Преобразователь перемещений в код / В. А. Кузнецов. –
№ 3974816/24-24; заявл. 10.11.85; опубл. 30.12.87, Бюл. № 48. – 3 с. диуса навивки последнего витка талевого каната [3, 4]. у
р
Вариатор не может иметь большое передаточное отношение. Если принять
Кв = 1,5, передаточное отношение редуктора составит Кр = 20,5, что легко дости-
гается двухступенчатой зубчатой передачей. Второй вариант корректирующего устройства основан на применении элек-
тронного способа измерения коэффициента передачи измерительной цепи. На рисунке 4 представлена структурная схема электрического устройства для
автоматической коррекции погрешностей, связанных с измерением радиуса навив-
ки талевого каната на
барабане лебедки. На рисунке 4 представлена структурная схема электрического устройства для
автоматической коррекции погрешностей, связанных с измерением радиуса навив-
ки талевого каната на
барабане лебедки. Устройство содержит
подпружиненный рычаг
1, по которому свободно
скользит скоба 2, прижа-
тая к последнему витку
навивки талевого каната,
зубчатую
передачу
3,
механически связанную с
валом лебедки 4, преоб-
разователь частоты вра-
щения
в
число-
импульсный код 5, пре-
образователь
линейных
перемещений в код 6 и
делитель
частоты
им-
пульсов 7. На выходе преобразователя 5 генерируются электрические импульсы, число
б
б
б
А
Рис. 4. Структурная схема электрического
устройства для автоматической
коррекции погрешностей измерения Рис. 4. Структурная схема электрического
устройства для автоматической
коррекции погрешностей измерения Рис. 4. Структурная схема электрического
устройства для автоматической
коррекции погрешностей измерения Устройство содержит
подпружиненный рычаг
1, по которому свободно
скользит скоба 2, прижа-
тая к последнему витку
навивки талевого каната,
зубчатую
передачу
3,
механически связанную с
валом лебедки 4, преоб-
разователь частоты вра-
щения
в
число-
импульсный код 5, пре-
образователь
линейных
перемещений в код 6 и
делитель
частоты
им-
пульсов 7. Устройство содержит
подпружиненный рычаг
1, по которому свободно
скользит скоба 2, прижа-
тая к последнему витку
навивки талевого каната,
зубчатую
передачу
3,
механически связанную с
валом лебедки 4, преоб-
разователь частоты вра-
щения
в
число-
импульсный код 5, пре-
образователь
линейных
перемещений в код 6 и
делитель
частоты
им-
пульсов 7. Рис. 4. Структурная схема электрического
устройства для автоматической
коррекции погрешностей измерения у
На выходе преобразователя 5 генерируются электрические импульсы, число
которых пропорционально углу поворота барабана лебедки. Автоматическая кор-
рекция погрешностей измерения, связанных с изменением радиуса навивки тале-
вого каната, осуществляется посредством измерения коэффициента деления дели- Нефть и газ
№ 5, 2017 70 № 5, 2017 Нефть и газ теля числа импульсов 7. Коэффициент деления задается преобразователем линей-
ных перемещений в код 6. Выходной код этого преобразователя определяется ра-
диусом навивки последнего витка талевого каната. теля числа импульсов 7. Коэффициент деления задается преобразователем линей-
ных перемещений в код 6. Выходной код этого преобразователя определяется ра-
диусом навивки последнего витка талевого каната. диуса навивки последнего витка талевого каната [3, 4]. Если применить в качестве преобразователя 5 преобразователь ВЕ-175, генери-
рующий 1 000 импульсов за один оборот вала, то есть, имеющий коэффициент
преобразования Кп = 159,24 ИМП/рад, то с учетом (2) количество выходных им-
пульсов при перемещении бурильной колонны на h определится выражением . Для приведенных ранее параметров талевой системы число выходных импуль-
сов при перемещении бурильной колонны на 1 см составит для первого слоя на-
вивки N1 = 50,71, для пятого слоя навивки N5 = 43,04. Таким образом, для получения выходных электрических импульсов «ценой» в
1 см коэффициент пересчета делителя 7 должен изменяться от N1 до N5. Коэффициент деления должен быть целым числом, поэтому количество целых
разрядов N1 и N5 увеличивается, но во столько же раз должно быть увеличено ко-
личество импульсов на выходе преобразователя 5. С этой целью в измерительную
цепь вводится зубчатый мультипликатор 3. у
у
р
В качестве преобразователя линейных перемещений в код 6 могут быть ис-
пользованы устройства, описанные в [5–7]. Задача коррекции погрешностей измерения упрощается, если для обработки
информации применяется микроконтроллер. В этом случае на вход микроконтрол-
лера подаются электрические импульсы с выхода преобразователя 5 и цифровой
код, соответствующий значению радиуса навивки талевого каната. С помощью
микроконтроллера определяются такие параметры, как скорость спуска и подъема
колонны труб, проходка, скорость бурения, длина колонны. Сведения об авторах р
Кузнецов Владимир Андреевич, к. т. н., доцент ка-
федры информационно-измерительной техники, Самар-
ский государственный технический университет, г. Самара, тел. 8(846)3370865 Kuznetsov V. A., Candidate of Engineering, Associate
Professor at the Department of Information and Measuring
Technology, Samara State Technical University, phone:
8(846)3370865 Mikheev S. A., Master’s Student at the Department of
Information and Measuring Technology, Samara State Tech-
nical
University,
phone:
89276039804,
e-mail:
serzh.mixeew@yandex.ru Михеев Сергей Андреевич, магистрант, кафедра
информационно-измерительной
техники,
Самарский
государственный технический университет, г. Самара,
тел. 89276039804, e-mail: serzh.mixeew@yandex.ru Михеев Сергей Андреевич, магистрант, кафедра
информационно-измерительной
техники,
Самарский
государственный технический университет, г. Самара,
тел. 89276039804, e-mail: serzh.mixeew@yandex.ru № 5, 2017 71 Нефть и газ
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https://openalex.org/W4287687211
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https://zenodo.org/records/3991789/files/7317ijdkp03.pdf
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English
| null |
TWO PARTY HIERARICHAL CLUSTERING OVER HORIZONTALLY PARTITIONED DATA SET
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Zenodo (CERN European Organization for Nuclear Research)
| 2,020
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cc-by
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nternational Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 nternational Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 1M.Tech (CSE) Student KIET Group of Institution, Ghaziabad, U.P ,
2Assistant Professor KIET Group of Institution, Ghaziabad, U.P 1M.Tech (CSE) Student KIET Group of Institution, Ghaziabad, U.P ,
2Assistant Professor KIET Group of Institution, Ghaziabad, U.P KEYWORD Two party computations, Partitioning, clustering, k-means algorithm, Hierarichal clustering. ABSTRACT Data mining is a task in which data is extracted from the large database to make itin an understandable
form or structure so that it can be used for further use. In this paper we present an approach by which the
concept of hierarchal clustering applied over the horizontally partitioned data set. We also explain the
desired algorithm like hierarichal clustering, algorithms for finding the minimum closest cluster. In this
paper wealso explain the two party computations. Privacy of any data is the most important thing in these
days hence we present an approach by which we can apply privacy preservation over the two party which
are distributing their data horizontally. We also explain about the hierarichal clustering which we are
going to apply in our present method. TWO PARTY HIERARICHAL CLUSTERING OVER
HORIZONTALLY PARTITIONED DATA SET Priya Kumari1 and Seema Maitrey2 1. INTRODUCTION Data mining is a very current research area in these days only because of its ability to extract the
data from a large data set very efficiently. Data mining is a field in which the main aim is to
extract or mine knowledge from a large amount of data [1]. In data mining generally the
processing is done over the large volume of data that is stored in a database and search for pattern
and relationships inside the data. Privacy is also the main point of focus in these days in between
the researchers all of us have some data that we don’t want to share with anyone hence whenever
the situation arises that we wants to secure our data from others then we use have some
approaches like association rule mining, classification and clustering. In this paper we are going
to use clustering approach. Clustering of data is a method by which the similar kind of cluster are grouped together and one
by one each attribute comes to any cluster in the end of the approach. When we are dealing with
some sensitive information then the privacy issue is a major concern because if any of the
information is leak or compromise then that may result to effect or harm to individual or financial
losses to any well stablished organisation. Clustering is used widely in many real time areas like
in financial affairs, marketing, medical, chemistry, insurance, machine cleaning, data mining etc
[2,3]. DOI: 10.5121/ijdkp.2017.7303 33 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 2. PRELIMINARIES In this section we are going to present some preliminaries. We first introduce the partitioning
approach by which we partition the data set. Then we explain about how to party works and after
then the main approach or algorithms which we are going to implement to fulfil our approach. 2.1 Two party computation Two party computationsare an approach in which mainly two party involve in computation. These two parties have their own data set in an equal amount but they don’t expose their data to
its corresponding party. In this way they a form an distance matrix and share their distance matrix
to each other not the original data set by merging the two distance matrix of each party we come
to a single matrix by which we can easily get the solution of our queries because these distance
matrices compute the smallest distance of each cluster with the help of cluster center so any user
get their result in a very reasonable time [4]. Two party computation query model which consist
of mainly four entities these are following:- 1. Randomizer. 2. Computing engine. 3. Query front end engine. 4. Individual Database. In this the randomizer and computing engine are comes in primary engine. The query front end
engine which is responsible for receiving all of the queries from the users and then it forward
these queries to the randomizer. An encoded query which is normally contains the type of query
to its computing engine. The computing engine coordinates that query to individual database for
computing the result of query.In our approach we apply hierarichal clustering over two party
computations which are using horizontally partitioned data. In this the randomizer and computing engine are comes in primary engine. The query front end
engine which is responsible for receiving all of the queries from the users and then it forward
these queries to the randomizer. An encoded query which is normally contains the type of query
to its computing engine. The computing engine coordinates that query to individual database for
computing the result of query.In our approach we apply hierarichal clustering over two party
computations which are using horizontally partitioned data. Figure1. Two Party computations Model
DB
DB
DB
DB
Randomizer
Computing Engine
Query Front End Engine
Two parties
Query Encoded query
Query Input
Response query Figure1. Two Party computations Model 34 34 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
2 2 Partitioning Of Database 2.2 Partitioning Of Database Partitioning of data base is the process in which we partition the data set in horizontal, vertical
and arbitrary. In the partitioning we apply several techniques to full fill the task of preserving the
data if we apply the perfect partitioning then we can easily make our approach good. First is
horizontally partitioning this means that the partitioning apply on the data set in which we are
deal with the data of a complete row we don’t have to worry about the column information a
complete row information comes in result. In vertically partitioning the data is distributed in columns if any of the query come that means
that the information of a complete row comes in result this is good for if we are requesting for the
data of a single attribute. The third partitioning approach is arbitrary partitioning that means that the horizontal and vertical
both type of approaches comes in this. When the query is requested from the database at that time
it is decided to apply either horizontal or vertical approach. In all of the approaches the arbitrary approach is good but there is not much exploration in this
field but this approach is applied by using k-means clustering. Table 1. Record of Students
S.No
Name
Branch
Id
1
Ram
CSE
234
2
Rohan
CSE
235
3
Geeta
CSE
223
4
Pooja
ECE
342
Table 2. Vertically Partitioned
S.No
Name
1
Ram
2
Rohan
3
Geeta
4
Pooja
Table 3.Horizontally Partitioned
S.No
Name
Branch
Id
1
Ram
CSE
234
2
Rohan
CSE
235
In our approach we are going to use horizontally partitioned database hence we have the data
which is organised by row [5]. If we are taking data by a single attribute we have the all
information about a single attribute in a same time hence this is good approach for the banks and
academics. For this partitioning we use hierarchical clustering and also we are using
agglomerative approach with some encryption techniques. Table 1. Record of Students
S.No
Name
Branch
Id
1
Ram
CSE
234
2
Rohan
CSE
235
3
Geeta
CSE
223
4
Pooja
ECE
342 Table 1. Record of Students In our approach we are going to use horizontally partitioned database hence we have the data
which is organised by row [5]. 4. HIERARCHICAL CLUSTERING Hierarichal clustering is one of the clustering approaches [10]. In hierarchal clustering is mainly
divided in two methods which are following:- Hierarichal clustering is one of the clustering approaches [10]. In hierarchal clustering is mainly
divided in two methods which are following:- 1. Agglomerative approach. 2. Divisive approach. 3. CLUSTERING Clustering is an approach which is best if we are dealing with some sensitive data or information. The privacy is the major issue because there are many chances that the information is leak. Hence
clustering is the most appropriate approach for making the privacy strong[6] [7] [8]. Clustering is mainly said that if we are clustering some data then we have to find the data which
is most similar in their properties hencethey are cluster in a single group. Each group is different
from the other groups either in size, number of objects and their dimension and also they have
different data types. Figure 2. Clustering of Database
Clustering is a data mining technique which is unsupervised data analysis [9]. It offers advanced
and more abstracted view to the dataset which is complex to handle if we are using simple
techniques. There are many clustering based privacy techniques which are given by researchers. Types of clustering are following:-
C11
C12
C13
C23
C232
222
C211
Database
Cluster 1
Cluster 2 Cluster 1 Figure 2. Clustering of Database Clustering is a data mining technique which is unsupervised data analysis [9]. It offers advanced
and more abstracted view to the dataset which is complex to handle if we are using simple
techniques. There are many clustering based privacy techniques which are given by researchers. Types of clustering are following:- Clustering is a data mining technique which is unsupervised data analysis [9]. It offers advanced
and more abstracted view to the dataset which is complex to handle if we are using simple
techniques. There are many clustering based privacy techniques which are given by researchers. Types of clustering are following:- 1. Hierarchical clustering. 2. K-means clustering. 3. Density based clustering. 4. Self-organised maps EM clustering. These are basic types of clustering algorithm which are mainly used in these days in many
approaches. These are basic types of clustering algorithm which are mainly used in these days in many
approaches. 2.2 Partitioning Of Database If we are taking data by a single attribute we have the all
information about a single attribute in a same time hence this is good approach for the banks and
academics. For this partitioning we use hierarchical clustering and also we are using
agglomerative approach with some encryption techniques. 35 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 4.2 Divisive Approach In divisive approach hierarichal clustering is applied over the database from top to bottom. Hence
this approach is called as top to bottom approach. Figure 4. Divisive Approach
abcde
b
a
c
d
e Figure 4. Divisive Approach
abcde
b
a
c
d
e Figure 4. Divisive Approach Figure 4. Divisive Approach 4.1 Agglomerative Approach Agglomerative approach is one of the hierarichal clustering approach which is applied over the
database. In agglomerative approach this make cluster of database from its bottom to its top 36 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
hence it is also called as bottom up approach. This is the most commonly used approach in the
field of clustering of data sets. Figure 3. Agglomerative Approach
A
B
C
D
E
Ab
de
abcde
cde Figure 3. Agglomerative Approach Figure 3. Agglomerative Approach 1. SHA1
2. MD5 1. SHA1
2. MD5 These two algorithms are applied over the data of two parties. Each of the party uses a different
encryption technique. Hence it is hard to understand for each of the party about the original
dataset. The main advantage of this approach is that one dataset is damage then that will not affect the
other dataset. 5.1 K-Means clustering. In our approach we are using k-means clustering algorithm for partitioning of datasets. The
followings steps are followed:
•
Let X={X1,X2,……Xn} are the data elements and v={v1, v2,…….vc} are the set of
cluster center. In our approach we are using k-means clustering algorithm for partitioning of datasets. The
followings steps are followed: followings steps are followed:
•
Let X={X1,X2,……Xn} are the data elements and v={v1, v2,…….vc} are the set of
cluster center. Fi
d
l
l
‘ ’ l g
p
•
Let X={X1,X2,……Xn} are the data elements and v={v1, v2,…….vc} are the set of
cluster center. •
First randomly select ‘c’ cluster center. •
Calculate the distance between each of data element and cluster center. Calculate the distance between each of data element and cluster center. Assign the minimum distance of each element from the entire cluster center. •
These steps are repeated until all of the elements come to a cluster. 37 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
5.2 Euclidian Distance Matrix International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 Output: •
Assign all of the elements to a cluster. •
P1 compute k-cluster center (c1, c2, c3….ck) from the first attributes. •
P2 compute in the same manner the left attributes (ck+1, ck+2 ….c2k). •
P1 and P2 compute their cluster center and distance matrix as MP1 and MP2 respectively. •
P1 and P2 randomly share their cluster center and distance matrix. We use permute share
algorithm for sharing this information between them. •
P1 and P2 make the all possible cluster from the existing cluster informationi.e. k2 cluster
will be formed. •
Make a closest cluster from each party. •
Find the minimum value of each row of X matrix to find closest cluster for each instance
that is if the ith column have minimum value in jth instance then that will become the
closest ith cluster. •
Place each instance to its appropriate closest cluster. •
Merge k2 cluster to make the final k cluster. 4 Algorithm for closest cluster. •
P1 compute k-cluster center (c1, c2, c3….ck) from the first attributes. •
P2 compute in the same manner the left attributes (ck+1, ck+2 ….c2k). •
P1 and P2 compute their cluster center and distance matrix as MP1 and MP2 respectively. •
P1 and P2 randomly share their cluster center and distance matrix. We use permute share
algorithm for sharing this information between them. •
P1 and P2 make the all possible cluster from the existing cluster informationi.e. k2 cluster
will be formed. •
Make a closest cluster from each party. •
Find the minimum value of each row of X matrix to find closest cluster for each instance
that is if the ith column have minimum value in jth instance then that will become the
closest ith cluster. •
Place each instance to its appropriate closest cluster. 2 •
Place each instance to its appropriate closest
2 •
Merge k2 cluster to make the final k cluster. •
Merge k2 cluster to make the final k cluster. 5.3 Privacy Preserving Hierarichal agglomerative clustering. Input: P1 have his cluster center and distance matrix and P2 have its own cluster center and
distance Matrix. 5.2 Euclidian Distance Matrix Euclidian distance matrix is an nxn matrix which represents the spacing of n points in any
Euclidian space. Let there are two party P1 and P2 these are distributing the database D. P1 have the distance
matrix of first nxk elements and P2 have another nxk+1. Now both of the parties have two set of data and the k-cluster center and distance matrix. Now both of the parties have two set of data and the k-cluster center and distance matrix. Before sharing the distance matrix we have to apply encryption over the data. In our approach we
use two encryption algorithms which are following: Before sharing the distance matrix we have to apply encryption over the data. In our approach we
use two encryption algorithms which are following: 5.4 Algorithm for closest cluster. Input: Given distance matrix of P1 and distance matrix of P2. Output: first assign the closest cluster for n instance. A matrix X( nxk2) that holds the
distance between each pair of n points and the k2 cluster center. Input: Given distance matrix of P1 and distance matrix of P2. Output: first assign the closest cluster for n instance. A matrix X( nxk2) that holds the
distance between each pair of n points and the k2 cluster center. Input: Given distance matrix of P1 and distance matrix of P2. Output: first assign the closest cluster for n instance. A matrix X( nxk2) that holds the
distance between each pair of n points and the k2 cluster center. 38 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 nternational Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 •
For a=1 to n
•
1=0
•
For b= 1to k
•
For c=1 to k
•
1=1+1
•
Xa1= pab +qac
•
end for
•
end for
•
end for
•
Return X •
For a=1 to n
•
1=0
•
For b= 1to k
•
For c=1 to k
•
1=1+1
•
Xa1= pab +qac
•
end for
•
end for
•
end for
•
Return X •
For a=1 to n
•
1=0
•
For b= 1to k
•
For c=1 to k
•
1=1+1
•
Xa1= pab +qac
•
end for
•
end for
•
end for
•
Return X 6. EFFICIENCY AND PRIVACY ANALYSIS P1 and P2 both compute k-cluster on their own data set and then they share it to each other after
encrypting the value of data in distance matrix. The encryption is done through two algorithm
which are SHA1 and MD5 which takes O(k) time for each party. In the next step computational
complexity for computing the distance matrix by each party is calculated as O(nk). In the next
step hierarichal k-clustering take O(k2) time for computation. The computational complexity for
closest cluster is O(nk2). In the last step for each instance run time is O(k2). So the total time complexity is O(nk2) Both of the party send or receive their k cluster independently but in an encrypted form. So the
information of the parties does not public to other including the opposite party. They share only
the distance matrix but this distance matrix is only the distance computed between the cluster
center and instance. So the information is not leak. After merging the final k-cluster center is
exposing to each other. Hence the privacy preserving by using hierarichal clustering algorithm
over two parties using horizontally partitioned data is secure and does not leak any information. 7. EXPERIMENTAL RESULT In the given approach we take a small database of 500 students. There records have weight and
height. These records are distributed in four clusters by using k-means clustering algorithm. After
this on two clusters we apply SHA-1 and on the other we apply MD-5 algorithm for encryption of
the data or information that these cluster have. After encryption these are shared between these
two party P1 and P2. The overall approach is explained briefly above. Here we give the comparison of our approach with some other techniques which are similar in
work but take more time in query processing. We take basically k-means clustering over
horizontally partitioned data , hierarichal clustering of two parties over vertically partitioned data
and k-means clustering over vertically partitioned data. 39 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
A brief description is given in figure that how the given approach is better than the existing
methods. Figure 5. Some of data set on which hierarichal clustering is applied Figure 5. Some of data set on which hierarichal clustering is applied Figure 6: After clustering the database is divided in two party and 6 clusters Figure 6: After clustering the database is divided in two party and 6 clusters Figure 6: After clustering the database is divided in two party and 6 clusters 40 nternational Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017 Graph 1: Cluster formed in Party1
Graph 2: Cluster formed in Party 2
73
65
59
75
67
61
77
60
62
74
70
66
0%
50%
100%
cluster 1
cluster 2
cluster 3
Party 1
Series 5
Series 4
Series 3
Series 2
Series 1
58
71
66
59
72
68
61
75
65
59
76
0
100
200
300
400
Cluster 1
Cluster 2
Cluster 3
Party 2
Series 4
Series 3
Series 2
Series 1 Graph 2: Cluster formed in Party 2
58
71
66
59
72
68
61
75
65
59
76
0
100
200
300
400
Cluster 1
Cluster 2
Cluster 3
Party 2
Series 4
Series 3
Series 2
Series 1 Graph 2: Cluster formed in Party 2 The above graph shows the representation of data after the original database is distributed in
between the two parties. Hence there are total six cluster are formed in our experiment. 8. CONCLUSION In this paper we analyse the privacy preserving
are various techniques used to solve the problems like adding noise or encryption data value. In
this paper a hierarichal clustering approach for horizontally
novel approach to secure data. In this paper we analyse the privacy preserving problems for horizontally partitioned data. There
are various techniques used to solve the problems like adding noise or encryption data value. In
this paper a hierarichal clustering approach for horizontally partitioned data for two
problems for horizontally partitioned data. There
are various techniques used to solve the problems like adding noise or encryption data value. In
partitioned data for two parties is a 9. FUTURE RESEARCH W
WORK The future research work can be to find solution for hierarichal clustering for multiparty which
can be apply over horizontal and vertically partitioned data. The
further enhancing for arbitrary partitioned data. can be to find solution for hierarichal clustering for multiparty which
can be apply over horizontal and vertically partitioned data. The hierarichal clustering can be
for arbitrary partitioned data. can be to find solution for hierarichal clustering for multiparty which
hierarichal clustering can be 7. EXPERIMENTAL RESULT On these
two different set of cluster we use two different encryption algorithms. Both the parties are
unaware of the encryption technique of other. Hence the privacy of data is high. 4
Figure 5.Encryption over party 1 using SHA-1
Figure 6.Encryption over party 2 using MD5 Figure 5.Encryption over party 1 using SHA-1 41 International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
Table 4. Comparison among different cluster approach
Types of
cluster
k-means
clustering over
HPD
No of
database
scanning
300
Running time
(sec)
3.6304
Fig
0
1
2
3
4
C1
International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
Comparison among different cluster approach
clustering over
Hierarichal with
HPD
k-means
clustering over
VPD
Hierarichal with
VPD
150
300
150
1.342
3.6309
1.451
Figure 7.Execution time of each approach
C2
C3
C4
K-means HPD
Hierarichal HPD
K-means VPD
Hierarichal VPD
International Journal of Data Mining & Knowledge Management Process (IJDKP) Vol.7, No.3, May 2017
Hierarichal with
means HPD
Hierarichal HPD
means VPD
Hierarichal VPD Fig
0
1
2
3
4
C1
Figure 7.Execution time of each approach
C2
C3
C4
K-means HPD
Hierarichal HPD
K-means VPD
Hierarichal VPD
means HPD
Hierarichal HPD
means VPD
Hierarichal VPD Fig
Figure 7.Execution time of each approach [1]
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Clinical Management of Potential Toxicities and Drug Interactions Related to Cyclin-Dependent Kinase 4/6 Inhibitors in Breast Cancer: Practical Considerations and Recommendations
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Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:35982167 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Published Version
doi:10.1634/theoncologist.2017-0142 Published Version
doi:10.1634/theoncologist.2017-0142 Citation Spring, Laura M., Mark L. Zangardi, Beverly Moy, and Aditya Bardia. 2017. “Clinical
Management of Potential Toxicities and Drug Interactions Related to Cyclin#Dependent
Kinase 4/6 Inhibitors in Breast Cancer: Practical Considerations and Recommendations.” The
Oncologist 22 (9): 1039-1048. doi:10.1634/theoncologist.2017-0142. http://dx.doi.org/10.1634/
theoncologist.2017-0142. Correspondence: Aditya Bardia, M.D., Massachusetts General Hospital Cancer Center, Harvard Medical School, Lawrence House 304, 10 North
Grove St., Boston, Massachusetts 02114, USA. Telephone: 617-643-2208; e-mail: Bardia.Aditya@mgh.harvard.edu
Received March 26, 2017;
accepted for publication June 8, 2017; published Online First on July 13, 2017. http://dx.doi.org/10.1634/theoncologist.2017-0142 ABSTRACT Aberrations of the cell cycle are pervasive in cancer, and
selective cell cycle inhibition of cancer cells is a target of
choice for a number of novel cancer therapeutics. Cyclin-
dependent kinases (CDKs) are key regulatory enzymes that
control cell cycle transitions and the commitment to cell divi-
sion. Palbociclib and ribociclib are both orally active, highly
selective reversible inhibitors of CDK4 and CDK6 that are
approved by the U.S. Food and Drug Administration (FDA) for
hormone receptor-positive metastatic breast cancer in combi-
nation with specific endocrine therapies. A third oral CDK4/6
inhibitor, abemaciclib, received Breakthrough Therapy desig-
nation status from the FDA and is also being developed in
breast cancer. The most common adverse events associated
with palbociclib and ribociclib are hematologic, particularly neutropenia. However, the
neutropenia
associated
with
CDK4/6 inhibitors is distinct from chemotherapy-induced neu-
tropenia in that it is rapidly reversible, reflecting a cytostatic
effect on neutrophil precursors in the bone marrow. Most
hematologic abnormalities seen with CDK4/6 inhibitors are
not complicated and are adequately managed with standard
supportive care and dose adjustments when indicated. Cyto-
penias are less prevalent with abemaciclib, although fatigue
and gastrointestinal toxicity is more common with this agent. This review focuses on the clinical management of potential
toxicities and drug interactions seen with the use of CDK4/6
inhibitors in breast cancer, with a focus on palbociclib and
ribociclib, and summarizes practical management strategies
for an oncologist. The Oncologist 2017;22:1039–1048 Implications for Practice: The emergence of modern cyclin-dependent kinase (CDK) inhibitors has changed the treatment paradigm
for metastatic hormone receptor (HR)-positive breast cancer. Palbociclib, ribociclib, and abemaciclib are highly selective reversible
inhibitors of CDK4 and CDK6. Palbociclib is U.S. Food and Drug Administration (FDA)-approved in the first- and second-line settings
in combination with endocrine therapy for HR-positive metastatic breast cancer. Ribociclib is FDA-approved in the first-line setting. Abemaciclib has received FDA Breakthrough Therapy designation status. This review focuses on the clinical management of
potential toxicities and drug interactions seen with the use of CDK4/6 inhibitors in breast cancer. Clinical Management of Potential Toxicities and Drug Interactions
Related to Cyclin-Dependent Kinase 4/6 Inhibitors in Breast Cancer:
Practical Considerations and Recommendations
LAURA M. SPRING,† MARK L. ZANGARDI,† BEVERLY MOY, ADITYA BARDIA
Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts, USA LAURA M. SPRING,† MARK L. ZANGARDI,† BEVERLY MOY, ADITYA BARDIA
Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts, USA
†Contributed equally Disclosures of potential conflicts of interest may be found at the end of this article. Key Words. Breast cancer
• Cyclin-dependent kinases
• Drug toxicity Disclosures of potential conflicts of interest may be found at the end of this article. Key Words. Breast cancer
• Cyclin-dependent kinases
• Drug toxicity Clinical Management of Potential Toxicities and Drug Interactions
Related to Cyclin-Dependent Kinase 4/6 Inhibitors in Breast Cancer:
Practical Considerations and Recommendations
Breast Cancer Food and Drug Administration (FDA) for
hormone receptor-positive metastatic breast cancer in combi-
nation with specific endocrine therapies. A third oral CDK4/6
inhibitor, abemaciclib, received Breakthrough Therapy desig-
nation status from the FDA and is also being developed in
breast cancer. The most common adverse events associated
with palbociclib and ribociclib are hematologic, particularly
neu
CD
tro
effe
hem
not
sup
pen
and
Thi
tox
inh
rib
for
Implications for Practice: The emergence of modern cyclin-dependent
for metastatic hormone receptor (HR)-positive breast cancer. Palbocic
inhibitors of CDK4 and CDK6. Palbociclib is U.S. Food and Drug Adminis
in combination with endocrine therapy for HR-positive metastatic brea
Abemaciclib has received FDA Breakthrough Therapy designation st
potential toxicities and drug interactions seen with the use of CDK4/6 in
INTRODUCTION
At a fundamental level, abnormal cellular division and migration
represents the hallmark of cancer. Cyclin-dependent kinases
(CDKs), including CDK1, CDK2, CDK4, and CDK6, are key regulatory
enzymes that control cell cycle transitions and the commitment to
cell division [1–4]. Cyclin-dependent kinase 4 and CDK6 regulate
the transition from the G1 to S phase, CDK2 regulates transition
through the S phase, and CDK1 regulates the transition from the
G2 to the M phase [5]. In animals and humans, much of the con-
trol over cell cycle entry is determined by CDK4 and CDK6 [6, 7]. div
(Rb
the
bin
[8]
tio
ing
CD
Correspondence: Aditya Bardia, M.D., Massachusetts General Hospital Cance
Grove St., Boston, Massachusetts 02114, USA. Telephone: 617-643-2208; e-m
accepted for publication June 8, 2017; published Online First on July 13, 2017. Clinical Management of Potential Toxicities and Drug Interactions
Related to Cyclin-Dependent Kinase 4/6 Inhibitors in Breast Cancer:
Practical Considerations and Recommendations
Breast Cancer Breast Cancer Clinical Management of P
Related to Cyclin-Depende
Practical Considerations a
LAURA M. SPRING,† MARK L. ZANGARDI,† BEVERLY
Massachusetts General Hospital Cancer Center, Ha
†Contributed equally
Disclosures of potential conflicts of interest may be found
Key Words. Breast cancer
• Cyclin-dependent kin
ABSTRACT
Aberrations of the cell cycle are pervasiv
selective cell cycle inhibition of cancer ce
choice for a number of novel cancer the
dependent kinases (CDKs) are key regulato
control cell cycle transitions and the commi
sion. Palbociclib and ribociclib are both or
selective reversible inhibitors of CDK4 an
approved by the U.S. Food and Drug Admin
hormone receptor-positive metastatic breast
nation with specific endocrine therapies. A
inhibitor, abemaciclib, received Breakthroug
nation status from the FDA and is also be
breast cancer. The most common adverse
with palbociclib and ribociclib are hemato
Implications for Practice: The emergence of m
for metastatic hormone receptor (HR)-positiv
inhibitors of CDK4 and CDK6. Palbociclib is U. in combination with endocrine therapy for H
Abemaciclib has received FDA Breakthroug
potential toxicities and drug interactions seen
INTRODUCTION
At a fundamental level, abnormal cellular divi
represents the hallmark of cancer. Cyclin-d
(CDKs), including CDK1, CDK2, CDK4, and CDK6
enzymes that control cell cycle transitions and t
cell division [1–4]. Cyclin-dependent kinase 4
the transition from the G1 to S phase, CDK2
through the S phase, and CDK1 regulates the
G2 to the M phase [5]. In animals and humans
trol over cell cycle entry is determined by CDK4
Correspondence: Aditya Bardia, M.D., Massachus
Grove St., Boston, Massachusetts 02114, USA. Te
accepted for publication June 8, 2017; published O
TheOncologist 2017;22:1039–1048 Clinical Management of Potential Toxic
Related to Cyclin-Dependent Kinase 4/6
Practical Considerations and Recomme
LAURA M. SPRING,† MARK L. ZANGARDI,† BEVERLY MOY, ADITYA BARDIA
Massachusetts General Hospital Cancer Center, Harvard Medical School, Bost
†Contributed equally
Disclosures of potential conflicts of interest may be found at the end of this article. Key Words. Breast cancer
• Cyclin-dependent kinases
• Drug toxicity
ABSTRACT
Aberrations of the cell cycle are pervasive in cancer, and
selective cell cycle inhibition of cancer cells is a target of
choice for a number of novel cancer therapeutics. Cyclin-
dependent kinases (CDKs) are key regulatory enzymes that
control cell cycle transitions and the commitment to cell divi-
sion. Palbociclib and ribociclib are both orally active, highly
selective reversible inhibitors of CDK4 and CDK6 that are
approved by the U.S. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility INTRODUCTION At a fundamental level, abnormal cellular division and migration
represents the hallmark of cancer. Cyclin-dependent kinases
(CDKs), including CDK1, CDK2, CDK4, and CDK6, are key regulatory
enzymes that control cell cycle transitions and the commitment to
cell division [1–4]. Cyclin-dependent kinase 4 and CDK6 regulate
the transition from the G1 to S phase, CDK2 regulates transition
through the S phase, and CDK1 regulates the transition from the
G2 to the M phase [5]. In animals and humans, much of the con-
trol over cell cycle entry is determined by CDK4 and CDK6 [6, 7]. Transition from G1 to the S phase, a critical phase in cell
division, is controlled by the tumor suppressor retinoblastoma
(Rb) protein [8]. Retinoblastoma is a key negative regulator of
the cell cycle because it prevents premature cell division by
binding the E2F transcription factors to inhibit G1/S transition
[8]. Inactivation of Rb, which occurs via sequential phosphoryla-
tion of the protein, allows cellular division to proceed [9]. Dur-
ing G1, growth signals allow cyclin D to complex with either
CDK4 or CDK6, leading to phosphorylation of Rb, which Correspondence: Aditya Bardia, M.D., Massachusetts General Hospital Cancer Center, Harvard Medical School, Lawrence House 304, 10 North
Grove St., Boston, Massachusetts 02114, USA. Telephone: 617-643-2208; e-mail: Bardia.Aditya@mgh.harvard.edu
Received March 26, 2017;
accepted for publication June 8, 2017; published Online First on July 13, 2017. http://dx.doi.org/10.1634/theoncologist.2017-0142 TheOncologist 2017;22:1039–1048 www.TheOncologist.com Oc AlphaMed Press 2017 Management of CDK4/6 Inhibitors in Breast Cancer 1040 Figure 1. Role of cyclin-dependent kinase 4/6 inhibitors in halting cellular division. Abbreviations: Akt, protein kinase B; CDK, cyclin-dependent kinase; E2F, E2 factor; G, growth; HER2, human epidermal growth factor
receptor 2; M, mitosis; mTOR, mechanistic target of rapamycin; P, phosphate; PI3K, phosphoinositide 3-kinase; RB, retinoblastoma tumor
suppressor protein; S, synthesis. Figure 1. Role of cyclin-dependent kinase 4/6 inhibitors in halting cellular division. Abbreviations: Akt, protein kinase B; CDK, cyclin-dependent kinase; E2F, E2 factor; G, growth; HER2, human epidermal growth factor
receptor 2; M, mitosis; mTOR, mechanistic target of rapamycin; P, phosphate; PI3K, phosphoinositide 3-kinase; RB, retinoblastoma tumor
suppressor protein; S, synthesis. releases E2F to drive expression of genes required for S-phase
entry and progression through the cell cycle (Fig. 1) [8]. where the estrogen effector is the cyclin D1-CDK4/6–Rb com-
plex [18–20]. Binding of estrogen to ER-alpha drives cyclin D1
transcription, with activation of CDK4/6 and phosphorylation
of Rb leading to subsequent cell cycling [21–23]. CDK4/6 Inhibitor Development in Breast Cancer CDK4/6 Inhibitor Development in Breast Cancer
The development of selective inhibitors of both CDK4 and
CDK6 has markedly changed the perception of CDKs as thera-
peutic targets in cancer after underwhelming results and unac-
ceptable toxicity were seen with pan-CDK inhibitors such as
flavopiridol (alvocidib) in the early 2000s [17]. Palbociclib is an
orally active pyridopyrimidine that is a potent first-in-class,
highly selective reversible inhibitor of CDK4 and CDK6 [25]. As
expected based on the biology of the G1–S transition, the
effects of palbociclib are dependent on the presence of a func-
tional Rb protein, and no activity was seen in Rb-deficient cells
[10, 25]. Parallel drug discovery efforts resulted in the develop-
ment of the drugs abemaciclib and ribociclib [26–31]. Abemaci-
clib and ribociclib are both orally bioavailable highly selective
small molecule reversible inhibitors of CDK4/6. The selectivity
of all three compounds is theorized to reflect binding to the
specialized ATP-binding pocket of CDK4 and CDK6 with specific
interactions with residues in the ATP-binding cleft [17]. INTRODUCTION Similarly,
HER2-induced cellular proliferation is mediated via the CDK4/
6–Rb axis, and knockdown of cyclin-D in mice makes them
resistant to tumors induced by the neu oncogene [24]. Cyclin-dependent kinase 4 and CDK6, both serine/threo-
nine kinases, are structurally related proteins with many bio-
chemical
and
biological
similarities
[6]. In
terms
of
therapeutics, CDK4 and CDK6 are considered functionally
equivalent in their ability to phosphorylate Rb, and inhibition of
both kinases is likewise of considerable interest [10]. This
review will focus on clinical management of potential toxicities
and drug interactions seen with the use of CDK4/6 inhibitors in
breast cancer and summarize practical management strategies
for an oncologist. Dysregulation of the CDK4/6–Rb pathway in breast
cancer Goserelin (luteinizing hormone-releasing hormone analog) is coadministered with fulvestrant to premenopausal women in PALOMA-3 and MON-
ARCH-2. Abbreviations: 3/1, 3 weeks on, 1 week off; ABC, advanced breast cancer; HER2, human epidermal growth factor receptor 2; HR, hazard ratio;
HR1, hormone receptor-positive; ORR, overall response rate; PFS, progression-free survival; Rb, retinoblastoma tumor suppressor protein. ARCH-2. Abbreviations: 3/1, 3 weeks on, 1 week off; ABC, advanced breast cancer; HER2, human epidermal growth factor receptor 2; HR, hazard ratio;
HR1, hormone receptor-positive; ORR, overall response rate; PFS, progression-free survival; Rb, retinoblastoma tumor suppressor protein. Finn and colleagues tested palbociclib in vitro on molecularly
characterized human breast cancer cell lines and found that sen-
sitivity to palbociclib varied based on molecular phenotype. Estrogen receptor-positive cell lines with luminal features were
found to be the most sensitive and basal cell lines were found
to be resistant [32]. The combination of palbociclib with tamoxi-
fen was tested in vitro in ER1 human breast cancer cell lines
and demonstrated a synergistic interaction [32]. Similar to
results seen with tamoxifen, the combination of palbociclib and
trastuzumab was synergistic in sensitive HER2-amplified cell lines
[32]. Furthermore, combination studies carried out in cell lines
and primary tumor explants have illustrated that CDK4/6 inhibi-
tion with palbociclib provides a complementary mechanism of
action to ado-trastuzumab emtansine, and efficiently suppresses
the proliferation of residual HER2-positive tumor cell populations
that survive ado-trastuzumab emtansine [33]. These preclinical
observations led to the development of several clinical trials
evaluating the combination of CDK4/6 inhibitors with endocrine
therapy, and, more recently, with HER2-directed therapies. untreated patients with metastatic HR-positive/HER2-negative
breast cancer were randomized to palbociclib plus letrozole or
letrozole alone. Palbociclib significantly increased progression-
free survival (PFS) compared with letrozole alone, with a median
PFS of 10.2 months for the letrozole group and 20.2 months for
the palbociclib plus letrozole group (hazard ratio [HR] 0.488;
one-sided p< .001) [35]. A phase 3 confirmatory trial, PALOMA-
2 (NCT01942135), confirmed these findings with a median PFS
of 24.8 versus 14.5 months, respectively, for patients treated
with palbociclib or placebo in addition to letrozole (HR 0.58;
p< .001) [36]. Initial overall survival (OS) results from PALOMA-
1 demonstrate a statistically non-significant trend towards an
improvement in OS in the palbociclib group (37.5 months vs. 33.3 months, respectively, HR 0.837, p5 .280) [44]. Palbociclib Early trials studying palbociclib in advanced solid tumors demon-
strated encouraging results in breast cancer [34, 40, 41]. The
PALOMA-1 trial was the basis for the U.S. Food and Drug Admin-
istration (FDA) granting palbociclib accelerated approval in Feb-
ruary 2015 after it had received Breakthrough Therapy
designation in April 2013 [42]. In this trial, 165 previously Clinical Experience Select phase II and phase III studies of CDK4/6 inhibitors in
breast cancer are outlined in Table 1. Dysregulation of the CDK4/6–Rb pathway in breast
cancer The PALOMA-3 trial was a double-blind placebo-controlled,
randomized, phase 3 study of palbociclib added to the ER
down-regulator fulvestrant in previously treated HR-positive,
HER2-negative metastatic breast cancer patients [37]. Both pre-
and postmenopausal women were included in the study. The
addition of palbociclib improved the median PFS as compared
with fulvestrant alone, from 4.6 months to 9.5 months (HR
0.46; p< .001) [37, 38]. Based on these results, the FDA also
approved palbociclib for use in combination with fulvestrant for
disease progression following endocrine therapy. A number of
ongoing studies are exploring palbociclib in a number of disease
settings for breast cancer, including adjuvant and neoadjuvant
studies, and in combination with other targeted agents [43]. Dysregulation of the CDK4/6–Rb pathway in breast
cancer The CDK4/6–Rb axis is critical to cell cycle entry, and therefore
it is to be expected that the vast majority of cancers disrupt this
axis to promote growth [2, 11]. The majority of estrogen
receptor-positive (ER1) and human epidermal growth factor
receptor 2 (HER2)-positive breast cancers maintain functioning
Rb, and, potentially, susceptibility to CDK4/6 inhibitors [12–14]. Cyclin D1 amplification occurs in an estimated 15% of breast
cancers, particularly ER1 breast cancer [15]. Loss of proteins in
the INK4 and Cip/Kip families, as well as amplification of CDK4
and CDK6, have also been noted in breast cancer [16, 17]. In
particular, in ER1 breast cancer, estrogen has been shown to
increase the rate of progression from the G1 to the S phase, Oc AlphaMed Press 2017 Spring, Zangardi, Moy et al. Spring, Zangardi, Moy et al. 1041 Table 1. Select phase II and III studies of cyclin-dependent kinase 4/6 inhibitors in breast cancer
Study
Population
Phase
n
Setting
Treatment
Results
Palbociclib
NCT01037790 [34]
Rb-protein
expression
positive ABC
II
37
Pretreated
Palbociclib 125 mg daily,
3/1 schedule
PFS 3.7 months
PALOMA-1/TRIO-18
(NCT00721409) [35]
Postmenopausal,
HR1/HER2- ABC
II
165
First-line
Letrozole plus palbociclib
or placebo
PFS 20.2 months
vs. 10.2 months
PALOMA-2
(NCT01942135) [36]
Postmenopausal,
HR1/HER2- ABC
III
666
First-line
Letrozole plus palbociclib
or placebo
PFS 24.8 months
vs. 14.5 months
PALOMA-3
(NCT01942135) [37, 38]
Pre- and
postmenopausal,
HR1/HER2- ABC
III
521
Second-line
or greater
Fulvestrant plus
palbocicliba or placeboa
PFS 9.5 months
vs. 4.6 months
Ribociclib
MONALEESA-2
(NCT01958021)
Postmenopausal,
HR1/HER2- ABC
III
668
First-line
Letrozole plus ribociclib
(600 mg daily, 3/1
schedule) vs. placebo
PFS not reached
vs. 14.7 months
(HR 0.56)
Abemaciclib
MONARCH 1
(NCT02102490) [39]
HR1/HER2- ABC
II
132
Third-line
or greater
Abemaciclib 200 mg every
12 hours, continuously
PFS 6 months;
ORR 19.7%
MONARCH 2
(NCT02107703) [40]
Pre- and
postmenopausal,
HR1/HER2- ABC
III
669
Failed endocrine
therapy in localized
or first-line
metastatic setting
Fulvestranta plus
abemaciclib 200 mg
every 12 hours,
continuously vs. placebo
PFS 16.4 months
vs. 9.3 months
Palbociclib dose was 125 mg daily orally on a 3/1 schedule in all studies. aGoserelin (luteinizing hormone-releasing hormone analog) is coadministered with fulvestrant to premenopausal women in PALOMA-3 and MON-
ARCH-2. Abbreviations: 3/1, 3 weeks on, 1 week off; ABC, advanced breast cancer; HER2, human epidermal growth factor receptor 2; HR, hazard ratio;
HR1, hormone receptor-positive; ORR, overall response rate; PFS, progression-free survival; Rb, retinoblastoma tumor suppressor protein. Ribociclib Ribociclib received FDA Breakthrough Therapy designation in
August 2016 and full approval in March 2017 based primarily Oc AlphaMed Press 2017 www.TheOncologist.com www.TheOncologist.com www.TheOncologist.com Management of CDK4/6 Inhibitors in Breast Cancer 1042 on the phase III MONALEESA-2 (NCT01958021) trial exploring
ribociclib in combination with letrozole as first-line therapy in
women with HR-positive/HER2-negative metastatic breast can-
cer [44]. The trial met its primary endpoint, with the median
duration of PFS not reached in the ribociclib group (95% confi-
dence interval [CI], 19.3 to not reached) versus 14.7 months
(95% CI, 13.0–16.5) in the placebo group (HR, 0.56; 95% CI,
0.43–0.72; p5 3.29 3 1026 for superiority) [44]. For HR-posi-
tive/HER2-negative metastatic breast cancer, MONALEESA-7
(NCT02278120) is a phase 3 study exploring ribociclib in combi-
nation with endocrine therapy (plus the gonadotropin-
releasing
hormone
agonist
goserelin)
in
premenopausal
women, and MONALEESA-3 (NCT02422615) is a phase 3 study
in men and postmenopausal women exploring fulvestrant with
or without ribociclib in both the first- and/or second-line meta-
static setting. A number of other ongoing studies are exploring
ribociclib in various disease settings for breast cancer and in
combination with other targeted agents [43]. Absorption and drug exposure were found to be low
in the fasted state in a portion of the population,
which was increased when administered with food. Therefore, taking palbociclib on an empty stomach
could reduce drug levels and may compromise effec-
tiveness in a subset of patients. In vitro and in vivo studies indicated that palbociclib under-
goes hepatic metabolism in humans and is primarily metabo-
lized by CYP3A and sulfotransferase (SULT) enzyme SULT2A1
[46]. Concomitant use of strong CYP3A inhibitors should be
avoided, but if patients must be coadministered a strong
CYP3A inhibitor, the recommendation is to reduce the palboci-
clib dose to 75 mg once daily [46]. If the strong inhibitor is dis-
continued, the dose of palbociclib can be increased (after 3–5
half-lives of the inhibitor) to the dose used prior to the initia-
tion of the strong CYP3A inhibitor [46]. Additionally, coadminis-
tration of a strong CYP3A inducer (rifampin) decreased the
plasma exposure of palbociclib in healthy subjects by 85%, and
therefore it is recommended to avoid coadministration of
strong CYP3A inducers [46]. In vivo, palbociclib is a weak, time-
dependent inhibitor of CYP3A. Abemaciclib Abemaciclib received FDA Breakthrough Therapy designation in
October 2015. Among heavily pretreated HR-positive breast
cancer patients (n 5 47) enrolled in a phase 1 study of abema-
ciclib monotherapy, the overall response rate was 31%, with
61% of patients achieving either response or stable disease last-
ing 6 months [45]. This study suggested abemaciclib may
have benefit as monotherapy [45]. In the phase 2 single-arm
study, MONARCH 1 (NCT02102490), women with metastatic
HR-positive/HER2-negative breast cancer with disease progres-
sion following both antiestrogen therapy and at least one, but
no more than two, lines of chemotherapy in the metastatic set-
ting, received abemaciclib as monotherapy [39]. The confirmed
objective response rate was 19.7% and median PFS was 6.0
months [39]. The MONARCH 3 (NCT02246621) and MONARCH
2 (NCT02107703) trials are both large phase 3, randomized,
double-blind, placebo-controlled trials further evaluating the
combination of abemaciclib plus endocrine therapy. Recent
results from MONARCH 2 exploring the addition of abemaciclib
to fulvestrant demonstrated a median PFS of 16.4 months for
abemaciclib plus fulvestrant compared with 9.3 months in the
placebo plus fulvestrant group (HR: 0.553; p< .001) [40]. Ribociclib Lastly, the dose of a sensitive
CYP3A substrate with a narrow therapeutic index may need to
be reduced because palbociclib could increase its exposure
(Table 2) [46]. Ribociclib The recommended initial dose of ribociclib is 600 mg taken
orally once daily (preferably in the morning) for 21 consecutive
days, followed by 7 days off treatment, constituting a total cycle
of 28 days [46]. Absorption is not affected by food; therefore, it
may be taken with or without food.The FDA approval for riboci-
clib for first-line treatment of metastatic HR-positive/HER2-neg-
ative breast cancer is in combination with any of the three
approved aromatase inhibitors (letrozole, anastrozole, or
exemestane), which are given continuously throughout the 28-
day cycle [48]. Ribociclib undergoes extensive hepatic metabolism, the
majority of which is mediated by CYP3A4. As such, its elimina-
tion may be affected by CYP3A4 inhibitors or inducers [48, 49]. Concomitant use of strong CYP3A inhibitors should be avoided
and alternative therapy should be sought. If a strong CYP3A
inhibitor must be used concomitantly with ribococlib, the dose
of ribociclib should be reduced to 400 mg once daily. If the
strong inhibitor is discontinued, the dose of ribociclib should be
increased (after 5 half-lives of the inhibitor) to the dose used
prior to the initiation of the strong CYP3A inhibitor [48]. Con-
comitant use of strong CYP3A inducers should be avoided
because plasma exposure of ribociclib was decreased by 89%
with coadministration of rifampin (a strong CYP3A4 inducer) in
healthy volunteers [48]. Ribociclib also inhibits CYP3A4 in a
time-dependent manner and is a reversible CYP1A2 inhibitor
[48]. Concomitant use of CYP3A substrates with a narrow ther-
apeutic index should be done with caution, and may require a
dose reduction of the CYP3A substrate, as ribociclib could
increase their exposure [48]. Palbociclib In all three PALOMA trials, palbociclib was dosed at 125 mg
daily for 3 weeks followed by 1 week off based on earlier work
determining the recommended phase II dose (RP2D) [40, 41]. The recommended initial dose of palbociclib is a 125 mg cap-
sule taken orally once daily, with food, for 21 consecutive days,
followed by 7 days off treatment, constituting a total cycle of
28 days [46]. Absorption and drug exposure were found to be
low in the fasted state in a portion of the population, which
was increased when administered with food. Therefore, taking
palbociclib on an empty stomach could reduce drug levels and
may compromise effectiveness in a subset of patients [46]. The
FDA approval for palbociclib for first-line treatment of meta-
static HR-positive/HER2-negative breast cancer was initially in
combination with letrozole 2.5 mg once daily, but has been
updated to include any of the three approved aromatase inhibi-
tors (letrozole, anastrozole, or exemestane) [48]. May result in increased
exposure of concomitant
agent. Treatment implications
Recommendation Avoid concomitant use
and consider
alternative therapy. Reduced exposure of
palbociclib or ribociclib. Reduced exposure of
palbociclib or ribociclib. Increased exposure of
palbociclib and ribociclib. Avoid concomitant use
and consider alternative
therapy. Itraconazole, ketoconazole,
posaconazole, voriconazole
Atazanavir, darunavir, indinavir,
lopinavir/ritonavir, nelfinavir,
ritonavir, saquinavir, telaprevir
Grapefruit or grapefruit juice,
nefazodone Sensitive CYP3A substrates
with a narrow therapeutic index Midazolam, alfentanil,
cyclosporine, dihydroergotamine,
ergotamine, everolimus, fentanyl,
pimozide, quinidine, sirolimus
and tacrolimus Monitor closely for signs
of toxicity of
concomitant agent. Dose of concomitant
agent may need to
be reduced. For ribociclib only
QT prolonging agentsa
Antiarrhythmics
Other For ribociclib only
QT prolonging agentsa
Antiarrhythmics QT prolonging agentsa
Antiarrhythmics
Amiodarone, disopyramide,
procainamide, quinidine, sotalol
QTc prolongation and
related consequences. Other
Chloroquine, halofantrine,
clarithromycin, haloperidol,
methadone, moxifloxacin, bepridil,
pimozide, ondansetron (IV)
aNot all inclusive. Refer to drug information resources regarding medications that may prolong the QT interval. Abbreviations: CYP, cytochrome P450; IV, intravenous. QT prolonging agentsa
Antiarrhythmics
Amiodarone, disopyramide,
procainamide, quinidine, sotalol
QTc prolongation and
related consequences. Avoid
with ri
Other
Chloroquine, halofantrine,
clarithromycin, haloperidol,
methadone, moxifloxacin, bepridil,
pimozide, ondansetron (IV)
aNot all inclusive. Refer to drug information resources regarding medications that may prolong the QT interval. Abbreviations: CYP, cytochrome P450; IV, intravenous. Avoid coadministration
with ribociclib Chloroquine, halofantrine,
clarithromycin, haloperidol,
methadone, moxifloxacin, bepridil,
pimozide, ondansetron (IV) aNot all inclusive. Refer to drug information resources regarding medications that may prolong the QT interval. Abbreviations: CYP, cytochrome P450; IV, intravenous. Abemaciclib neutropenia, leukopenia, fatigue, and nausea [37]. Overall, nine
(2.6%) patients in the palbociclib plus fulvestrant group discon-
tinued therapy due to adverse events, compared with three
(1.7%) patients in the fulvestrant group. Dosing for abemaciclib is 200 mg every 12 hours, given continu-
ously throughout the cycle [45]. Abemaciclib undergoes exten-
sive hepatic metabolism in humans. In vitro and in vivo studies
have identified CYP3A as the enzyme responsible for the major-
ity of the CYP-mediated metabolism of abemaciclib and its
metabolites [50]. This suggests that concomitant use of strong
CYP3A
inducers
or
inhibitors
should
be
avoided
with
abemaciclib. Ribociclib As with palbociclib, hematologic adverse events, including neu-
tropenia, are common with ribociclib; therefore, a 1-week rest-
ing period is incorporated into dosing regimens in most trials,
although continuous dosing continues to be explored [17, 43]. In MONALEESA-2, the most common adverse events of any
grade were neutropenia, nausea, infections, fatigue, and diar-
rhea (Table 3) [44]. The majority of the non-hematologic
adverse events were grade 1 or 2 [44]. The most common
grade 3 or 4 adverse events were neutropenia, leukopenia,
hypertension, increased alanine aminotransferase (ALT) level,
lymphopenia, and increased asparatate aminotransferase (AST)
level [44]. An increase of more than 60 msec from baseline in
the Fridericia’s correction formula (QTcF) interval occurred in
nine patients (2.7%) in the ribociclib group, and 11 patients Increased exposure of
palbociclib and ribociclib. Oc AlphaMed Press 2017 Spring, Zangardi, Moy et al. 1043 Avoid coadministration
with ribociclib Management of CDK4/6 Inhibitors in Breast Cancer Management of CDK4/6 Inhibitors in Breast Cancer 1044 Table 3. Common adverse events for the palbociclib plus letrozole group in PALOMA-2 [51] and the ribociclib plus letro-
zole group in MONALEESA-2 [44]
Palbociclib plus letrozole
(n 5 444)
Ribociclib plus letrozole
(n 5 334)
Adverse event
Any grade,
n (%)
Grade 3,
n (%)
Grade 4,
n (%)
Any grade,
n (%)
Grade 3,
n (%)
Grade 4,
n (%)
Any adverse event
439 (98.9)
276 (62.2)
60 (13.5)
329 (98.5)
221 (66.2)
50 (15.0)
Neutropenia
353 (79.5)
249 (56.1)
46 (10.4)
248 (74.3)
166 (49.7)
32 (9.6)
Leukopenia
173 (39.0)
107 (24.1)
3 (0.7)
110 (32.9)
66 (19.8)
4 (1.2)
Fatigue
166 (37.4)
8 (1.8)
0
122 (36.5)
7 (2.1)
1 (0.3)
Nausea
156 (35.1)
1 (0.2)
0
172 (51.5)
8 (2.4)
0
Anemia
107 (24.1)
23 (5.2)
1 (0.2)
62 (18.6)
3 (0.9)
1 (0.3)
Headache
95 (21.4)
1 (0.2)
0
74 (22.2)
1 (0.3)
0
Diarrhea
116 (26.1)
6 (1.4)
0
117 (35.0)
4 (1.2)
0
Thrombocytopenia
69 (15.5)
6 (1.4)
1 (0.2)
NA
NA
NA
Palbociclib dose was 125 mg daily orally, 3 weeks on, 1 week off. Ribociclib dose was 600 mg daily orally. Abbreviation: NA, no data. Table 3. Common adverse events for the palbociclib plus letrozole group in PALOMA-2 [51] and the ribociclib plus letro-
zole group in MONALEESA-2 [44] erse events for the palbociclib plus letrozole group in PALOMA-2 [51] and the ribociclib plus letro-
EESA-2 [44] (3.3%) had at least one average QTcF interval of more than 480
msec after baseline [44]. No cases of Torsades de Pointes were
reported in MONALEESA-2 [44]. A few considerations related to CDK4/6-induced neutrope-
nia should be noted. First, although neutropenia is a common
side effect of cytotoxic agents, the neutropenia associated with
CDK4/6 inhibitors is distinct in that it is rapidly reversible, reflect-
ing a cytostatic effect on neutrophil precursors in the bone mar-
row
[17]. Recent
work
to
explore
the
mechanism
of
hematologic toxicity induced by palbociclib utilized human bone
marrow mononuclear cells to demonstrate that palbociclib-
induced bone marrow suppression occurred through cycle
arrest with no apoptosis at clinically relevant concentrations and
resumed proliferation following palbociclib withdrawal, thereby
demonstrating pharmacologic quiescence [54]. In contrast,
exposure of the same cells to chemotherapeutic agents resulted
in apoptotic cell death [54]. Abemaciclib Abemaciclib is structurally distinct from palbociclib and riboci-
clib, demonstrating greater selectivity for CDK4 compared with
CDK6 [29]. Hematologic adverse events, including neutropenia,
appear to be less common with abemaciclib, while fatigue and
gastrointestinal-related toxicity are more predominant [17]. The most common treatment-related adverse events in the
MONARCH 1 (NCT02102490) study of abemaciclib monother-
apy in women with pretreated metastatic HR-positive/HER2-
negative breast cancer were creatinine increase (98.5%; grade
35 0.8%), diarrhea (90.2%; grade 35 19.7%), neutropenia
(87.7%;
grade
3/4
26.9%),
and
fatigue
(65.2%;
grade
35 12.9%) [39]. Abemaciclib-induced diarrhea was managea-
ble with conventional antidiarrheal agents or dose reduction
[39]. Diarrhea was experienced early on (median time to
onset5 7 days) and generally resolved quickly (median dura-
tion of grade 2 5 7.5 days, grade 35 4.5 days). Regarding the
creatinine increase, abemaciclib is a competitive inhibitor of
efflux transporters of creatinine (OCT2, MATE1, and MATE2-K);
therefore, cystatin C calculated glomerular fraction rate was
performed to more accurately assess renal function, and this
was not raised [39]. In general, dose interruptions and adjust-
ments are typically considered for grade 3 or higher toxicities. Complete blood count (CBC) with differential needs to be
monitored for all patients on CDK4/6 inhibitors. For palbociclib
and ribociclib, it is recommended to check a CBC with differen-
tial at baseline and every 2 weeks for the first two cycles. For
palbociclib, it is recommended to then check prior to each 28-
day cycle and as clinically indicated. For ribociclib, it is recom-
mended to check at the beginning of each subsequent four
cycles, and then as clinically indicated. Palbociclib and ribociclib
should be held at an absolute neutrophil count (ANC) less than
1,000/mm3 (grade 3) on the first day of each cycle. Further
details on managing day 1 and day 15 neutropenia are included
in Figure 2. Neutropenia is considered complicated if it is asso-
ciated with a documented infection or fever (typically greater
than or equal to 38.58C/100.48F). Criteria for retreatment with
palbociclib or ribociclib following treatment interruption for
neutropenia has generally been when the ANC is greater than
1,000/mm3 with no fever. Management of palbociclib- and Management of CDK4/6 Inhibitors in Breast Cancer Accordingly, palbociclib and riboci-
clib are dosed intermittently to accommodate a break for hema-
tological recovery. Second, while neutropenia is a common
adverse event, the incidence of febrile neutropenia is very low
(0% in PALOMA-1, 0.6% in PALOMA-3, 1.5% in MONALEESA-2). Third, the median duration of grade 3/4 neutropenia is around
7 days, and typically resolves with drug hold [55]. Fourth, neu-
tropenia is proportional to exposure, and often decreases with
subsequent cycles, suggesting a lack of cumulative toxicity and
effective early dose reductions when indicated. Palbociclib In PALOMA-1, the most common adverse events reported for
the palbociclib plus letrozole group were neutropenia, leukope-
nia, and fatigue. Overall, 27 (33%) patients in the palbociclib
plus letrozole group had dose interruptions because of adverse
events, compared with only three (4%) patients in the letrozole
group. Adverse events in the PALOMA-2 trial are outlined in
Table 3. In PALOMA-3, the most common adverse events
reported for the palbociclib plus fulvestrant group were www.TheOncologist.com Oc AlphaMed Press 2017 Palbociclib and Ribociclib: Hematologic Adverse Events
and Management Hematologic adverse events are common with CDK4/6 inhibi-
tors. Cyclin-dependent kinase 6 is particularly important in pro-
moting the proliferation of hematological precursors, and its
inhibition is an on-target effect that results in the cytopenias
seen with CDK4/6 inhibition [52, 53]. In general, most hemato-
logic abnormalities seen with CDK4/6 inhibitors can be
adequately managed with standard supportive care [40]. Oc AlphaMed Press 2017 Spring, Zangardi, Moy et al. 1045 Figure 2. Management of palbociclib and ribociclib-related neutropenia. Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count. Figure 2. Management of palbociclib and ribociclib-related neutropenia. Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count. Figure 2. Management of palbociclib and ribociclib-related neutropenia. Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count. Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count. ribociclib-induced neutropenia is outlined in Figure 2. In
PALOMA-3, if a patient was already receiving palbociclib 75 mg
per day and required another dose reduction, 75 mg daily on a
2 weeks on, 2 weeks off schedule could be utilized [56]. For
grade 3 (<50,000–25,000/mm3) and grade 4 (<25,000 mm3)
thrombocytopenia, treatment was held until recovery to
50,000/mm3 and resumed at one dose level lower. If recurrent
grade 3 or 4 thrombocytopenia occurred, or if recovery of pla-
telet count to 50,000/mm3 took greater than 2 weeks, two
dose reductions were instituted [56]. Similarly, for grade 3
(hemoglobin <8.0 g/dL) and grade 4 (life-threatening conse-
quences; urgent intervention indicated) anemia, treatment
should be held until improvement to grade 2 (hemoglobin
<10.0–8.0 g/dL) or better and resumed at one dose level
lower. Packed red blood cell transfusions should be per institu-
tional protocol. coprescribing antiemetics with ribociclib due to the risk of QT
prolongation. In the absence of signs of infection, diarrhea can
be safely managed with standard non-pharmacologic (hydra-
tion, dietary modification, avoidance of other offending agents)
and pharmacologic (anti-diarrheal agents such as loperamide,
deodorized tincture of opium, diphenoxylate/atropine, octreo-
tide) interventions [58]. Prophylactic treatment can be consid-
ered for more significant diarrhea. Nausea and vomiting should be treated with routine
antiemetics, such as metoclopramide, prochlorpera-
zine, haloperidol, or serotonin 5-HT3 antagonists on an
as-needed basis. Caution should be taken when copre-
scribing antiemetics with ribociclib due to the risk of
QT prolongation. Management of CDK4/6 Inhibitors in Breast Cancer Management of CDK4/6 Inhibitors in Breast Cancer 1046 Table 4. Ribociclib: Dose modification and management for hepatobiliary toxicity [48]
Grade 1
(>ULN to 33 ULN)
Grade 2
(>3 to 53 ULN)
Grade 3
(>5 to 203 ULN)
Grade 4
(>203 ULN)
AST and/or ALT elevations
from baseline,
total bilirubin < 23 ULN
No dose adjustment
required
Dose interruption until
recovery to baseline
grade, then resume
at same dose levela
If grade 2 recurs, resume
at next lower dose level
Dose interruption until
recovery to baseline grade,
then resume at next lower
dose level
If grade 3 recurs, discontinue
ribociclib
Discontinue
ribociclib
Elevation in AST and/
or ALT with total
bilirubin increaseb
Discontinue ribociclib,
irrespective of baseline
grade
aNo dose interruption if at grade 2 at baseline. bIn the absence of cholestasis. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. Table 4. Ribociclib: Dose modification and management for hepatobiliary toxicity [48] abnormalities. Treatment modifications for hepatobiliary toxic-
ity are outlined in Table 4. Similar to palbociclib, alopecia is pos-
sible with ribociclib and is typically grade 1. Ribociclib may
cause embryo-fetal toxicity and effective contraception is rec-
ommended [50]. risk for the patient. Palbociclib and ribociclib should then be
resumed at the next lower dose. Ribociclib: Other Toxicities Fatigue, transaminase elevation, and QTc prolongation are
other notable toxicities reported with ribociclib [44, 48]. Riboci-
clib prolongs the QT interval in a concentration-dependent
manner. A baseline electrocardiogram (ECG) should demon-
strate a QTcF of less than 450 msec prior to initiating therapy. Patients that have or are at a significant risk of developing QTc
prolongation should not receive ribociclib. The QT interval
should be assessed via ECG at baseline, approximately day 14,
the beginning of cycle two, and then as clinically necessary. Avoidance of QT-prolonging agents, as well as appropriate sup-
plementation for electrolyte abnormalities, is recommended. Serum electrolytes should be monitored at baseline, prior to
the first six cycles, and as clinically indicated. Dose Adjustments for Palbociclib Dose Adjustments for Palbociclib
Following dose interruption, the palbociclib dose may need to
be reduced when treatment is resumed. The first dose reduc-
tion is to 100 mg/day and the second and final dose reduction
is to 75 mg/day. Once a dose has been reduced for a given
patient, dose re-escalation should typically be avoided. No spe-
cific dose adjustments are needed for geriatric use, mild
hepatic impairment, or mild to moderate renal impairment. Palbociclib has not been studied in patients with moderate or
severe hepatic impairment (total bilirubin >1.5 3 ULN and any
AST) or in patients with severe renal impairment (CrCl <30 mL/
min). Minimal drug is recovered in the urine, suggesting renal
impairment would not significantly impact drug levels. A study
of palbociclib in renal impairment has been completed, with
results pending (NCT02085538). Palbociclib and Ribociclib: Surgical Procedures Palbociclib and Ribociclib: Surgical Procedures
Count nadir should be avoided at the time of surgery. For pal-
bociclib, it is recommended that patients pursuing surgery hold
therapy for 7 days before the surgery and up to 3 weeks follow-
ing surgery, based on wound healing and recovery. Palbociclib
can be resumed once satisfactory wound healing and recovery
have occurred. Patients should continue endocrine therapy if
palbociclib is held for surgery. There are no specific recommen-
dations for ribociclib surrounding surgery, although we recom-
mend following a similar algorithm to that outlined for
palbociclib. Oc AlphaMed Press 2017 Palbociclib: Other Toxicities A variety of other non-hematologic and non-gastrointestinal
events have been reported with palbociclib, although the vast
majority are grade 1–2 (Table 3).The other most common toxic-
ity seen with palbociclib is fatigue, which is often mild [37]. Thromboembolic events have rarely been reported with palbo-
ciclib, although it is notably listed as precaution in the prescrib-
ing information. In PALOMA-1, two patients (0.6%) had a non-
serious event, and four patients (1.2%) had a serious event
(three pulmonary emboli and one deep-vein thrombosis) in the
palbociclib plus fulvestrant group, versus none in the fulves-
trant alone group [37]. Also, palbociclib may cause embryo-
fetal toxicity and effective contraception is recommended [46]. While typically grade 1, the possibility of alopecia is notable,
with grade 1 alopecia seen in 33% of patients in the palbociclib
group compared with 16% in the letrozole alone group in
PALOMA-2. As noted above, dose hold and subsequent dose
decrease are typically considered for grade 3 or higher
toxicities. Palbociclib and Ribociclib: Gastrointestinal Adverse
Events and Management Nausea and diarrhea can occur with the use of palbociclib and
ribociclib, although rates of grade 3 or 4 gastrointestinal toxic-
ities are low (Table 3). Palbociclib and ribociclib both carry a
minimal to low emetic risk, thus prophylactic antiemetics are
not routinely indicated [57]. Nausea and vomiting should be
treated with routine antiemetics, such as metoclopramide, pro-
chlorperazine, haloperidol, or serotonin 5-HT3 antagonists on
an as-needed basis [57]. Caution should be taken when Other Toxicities: General Management
In general, for non-hematologic toxicities, no dose hold or dose
adjustment are required for grade 1 or 2 toxicities unless they
are persistent and bothersome to the patient. For grade 3 or
higher toxicities that persist despite medical treatment, palbo-
ciclib and ribociclib should be held until symptoms resolve
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interruption for toxicity. The first dose reduction is to 400 mg/
day and the second dose reduction is to 200 mg/day. Ribociclib
should be discontinued if a dose reduction below 200 mg/day
is required. Dose re-escalation is typically not recommended
following modification. Ribociclib is commercially available as
200 mg tablets. No specific dose adjustments are needed for
geriatric use, mild hepatic impairment, or mild to moderate Ribociclib has been associated with hepatobiliary toxicity,
manifesting as transaminase (AST and/or ALT) elevation. Liver
function tests (LFTs) should be checked at baseline, every 2
weeks for the first two cycles, prior to each subsequent four
cycles, and as clinically indicated thereafter. More frequent
monitoring is recommended in the setting of grade 2 Oc AlphaMed Press 2017 Spring, Zangardi, Moy et al. 1047 renal impairment. The recommended starting dose is 400 mg/
day for patients with moderate (Child-Pugh B) or severe (Child-
Pugh C) hepatic impairment.The impact of severe renal impair-
ment (CrCl <30 mL/min) on ribociclib is unknown; however,
the renal route contributes to only a small portion of ribociclib
elimination. A study of ribociclib in healthy patients with vari-
ous degrees of renal impairment is currently recruiting partici-
pants (NCT02431481). renal impairment. The recommended starting dose is 400 mg/
day for patients with moderate (Child-Pugh B) or severe (Child-
Pugh C) hepatic impairment.The impact of severe renal impair-
ment (CrCl <30 mL/min) on ribociclib is unknown; however,
the renal route contributes to only a small portion of ribociclib
elimination. A study of ribociclib in healthy patients with vari-
ous degrees of renal impairment is currently recruiting partici-
pants (NCT02431481). modification. Gastrointestinal toxicity and fatigue are more
prevalent with abemaciclib. Additional research is needed to
better understand the toxicity profile and develop manage-
ment strategies to minimize drug interruptions to optimize the
highest possible therapeutic efficacy for patients with breast
cancer. ACKNOWLEDGMENTS Dr. Bardia is supported by National Cancer Institute grant
5K12CA087723-13. Dr. Spring is supported by National Cancer
Institute grant 5T32CA071345-19. Dr. Bardia is supported by National Cancer Institute grant
5K12CA087723-13. Dr. Spring is supported by National Cancer
Institute grant 5T32CA071345-19. CONCLUSION CDK4/6 inhibitors are generally well-tolerated oral agents. Pal-
bociclib is currently approved for the first-line treatment of
metastatic HR-positive breast cancer in combination with letro-
zole and in the second-line setting in combination with fulves-
trant. Ribociclib is approved for the first-line treatment of
metastatic HR-positive breast cancer in combination with any
aromatase inhibitor. Abemaciclib has been granted Break-
through Therapy designation by the FDA. The most common
adverse event associated with palbociclib and ribociclib is neu-
tropenia; however, febrile neutropenia is rare and the neutro-
penia is generally easily reversible when the drug is held. Ribociclib is associated with hepatobiliary toxicity and QT pro-
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lence of retinoblastoma protein loss in triple- AUTHOR CONTRIBUTIONS AUTHOR CONTRIBUTIONS
Conception/design: Laura M. Spring, Aditya Bardia
Provision of study material or patients: Laura M. Spring, Mark Zangardi, Aditya
Bardia
Collection and/or assembly of data: Laura M. Spring, Mark Zangardi
Data analysis and interpretation: Laura M. Spring, Mark Zangardi, Beverly Moy,
Aditya Bardia
: Laura M. Spring, Mark Zangardi, Aditya Bardia Conception/design: Laura M. Spring, Aditya Bardia
Provision of study material or patients: Laura M. Spring, Mark Zangardi, Aditya
Bardia Data analysis and interpretation: Laura M. Spring, Mark Zangardi, Beverly Moy,
Aditya Bardia
L
M S
i
M k Z
di Adi
B di Manuscript writing: Laura M. Spring, Mark Zangardi, Aditya Bardia
Final approval of manuscript: Laura M. Spring, Mark Zangardi, Beverly Moy,
Aditya Bardia Manuscript writing: Laura M. Spring, Mark Zangardi, Aditya Bardia
Final approval of manuscript: Laura M. Spring, Mark Zangardi, Beverly Moy,
Aditya Bardia www.TheOncologist.com www.TheOncologist.com Oc AlphaMed Press 2017 Management of CDK4/6 Inhibitors in Breast Cancer 1048 kinase inhibitor, administered in 3-week cycles
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English
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Analyzing Visual Metaphor and Metonymy to Understand Creativity in Fashion
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Frontiers in psychology
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cc-by
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Analyzing Visual Metaphor and
Metonymy to Understand Creativity
in Fashion Ryoko Uno1*, Eiko Matsuda2 and Bipin Indurkhya3 1 Division of Language and Culture Studies, Institute of Engineering, Tokyo University of Agriculture and Technology, Tokyo,
Japan, 2 Division of Advanced Information Technology and Computer Science, Institute of Engineering, Tokyo University
of Agriculture and Technology, Tokyo, Japan, 3 Department of Cognitive Science, Institute of Philosophy, Jagiellonian
University, Kraków, Poland The role of figurative language such as metaphor and metonymy in creativity has been
studied in cognitive linguistics. These methods can also be applied to analyze non-
linguistic data such as pictures and gestures. In this paper, we analyze fashion design
by focusing on visual metaphor and metonymy. The nature of creativity in fashion design
has not been fully studied from a cognitive perspective compared to other related fields
such as art. We especially focus on fashion design as a communication tool between
the designer and audience in conveying a designer’s image of human beings. Photos
from two fashion shows were analyzed. We carried out an experiment to compare how
human images in two shows are interpreted by those who are familiar with fashion and
those who are not. We obtained three results: (1) As far as figurative (metaphorical and
metonymic) interpretations of human images are concerned, two groups with different
levels of familiarity with fashion had significantly different patterns of responses to two
shows. (2) For the non-figurative interpretations (such as physical or personal attributes),
no significant difference in the pattern of response to the show was observed between
the two groups. However, the participants as a whole responded to the two shows
differently. (3) In addition, for the non-figurative interpretations, the fashion experts found
significantly more attributes in human images than the other group. The results show
that the analysis of figurative interpretations is effective in understanding how familiarity
with fashion affects the mode of seeing fashion shows. *Correspondence:
Ryoko Uno Specialty section:
This article was submitted to
Human-Media Interaction,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Human-Media Interaction,
a section of the journal
Frontiers in Psychology
Received: 14 August 2018
Accepted: 27 November 2018
Published: 07 January 2019
Citation:
Uno R, Matsuda E and
Indurkhya B (2019) Analyzing Visual
Metaphor and Metonymy
to Understand Creativity in Fashion. Front. Psychol. 9:2527. doi: 10.3389/fpsyg.2018.02527 ORIGINAL RESEARCH
published: 07 January 2019
doi: 10.3389/fpsyg.2018.02527 Keywords: fashion design, creativity, cognitive linguistics, metaphor, metonymy INTRODUCTION Received: 14 August 2018
Accepted: 27 November 2018
Published: 07 January 2019 An influential designer in the fashion industry today, Karl Lagerfeld, once said “Fashion is a
language that creates itself in clothes to interpret reality.” Fashion design is an act of creating what
people put on, and people are always presupposed in this process. Thus, to design fashion is to
design the human image that sustains this fashion. Lagerfeld’s declaration that fashion interprets
reality also suggests that fashion proposes an image of the human whose fashion reflects reality. When we focus on fashion as a creation of human images, we make a case to examine fashion
within cognitive science. Edited by:
Gerrit C. Van Der Veer,
University of Twente, Netherlands Edited by:
Gerrit C. Van Der Veer,
University of Twente, Netherlands
Reviewed by:
Danzhu Li,
University of Twente, Netherlands
Barbara
Lewandowska-Tomaszczyk,
State University of Applied Sciences
in Konin, Poland
*Correspondence:
Ryoko Uno
ryokouno@cc tuat ac jp Reviewed by:
Danzhu Li,
University of Twente, Netherlands
Barbara
Lewandowska-Tomaszczyk,
State University of Applied Sciences
in Konin, Poland *Correspondence:
Ryoko Uno
ryokouno@cc.tuat.ac.jp Citation: For example, in the Cambridge Handbook of Creativity
(Kaufman and Sternberg, 2010), while there are independent
chapters on art (Locher, 2010) and product design (Cropley and
Cropley, 2010), fashion does not even appear in the index. Not many studies of fashion creation take on a cognitive
perspective, although some have employed a sociological
perspective. Kawamura (2005) proposed a field of study called
“fashion-ology,” pointing out that fashion and clothing are two
different things: the former, which is invisible, is realized through
the latter, which is visible. The origin of the term “fashion,” as it
is currently used, can be traced to 1489 (Brenninkmeyer, 1963). Though fashion was limited to the upper class gentry in the
15th century, it became more democratic in the 20th century. This is only one of the many aspects telling us that the meaning
of fashion has shifted over years. Nonetheless, “change” and
“novelty” are still regarded as two main characteristics of fashion
(Kawamura, 2005). Even in the sociological context, there is no consensus on
whether fashion can be classified as art (Miller, 2007). Moreover,
Skov et al. (2009) argue that even when we see fashion as art,
there are two different interpretations. On one hand, fashion can
be seen as an everyday art form. For example, Wilson (1985)
describes fashion as a way to express modernity, even by ordinary
people. On the other hand, we can see fashion designers as artists. In this perspective, the most important event in fashion is the
fashion show: it is an event where the fashion designer is in
complete control of esthetic concepts. Fashion shows began by
conveying the image of fashion to end customers. However, their
role has changed since the 1960s, with the rise of fashion photos
and magazines (Moeran, 2006), and the press became the main
audience of these shows, freeing them from having to promote
sales and making them autonomous (Skov et al., 2009). In figurative language, metaphor and metonymy are the
most widely studied phenomena. Metonymy is also a non-literal
expression, but here, the shift from the literal meaning to a
figurative meaning is based on contiguity, not similarity. For
example, when we say, “We need more hands,” we mean that we
need more people who can help us, not only their hands. We
can refer to humans as a whole by their parts such as hands or
heads because they are close. Citation: January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org Metaphor and Metonymy in Fashion Uno et al. uses a form to express a meaning that is similar to the literal
meaning, an example being “time is money.” Literally, time is
not money. The reason why time is said to be money in this
expression is because there is a common property between them:
being “valuable.” Thus, to understand the concept of time, we
use the concept of money. It is a mechanism to expand our
understanding of things by utilizing already acquired knowledge. Lakoffand Johnson (1980) point out that mapping the knowledge
structure of money to that of time is happening at the conceptual
level, not the verbal level. To confirm this point, Thibodeau
and Boroditsky (2011, 2013) carried out experiments to show
that by presenting different metaphors, we can affect people’s
choice of actions. As Forceville (2009, 2016) notes, if metaphor
is conceptual, it cannot be limited to language. Among cases
of non-verbal metaphor, the most studied are those in pictures
and gestures. Visual metaphor has been studied in comics,
advertisements, and films but not in fashion. For example,
in Japanese comics, background drawings can metaphorically
represent the mental state of the protagonist (Shinohara and
Matsunaka, 2009). Consider an example of visual metaphor in
advertisement from Indurkhya and Ojha (2017). In the image
shown in Indurkhya and Ojha (2017), 29, the man at the center
is wearing headphones and is listening to music at an airport. He is surrounded by people whose heads are replaced with loud
speakers. Here, the visual metaphor is formed by mapping loud
speakers to human heads. This suggests that the sound emitted
by people is as loud as the sound from a loud speaker. In
other words, people are noisy. The property of a loud speaker
(“emitting loud noise”) can be naturally projected onto a human
being (“noisy person”). Following Lakoffand Johnson (1980), the role of figurative
language, especially metaphor, in creative thought has been
widely studied. Not only verbal but also visual metaphor and
metonymy have been analyzed to reveal dynamism in conceptual
structures (Indurkhya and Ojha, 2013). This paper seeks to
reveal creativity in fashion by focusing on visual metaphor and
metonymy. Compared to related fields such as art or product
design, creativity in fashion has not received much scholarly
attention. Citation: Another technique of metonymy
is referring to a person by what he or she is wearing. The
famous “little red riding hood” is a good example. It refers to
the girl, not her hood. What we put on is close to us. While
metaphor functions by expanding our understanding, metonymy
functions via landmarks (Langacker, 2008; Nishimura, 2008). The
metonymy can also be observed in other modalities: Catalano and
Waugh (2013) analyzed both verbal and visual metonymies and
showed how they contributed to shaping public opinion. In this paper, we take a cognitive scientist’s point of view to
capture the essence of fashion, i.e., the creation of human images,
and follow the abovementioned position that regards the designer
as an artist. We use photos from fashion shows, for fashion shows
reflect the designer’s images directly, as argued by Skov et al. (2009). We understand the show as a setting to communicate
human images from the designer to the audience through the
design medium. In this case, the designer and the trained
audience share creativity in fashion. In the research presented
here, by focusing on visual metaphor and metonymy, we examine
how photos from fashion shows are variously interpreted by those
who are familiar with fashion compared to those who are not. In brief, both metaphor and metonymy are seen in visual
modality and contribute to changing the cognitive state of
people. However, they have not been applied to analyze human
images in fashion shows. In the present research, our goal is to
investigate how an expert fashion show viewer understands the
fashion show differently from a non-expert viewer by focusing on
figurative (i.e., metaphorical and metonymic) and non-figurative
interpretations. Our results show that it is only by paying
attention to the figurative interpretations that we can understand
the characteristics of the way people who are familiar with fashion
see human images in fashion shows. January 2019 | Volume 9 | Article 2527 Visual Metaphor, Visual Metonymy Visual Metaphor, Visual Metonymy
In cognitive linguistics, the role of figurative language, especially
metaphor, in creativity has been studied (Lakoffand Johnson,
1980; Indurkhya, 1992). Metaphor is a non-literal expression that January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org 2 Metaphor and Metonymy in Fashion Uno et al. MATERIALS AND METHODS of them have been in employment. The participants were
all Japanese except for two Korean students in the fashion
group and one Chinese student in the non-fashion group. We did not check the personal cultural backgrounds of the
participants in the experiment, but the Japanese students in both
schools were mostly brought up in Japan. All participants were
proficient in Japanese, which ensured that they understood the
instructions. They participated in the experiment in their own
classrooms. Ethics Statement This study was approved by the Institutional Review Board
(IRB) of Tokyo University of Agriculture and Technology,
Tokyo, Japan. All participants gave written informed consent in
accordance with the Declaration of Helsinki. Participants Ninety-six participants took part in the experiment. This
included 24 students from a private fashion design school (62.5%
female) and 72 university students majoring in scientific fields
(47.22% female). Henceforth, we call the former set of students
the fashion group and the latter the non-fashion group. The
fashion group participants were between 16 and 30 years old
(average 25.65 years), and the non-fashion group participants
were between 19 and 22 years old (average 19.68 years). The
difference in the age groups between the two sets of participants
reflects the differences between their schools. In Japan, most
students enter undergraduate school just after finishing high
school, which is at the age of 18. Conversely, in fashion
schools, students come from various backgrounds, and many Stimuli Two sets of pictures, each from different fashion shows, were
chosen and shown on the question sheet (permission was
obtained from the copyright holders). In each picture, a model
walking the runway was shown from his or her front or
back. One show was the 2009–2010 Autumn Winter Collection
by writtenafterwards with the designer Yoshikazu Yamagata. Figure 1A shows the photos chosen from this show. The
other show was the 2014–2015 Autumn Winter Collection by
Mikio Sakabe with designers Mikio Sakabe and Shueh Jen-Fang. Figure 1B shows the photos used in the questionnaire from this
show. Both shows were held during the Japan Fashion Week in FIGURE 1 | Photos presented in the experiment: Photos from the two fashion shows: the garbage show (A) the letter show (B). The larger photos are the target
photos. These images are being reproduced with the permission of the copyright holder [(A) writtenafterwards inc; (B) Fashionsnap.com]. The original sources
appeared in (A) http://www.writtenafterwards.com; (B) https://www.fashionsnap.com/. FIGURE 1 | Photos presented in the experiment: Photos from the two fashion shows: the garbage show (A) the letter show (B). The larger photos are the target
photos. These images are being reproduced with the permission of the copyright holder [(A) writtenafterwards inc; (B) Fashionsnap.com]. The original sources
d i
(A) htt
//
itt
ft
d
(B) htt
//
f
hi
/ FIGURE 1 | Photos presented in the experiment: Photos from the two fashion shows: the garbage show (A) the letter show (B). The larger photos are the target
photos. These images are being reproduced with the permission of the copyright holder [(A) writtenafterwards inc; (B) Fashionsnap.com]. The original sources
appeared in (A) http://www.writtenafterwards.com; (B) https://www.fashionsnap.com/. FIGURE 1 | Photos presented in the experiment: Photos from the two fashion shows: the garbage show (A) the letter show (B). The larger photos are the target
photos. These images are being reproduced with the permission of the copyright holder [(A) writtenafterwards inc; (B) Fashionsnap.com]. The original sources
appeared in (A) http://www.writtenafterwards.com; (B) https://www.fashionsnap.com/. January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org 3 Metaphor and Metonymy in Fashion Uno et al. Tokyo. All three designers teach at the private fashion school
where the fashion group participants studied. photos from one of the two collections, as shown in Figure 1. The photo images were all in color. Ratings for Figurative and Non-figurative
Interpretations p
After all the data were collected, the answers (freely written
descriptions of human images represented by the two target
photos) were rated in six categories by three coders who worked
separately. There were two groups and six types of categories to
rate. The first group included categories that showed how the
figurative interpretations of the human images were working. The figurative group included the categories “metonymic” and
“metaphorical.” These were the two main categories observed in
this research. To determine how figurative interpretations work,
we also put together the non-figurative interpretations group. This second group comprised four categories characterizing
human personality: physical, personal, relational, and social role. This categorization was based on Smilek et al. (2007), who
analyzed the characteristics of a personality description of an
object by a participant with synesthesia. The details of the ratings
by each coder are shown in the Supplementary Material. y
We chose two pictures that met these criteria as target pictures. To begin with, Yamagata and Sakabe (2013), who were the
designers of the target photos, explicitly mentioned in a book
they co-authored that they present human images at fashion
shows: on the cover of this book is written, “Beyond making
clothes, we make a new human.” Moreover, these pictures
can be interpreted both metaphorically and metonymically. Metonymic interpretations of clothes are not difficult because the
closeness between the clothes and the person wearing them is
apparent. However, not many fashion photos can easily induce
metaphorical interpretation. In the first target photo (the large
photo in Figure 1A), instead of ordinary clothes, the model
is wearing some garbage that was originally collected from
architecture departments at universities. We can say that the
garbage replaces the clothes, just like speakers replace heads as
in the example presented before. In the second target photo (the
large photo in Figure 1B), we can say that instead of clothes, the
model is wearing a letter on his back. This is a Chinese letter
used in Japanese and means “love.” Finally, the two shows are
distinctive. When we compare garbage and a letter, the former is
a substance (or a texture), and the latter is an object. In addition,
the former is concrete, and the latter is abstract. RESULTS Each set contained a target photo: the participants were asked
to describe the human image in the photo. A further three photos
in the set were presented to the participants so as to contextualize
the target photo. We used three criteria to choose the target
pictures. First, the show must target displays of the human image
and not just clothes. Second, since the current research analyzes
the distribution of metaphorical and metonymic interpretations,
the picture must be open to both kinds of interpretations. Third,
two target photos must be distinctly different so as to determine
whether the participants responded differently to them. Ratings for Figurative and Non-figurative
Interpretations Among the answers by the participants, those that did not
describe a human image at all were excluded: for example,
answers such as “I don’t know” or comments about the designer. For the remaining answers, the coders were asked to rate how
well each answer (the description of human image) matched the
attributes in the six categories. The rating was carried out using a
six-point Likert scale (1, matches completely; 6, does not match at
all). Each category was explained to the coders via an instruction
sheet. The descriptions of the six categories provided to the
coders are given below. Some examples from the participants’
answers are also shown below after the descriptions of each
category. These eight examples were all recognized by the coders
to generally match their respective categories. Two coders gave a
1, and one coder gave a 2 for the answers in (2, 3, and 4). The
coders rated all the remaining examples as 1. Henceforth, we call the former show the garbage show and
the latter show the letter show. Among the participants, only one
from the non-fashion group knew of the garbage show before the
experiment. No one knew the letter show. Conversely, among the
fashion group, twelve participants knew about both shows, four
knew only the garbage show, and one knew only the letter show. For the transcription of the Japanese examples, the Hepburn
system of Romanization was used. The following abbreviations
were used in the word-to-word translations: ACC for accusative,
COMP for complementizer, GEN for genitive, NOM for
nominative, PASS for passive, PAST for past tense, and POL for
polite. Stimuli Two types of sheets were
prepared: the first type presented the garbage show first, while the
other presented the letter show first. We randomly assigned either
one of the sheets to the participants. For each set of pictures,
we asked: “The following four pictures are from a fashion show. Look at the third large photo and answer what kind of human
is represented by this photo.” We had additional information
on the questionnaire for the participants. For the garbage show
photos, there was an explanation that the material used for the
clothes was garbage. For the letter show photos, there was an
explanation that read that what was on the back of the model was
a letter. Though the designers featured in the two sets of pictures
mainly sell their products in Japan, they are internationally
recognized. For example, Yamagata and Sakabe were shortlisted
for the LVMH Prize in 2015 and 2016, respectively. The LVMH
prize is launched by LVMH Moët Hennessy Louis Vuitton SE
in order to honor young fashion designers around the world. In
addition, all three designers were educated in Europe. Yamagata
studied at Central Saint Martins College of Arts and Design
(current Central Saint Martins, University of the Arts London) in
the United Kingdom, Sakabe at the Royal Academy of Fine Arts,
Antwerp, Belgium, and Shueh at La Cambre in Brussels, Belgium. Yamagata and Sakabe both graduated at the top of their respective
classes. (2) Metonymic (3) Metaphorical
shakai no haguruma to-shite tsukai sute-rareru hito
society GEN cog as use discard-PASS person
“A person who is used and discarded as a cog in a wheel in
society.” (2) Metonymic (2) Metonymic
mukashi no bushoh no kabuto no ai no
old.days GEN warrior GEN helmet GEN love GEN
imeeji ga tsuyoku isamashii hito ni kanji-ta
image NOM strong gallant person as feel-PAST
“The letter ‘ai ( = love)’ strongly reminds me of the letter
on the warrior’s helmet in old days, so I felt him as a gallant
person.” (8) Social role
yakuza
gangster
“A gangster.” The second example requires some explanation. In old Japan,
military commanders wore a helmet with a Chinese character on
the front as a part of the design. One of the famous commanders
wore a helmet with the letter ai. The answer in (2) refers to this
helmet. After all the answers were rated by the coders, two procedures
were followed before the analysis. First, we converted six-point
ratings into binary values. Ratings 1–3, which were affirmative to
the matching, were replaced with the value 1. Ratings 4–6, which
were negative evaluations of the matching, were replaced with the
value 0. Next, we merged the three ratings (one by each coder)
for each answer as follows. When there was a 1 by any coder in a
given category, the final rating was set to 1. Only when the ratings
by all the three coders were 0 was the final rating set to 0. After all the answers were rated by the coders, two procedures
were followed before the analysis. First, we converted six-point
ratings into binary values. Ratings 1–3, which were affirmative to
the matching, were replaced with the value 1. Ratings 4–6, which
were negative evaluations of the matching, were replaced with the
value 0. Next, we merged the three ratings (one by each coder)
for each answer as follows. When there was a 1 by any coder in a
given category, the final rating was set to 1. Only when the ratings
by all the three coders were 0 was the final rating set to 0. Next, for the metaphorical category, the coders were
instructed that the answer would match the attribute in the
metaphorical category when it expressed a person who is similar
to garbage or the letter (or the concept of love). The answer in (3)
is an example that was judged by a coder to describe a person as
being similar to garbage, i.e., someone who can be abandoned by
society. Frontiers in Psychology | www.frontiersin.org Procedure The participants were asked to answer two questions on the
questionnaire by writing freely. Each side of the paper had four January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org 4 Metaphor and Metonymy in Fashion Uno et al. synesthesia. The instruction sheet provided some examples of
each category to the coders as follows: Physical attributes include
features such as male, in 40s, black hair, Japanese, etc. Personal
attributes include serious, optimistic, mischievous, etc. Relational
attributes include friendly, dominant, etc. Finally, social role
attributes include younger brother, god, leader, etc. The following
are some examples judged by the coders as belonging to each
category: synesthesia. The instruction sheet provided some examples of
each category to the coders as follows: Physical attributes include
features such as male, in 40s, black hair, Japanese, etc. Personal
attributes include serious, optimistic, mischievous, etc. Relational
attributes include friendly, dominant, etc. Finally, social role
attributes include younger brother, god, leader, etc. The following
are some examples judged by the coders as belonging to each
category: For the metonymic category, the instruction sheet for the
coders instructed that the answer matches this category when it
expresses a person who is close (or contiguous) to garbage or
a letter. It is also noted that this “closeness” is physical, such
as spatiotemporal proximity or causation. Thus, closeness does
not include similarity. Another noteworthy point is that for the
letter collection, not only should we consider the closeness to the
letter but also the closeness to the concept of “love” (which is the
meaning of the letter). This category includes someone who has
garbage or the letter (or the concept of love) on his or her body
or someone who is staying close to garbage or the letter (or the
concept of love), etc. Example (1) expresses a person who is close
to garbage and (2) expresses a person who is close to the letter. Procedure (5) Physical
kireina onna-no-hito
beautiful woman
“A beautiful woman.” (5) Physical
kireina onna-no-hito
beautiful woman
“A beautiful woman.” (5) Physical
kireina onna-no-hito
beautiful woman
“A beautiful woman.” (1) Metonymic
tsukaeru mono wa hirot-teshimau youna hito to kanji-masu
useful thing TOP pick.up-PERF like person COMP feel-POL
“I feel that she is a kind of person who picks up whatever she
finds useful.” (6) Personal
kurai hito
dark person
“A gloomy person.” (7) Relational
mawari to no kyohchoh yori-mo
people.around with GEN cooperation rather.than
jishin no ishi o sonchoh-suru kanji ga shi-masu. oneself GEN will ACC respect feeling NOM do-POL
“I feel that this person respects her will more than
cooperating with people around her.” (7) Relational
mawari to no kyohchoh yori-mo
people.around with GEN cooperation rather.than
jishin no ishi o sonchoh-suru kanji ga shi-masu. oneself GEN will ACC respect feeling NOM do-POL
“I feel that this person respects her will more than
cooperating with people around her.” (2) Metonymic
mukashi no bushoh no kabuto no ai no
old.days GEN warrior GEN helmet GEN love GEN
imeeji ga tsuyoku isamashii hito ni kanji-ta
image NOM strong gallant person as feel-PAST
“The letter ‘ai ( = love)’ strongly reminds me of the letter
on the warrior’s helmet in old days, so I felt him as a gallant
person.” Analysis of Figurative Interpretations In this part, we focus on the figurative interpretations, namely,
metonymic and metaphorical categories. As noted above, the
answers to each question were converted into binary values for
each of the two categories (metonymic or metaphorical). We
refer to these binary values as “category degrees” for further
analysis. The following example (4) was judged by a coder to describe a
person who is similar to the letter: Taking the category degree for the objective variable, a three-
way ANOVA was performed for the photos (garbage or letter),
participant groups (fashion or non-fashion), and category types
(metonymic or metaphorical). There were significant interactions
among the photos, the participant groups, and the category
types [α = 5%, F(1, 370) = 9.04, p = 0.0028], and between the
photos and the category types [F(1, 370) = 4.11, p = 0.043]. No
significant interactions were observed between the photos and 4) Metaphorical
shuchoh no tsuyoi hito
assersion GEN strong person
“An assertive person.” (4) Metaphorical
shuchoh no tsuyoi hito shuchoh no tsuyoi hito assersion GEN strong person assersion GEN strong person
“An assertive person.” “An assertive person.” For the non-figurative group, we followed the categorization
used in the study by Smilek et al. (2007), which analyzed
the personification of inanimate objects by a participant with January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org 5 Metaphor and Metonymy in Fashion Uno et al. the participant groups [F(1, 370) = 1.35, p = 0.25] or between
the participant groups and the category types [F(1, 370) = 2.40,
p = 0.12]. Therefore, we analyzed the data from the fashion
and non-fashion participant groups separately so as to find the
sub-effects between the photos and category types. For each
participant group, a two-way ANOVA was conducted for the
photos and category types. physical, personal, relational, and social role. Similar to the
figurative interpretations, we used the category degree for our
analysis. Taking the category degree for the objective variable, a three-
way ANOVA was performed on the photos (garbage or letter),
participant groups (fashion or non-fashion), and category types
(physical, personal, relational, and social role). Analysis of Figurative Interpretations There were no
significant interactions among the photos, the participant groups,
and the category types [α = 5%, F(3, 740) = 0.72, p = 0.54],
between the photos and the participant groups [F(1, 740) = 0.39,
p = 0.53], or between the participant groups and the category
types [α = 5%, F(3, 740) = 1.80, p = 0.15]. A significant interaction
was observed between the photos and the category groups [F(3,
740) = 8.03, p < 0.0001]. Significant main effects were observed
in the photos [F(1, 740) = 6.4678, p = 0.011], the participant
groups [F(1, 740) = 16.86, p < 0.0001], and the category types
[F(3, 740) = 81.58, p < 0.0001]. Regarding
the
fashion
group,
there
was
a
significant
interaction between the photos and the category types [α = 5%,
F(1,88) = 9.2933, p = 0.0030]. We therefore proceeded to analyze
the simple main effects of the interaction. Figure 2 shows
the mean category degree for metonymic and metaphorical
categories, respectively (Figure 2 upper: metonymic, lower:
metaphorical). The x-axis denotes photos (garbage or letter),
and the y-axis indicates the mean category degree. The error
bars denote SEM. The results of the simple main effects
show a significant difference for the metonymic category [F(1,
88) = 9.6561, p = 0.0025] but not for the metaphorical category
[F(1, 88) = 1.4491, p = 0.2319], which are summarized in Figure 2
by the notation, ∗∗∗p < 0.01, n.s. p ≥0.05. We therefore focused on the simple interaction between the
photos and the category degrees without taking the participant
groups into account. Figure 3 depicts the mean category degree
for each category (a: physical, b: personal, c: relational, d: social
role). The x-axis denotes photos (garbage or letter), and the y-axis
indicates the mean category degree. The error bars denote SEM. The results of the simple main effects show a significant difference
for the physical category [α = 5%, F(1,740) = 5.4153, p = 0.0202],
the personal category [F(1,740) = 3.9691, p = 0.0467], and the
relational category [F(1, 740) = 20.3284, p < 0.0001], but not
for the social role category [F(1, 740) = 0.8327, p = 0.3618]. The
results are summarized in Figure 3 by the notation, ∗∗∗p < 0.01,
∗p < 0.05, n.s. p ≥0.05. Regarding the non-fashion group, the mean category degree
for each statistical group is depicted in Figure 1B. Analysis of Figurative Interpretations There was
neither any significant interaction between the photos and
category types [α = 5%, F(1, 282) = 0.9567, p = 0.3289] nor main
effects in the photos [F(1, 282) = 0.0096, p = 0.9222] and category
types [F(1, 282) = 3.748, p = 0.0539]. Analysis of Non-figurative Interpretations In this section, we focus on the remaining categories reflected
in the non-figurative interpretations. The categories were FIGURE 2 | Mean tag degree across all participants. The x-axis denotes the photo (garbage or letter), while the y-axis indicates the mean category degree. The error
bars indicate SEM. The results of two-way ANOVA are summarized by the notation ∗∗∗p < 0.01, n.s. p ≥0.05. (A) Results from the fashion group. (B) Results from
the non-fashion group. FIGURE 2 | Mean tag degree across all participants. The x-axis denotes the photo (garbage or letter), while the y-axis indicates the mean category degree. The error
bars indicate SEM. The results of two-way ANOVA are summarized by the notation ∗∗∗p < 0.01, n.s. p ≥0.05. (A) Results from the fashion group. (B) Results from
the non-fashion group. January 2019 | Volume 9 | Article 2527 6 Frontiers in Psychology | www.frontiersin.org Metaphor and Metonymy in Fashion Uno et al. Uno et al. FIGURE 3 | Mean tag degree across all participants. The x-axis denotes the
photo (garbage or letter), while the y-axis indicates the mean category degree. The error bars indicate SEM. Each sub-figure shows the results for each
category: (A), physical; (B), personal; (C), relational; and (D), social role. The
results of two-way ANOVA are summarized by the notation ∗∗∗p < 0.01,
∗p < 0.05, n.s. p ≥0.05. FIGURE 4 | Mean tag degree across all participants. The x-axis denotes the
participant group (fashion or non-fashion), while the y-axis indicates the mean
tag degree. The error bars indicate SEM. The results of two-way ANOVA are
summarized by the notation ∗∗∗p < 0.01, n.s. p ≥0.05. FIGURE 4 | Mean tag degree across all participants. The x-axis denotes the
participant group (fashion or non-fashion), while the y-axis indicates the mean
tag degree. The error bars indicate SEM. The results of two-way ANOVA are
summarized by the notation ∗∗∗p < 0.01, n.s. p ≥0.05. in how the two groups responded to the two shows. The
fashion group had more metonymic interpretations for the
letter show, but there was no significant difference between
the metaphoric and metonymic interpretations for the non-
fashion group. Regarding the non-figurative interpretations, no
significant difference was found between the two groups in
interpreting the two shows. Overall, the participants found more
outer attributes in the garbage show photos and more inner
attributes (personal and relational) in the letter show photos. Analysis of Non-figurative Interpretations In
addition, the fashion group saw more non-figurative attributes in
both shows compared to the non-fashion group. Our general conclusion is that to see how people who are
familiar with fashion understand photos from fashion shows,
simply analyzing the straightforward attributes (non-figurative
interpretations) of the depiction of humans in fashion images
is not enough. The distinctive feature of the fashion group in
understanding a fashion show is reflected in the metaphorical and
metonymic interpretations. The core of figurative interpretation
is how people derive new meanings from a form (Indurkhya,
2007). It is a way to have rich meaning with a limited number
of forms. FIGURE 3 | Mean tag degree across all participants. The x-axis denotes the
photo (garbage or letter), while the y-axis indicates the mean category degree. The error bars indicate SEM. Each sub-figure shows the results for each
category: (A), physical; (B), personal; (C), relational; and (D), social role. The
results of two-way ANOVA are summarized by the notation ∗∗∗p < 0.01,
∗p < 0.05, n.s. p ≥0.05. We further analyzed the main effects of the participant groups. Figure 4 depicts the difference in category degree across the
participant groups. More specifically, we argue that the two shows differ
significantly in the following ways. First, with respect to figurative
interpretations,
the
fashion
group
had
more
metonymic
interpretations for the letter collection. We suggest that this is
because the fashion group focused on the shape of the letter
and the texture of the garbage. The difference between the
shape and the texture is reflected in the language system as the
difference between countable and uncountable nouns or the
difference between nouns in English and Japanese (Nomura,
1996). Moreover, some studies have shown that young children
who just acquired a language are sensitive to the differences
between shape and texture (Soja et al., 1991), and based on the
acquired language, there is a difference in the importance of
features (Imai and Gentner, 1997). It is possible that people who
are familiar with fashion are more sensitive to shape. Frontiers in Psychology | www.frontiersin.org DISCUSSION While creativity in art and design has been studied in cognitive
science, fashion design has received little attention. In this study,
by applying studies in visual metaphor and metonymy, we
investigated the creativity of people familiar with fashion design
in watching fashion shows. While creativity in art and design has been studied in cognitive
science, fashion design has received little attention. In this study,
by applying studies in visual metaphor and metonymy, we
investigated the creativity of people familiar with fashion design
in watching fashion shows. We analyzed the interpretations of the human images
represented in photos from fashion shows presented to two
groups: people with and without knowledge of fashion. When we focused on figurative (metaphorical and metonymic)
interpretations of these photos, we found some key differences January 2019 | Volume 9 | Article 2527 Frontiers in Psychology | www.frontiersin.org 7 Metaphor and Metonymy in Fashion Uno et al. (Schmid, 2008, 2016). However, most linguistic analyses focus
on communication as an exchange of information. Even in the
framework of experimental semiotics (Galantucci, 2005; Steels,
2006; Kirby et al., 2008; Scott-Phillips and Kirby, 2010; Galantucci
and Garrod, 2012), where the aim is to understand the emergence
of communication or language by observing the usage of artificial
communication tools, the main focus is on communication or
a language that delivers information (Uno et al., 2012). As
Clark (1997) points out, humans can extend their cognitive
capacity using artifacts such as mobile phones, search engines, or
language. Fashion is also an artifact for us to extend ourselves. By analyzing metaphor and metonymy in fashion, we can arrive
at clues to analyze fashion as a phenomenon that reflects our
communication capacity. We can expand our study of fashion
design to reveal the mechanisms underlying our communicative
abilities in general. Second, in the non-figurative interpretations, the participants
characterized the garbage show photos with outer traits and
the letter show photos with inner traits. We suggest that this
difference emanates from the concrete versus abstract contrast
between the two shows rather than the texture versus shape
contrast. Needless to say, this research has some limitations. For
example, by using four pictures from each show, we were able
to capture only some parts of the information related to fashion
design in a fashion show. ACKNOWLEDGMENTS From the perspective of human communication, the target
of this analysis is interesting because while we saw fashion
shows as a form of communication between the designer and
the audience in conveying human images, the purpose of this
communication was not to convey the image in a precise manner. The purpose was communication itself, which was done by
sharing the human image. Even with natural languages, we
do not always try to convey messages precisely. For example,
when we are playing with a language (Crystal, 1998), we are
simply communicating for the sake of communicating. This
type of activity can often lead to the creation of new words We would like to thank Daisuke Nonaka, Kazuko Shinohara, and
Shiro Yano for helpful comments. We would like to thank Daisuke Nonaka, Kazuko Shinohara, and
Shiro Yano for helpful comments. DISCUSSION Also, since we only targeted one fashion
school, where the focus is on creative aspects of fashion, we were
unable to ascertain what would have happened had we included
participants from other fashion schools that teach more practical
or commercial aspects of fashion. These points should be taken
into consideration in the following studies. For future research, we plan to study two points: (1) how the
shape versus texture and the abstract versus concrete contrasts
work; (2) whether the different responses to the shows by the
fashion group with figurative interpretations were related to
having more non-figurative interpretations. FUNDING This work was supported by MEXT/JSPS Grant-in-Aid for
Scientific Research on Innovative Areas #4903 (Evolinguistics)
grant no. JP18H05062. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpsyg. 2018.02527/full#supplementary-material AUTHOR CONTRIBUTIONS RU, EM, and BI conceived the experiments. RU performed
the experiments. EM carried out the data analyses. All authors
discussed and interpreted the results and contributed to drafts of
this manuscript. Finally, we would like to make two comments based on our
results from a more global perspective. One is from a human-
agent interaction perspective and the other is a perspective from
communication studies. How people interpret a rich human image from the photo
of a human, i.e., without interacting with the human, seems to
share the same mechanism as how people see a non-human as
a human (personification). Many researchers have explored why
we personify computers (Nass, 1996; Colburn and Shute, 2008;
Nass and Yen, 2010), robots, and androids (Straub et al., 2010). Our results suggest that by manipulating clothes or appearances
as well as by training the human who interacts with agents, we
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distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
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terms. Nishimura, Y. (2008). Metonymy underlying grammar. ENERGEIA 33, 15–26. Nomura, M. (1996). The ubiquity of the fluid metaphor in Japanese: a case study. Poetica 46, 41–75. Schmid, H. J. (2008). New words in the mind: concept-formation and
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https://openalex.org/W2063064552
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https://www.scielo.br/j/rbcs/a/fRBzJ4CZN8fC9SrTHYKMrqk/?lang=pt&format=pdf
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Portuguese
| null |
Decomposição e liberação de N, P e K de esterco bovino e de cama de frango isolados ou misturados
|
Revista Brasileira de Ciência do Solo
| 2,014
|
cc-by
| 8,154
|
Valéria Borges da Silva(2), Alexandre Paiva da Silva(3), Bruno de Oliveira Dias(3),
Josinaldo Lopes Araujo(4), Djail Santos(3) & Renata Pinto Franco(2) Valéria Borges da Silva(2), Alexandre Paiva da Silva(3), Bruno de Oliveira D
Josinaldo Lopes Araujo(4), Djail Santos(3) & Renata Pinto Franco(2) Valéria Borges da Silva(2), Alexandre Paiva da Silva(3), Bruno de Oliveira Dias(3),
Josinaldo Lopes Araujo(4), Djail Santos(3) & Renata Pinto Franco(2) (1) Parte da Dissertação de Mestrado apresentada pelo primeiro autor ao Programa de Pós-Graduação em Manejo de Solo e Água
- PPGMSA, da Universidade Federal da Paraíba - UFPB. Recebido para publicação em 30 de julho de 2013 e aprovado em 16
de junho de 2014.
(2) Tecnóloga em Fruticultura Irrigada, Mestre em Manejo de Solo e Água, UFPB. Campus II. CEP 58.397-000 Areia (PB). E-mail:
valeria_borgess@hotmail.com, renatapintof@hotmail.com
(3) Professor Adjunto, Centro de Ciências Agrárias - CCA, Universidade Federal da Paraíba - UFPB. Campus Universitário do
CCA, s/n. CEP 58.397-000 Areia (PB). E-mail: paivadasilva@gmail.com, b2dias@yahoo.com.br, santosdj@cca.ufpb.br
(4) Professor Adjunto, Centro de Ciências e Tecnologia Agroalimentar - CCTA, Universidade Federal de Campo Grande - UFCG.
Rua Jario Vieira Feitosa, 1770, Bairro dos Pereiros. CEP 58.840-000 Pombal (PB). E-mail: jhosinal_araujo@yahoo.com.br R. Bras. Ci. Solo, 38:1537-1546, 2014 (1) Parte da Dissertação de Mestrado apresentada pelo primeiro autor ao Programa de Pós-Graduação em Manejo de Solo e Água
- PPGMSA, da Universidade Federal da Paraíba - UFPB. Recebido para publicação em 30 de julho de 2013 e aprovado em 16
de junho de 2014. RESUMO Termos de indexação: adubação orgânica, mineralização, resíduos orgânicos. SUMMARY: DECOMPOSITION AND MINERALIZATION OF N, P AND K OF
CATTLE MANURE AND POULTRY LITTER ISOLATED OR
MIXED Menezes, 2007; Silva, 2008; Pitta et al., 2012). Em
algumas situações, entretanto, a eficiência desses como
fonte de nutrientes é baixa em razão do manejo
inadequado (coleta, armazenagem e formas de
aplicação) e da ausência de critérios técnicos para
subsidiar o estabelecimento das doses (Lupwayi et al.,
2000; Hartz et al., 2000; Eghball et al., 2002; Fatondji
et al., 2009; Azeez & van Averbeke, 2010; Pitta et al.,
2012). RESUMO A utilização de resíduos orgânicos de origem animal na adubação das culturas
demanda informações sobre a dinâmica de decomposição e mineralização de
nutrientes neles contidos. Este trabalho objetivou avaliar a decomposição e a
liberação de N, P e K dos resíduos orgânicos esterco bovino e cama de frango
isolados ou misturados, em Argissolo Vermelho-Amarelo, da região de Tabuleiros
Costeiros paraibanos. O experimento foi conduzido em delineamento experimental
de blocos casualizados, utilizando-se o método das sacolas de decomposição
(litterbag), com os tratamentos arranjados em esquema fatorial 3 × 2 × 6, referentes
a três resíduos orgânicos: esterco bovino (EB), cama de frango (CF) e a mistura
desses resíduos na proporção de 1:1, denominada de esterco misto (EM); duas
camadas de incorporação (0-10 e 10-20 cm); e seis períodos de avaliação (0, 30, 90,
150, 210 e 270 dias após a aplicação - DAA) com três repetições. Os resultados
revelaram que a profundidade de incorporação não influenciou a decomposição
dos resíduos EB e EM, que essa foi mais rápida na CF (0,0035 g dia-1), mais lenta no
EB (0,0010 g dia-1) e ocorreu numa velocidade intermediária no EM (0,0020 g dia-1). Houve liberação mais rápida de N no EM (0,0011 g dia-1), de P no EB (0,0040 g dia-1)
e de K na CF (0,0025 g dia-1), com tendência de aumento na liberação de N na CF e
de P em todos os resíduos com a incorporação desses na camada de 10-20 cm. Após
270 DAP, estimou-se que a liberação de nutrientes pelos resíduos EB, CF e EM, em
relação ao teor inicial, foi de 5,0; 15,0; e 28,0 %, para N; 80,0; 68,0; e 47,0 %, para P; e
20,0; 53,0; e 32,0 %, para K. Em valores absolutos, esses percentuais representaram, R. Bras. Ci. Solo, 38:1537-1546, 2014 1538 Valéria Borges da Silva et al. respectivamente, 19,0; 103,5; e 149,5 kg ha-1 de N; 28,0; 18,0; e 15,0 kg ha-1 de P; e 75,0;
493,0; e 209,0 kg ha-1 de K. Os resíduos orgânicos se evidenciaram fontes
inadequadas para suprir isoladamente a demanda nutricional de culturas de ciclo
curto nesses solos. respectivamente, 19,0; 103,5; e 149,5 kg ha-1 de N; 28,0; 18,0; e 15,0 kg ha-1 de P; e 75,0;
493,0; e 209,0 kg ha-1 de K. Os resíduos orgânicos se evidenciaram fontes
inadequadas para suprir isoladamente a demanda nutricional de culturas de ciclo
curto nesses solos. SUMMARY: DECOMPOSITION AND MINERALIZATION OF N, P AND K OF
CATTLE MANURE AND POULTRY LITTER ISOLATED OR
MIXED The use of organic residues in the crop fertilizations demand information about the
dynamics of decomposition and release of nutrients contained therein. This study aimed to
evaluate the decomposition and release of N, P and K from organic residues cattle manure and
poultry litter isolates or mixed, using the method of decomposition bags (litterbag) in Alfissol
of Coast Tableland in Paraiba State. The experiment was conducted in randomized block
with treatments arranged in a factorial 3 × 2 × 5 refers to three organic residues [(cattle manure
(CM), poultry litter (PL) and the mixture of these residues in the ratio of 1: 1, called mixed
manure (MM)], the incorporation of two layers (0-10 and 10-20 cm) and six evaluation periods
(0, 30, 90, 150, 210 and 270 days after application) and three replications. From the results it
was observed that the depth did not affect the decomposition of organic residues CM and MM
and that it was more rapid in PL (0.0035 g day-1), more slow in CM (0.0010 g day-1 ) and
occurred at a speed intermediate in MM (0.0020 g day-1). It was release more faster of N in MM
(0.0011 g day-1), of P in CM (0.0040 g day-1) and K on PL (0.0025 g day-1), with an upward
trend in the release of N in the PL and P in all residues with the incorporation at 10-20 cm. After 270 daa, it was estimated that the release of nutrients in the residues CM, PL and MM in
relation to the initial content was 5.0, 15.0 and 28.0% for N; 80.0, 68.0 and 47.0% for P; and
20.0, 53.0 and 32.0% for K. In absolute terms, these percentages represented, respectively, 19.0,
103.0 and 149.5 kg ha-1 N, 28.0, 18.0 and 15.0 kg ha-1 P, and 75.0, 493.0 and 209.0 kg ha-1
K. The organic residues proved inadequate to meet the demand alone nutritional short cycle
crops in these soils. Index terms: organic manure, mineralization, organic residues. Menezes, 2007; Silva, 2008; Pitta et al., 2012). Em
algumas situações, entretanto, a eficiência desses como
fonte de nutrientes é baixa em razão do manejo
inadequado (coleta, armazenagem e formas de
aplicação) e da ausência de critérios técnicos para
subsidiar o estabelecimento das doses (Lupwayi et al.,
2000; Hartz et al., 2000; Eghball et al., 2002; Fatondji
et al., 2009; Azeez & van Averbeke, 2010; Pitta et al.,
2012). Index terms: organic manure, mineralization, organic residues. MATERIAL E MÉTODOS Quadro 1. Características químicas e físicas do solo da
área experimental, na profundidade de 0-20 cm,
antes da instalação do experimento Quadro 1. Características químicas e físicas do solo da
área experimental, na profundidade de 0-20 cm,
antes da instalação do experimento Este trabalho foi realizado de março a dezembro
de 2010 na Quandu Itapororoca, localizada no
município de Itapororoca, mesorregião da Mata
paraibana, região de Tabuleiros Costeiros do Estado
da Paraíba, que está definido pelas coordenadas
geográficas 6o 49’ 48" S, 35o 14’ 49" W e altitude de
81 m. Pela classificação de Köppen, o clima
predominante é do tipo As’ (quente e úmido), com
chuvas de outono-inverno e período de estiagem de
cinco a seis meses (Brasil, 1972). Na figura 1,
constam-se os dados de precipitação pluvial e de
temperatura do ar registrados durante o experimento. Atributo
Valor
pH(H2O) 1:2,5
5,5
MO (g dm-3)
8,7
P (mg dm-3)
3,3
K+ (cmolc dm-3)
0,19
Ca2+ (cmolc dm-3)
3,0
Mg2+ (cmolc dm-3)
1,2
SB (cmolc dm-3)
4,7
Na+ (cmolc dm-3)
0,26
H+Al (cmolc dm-3)
11,2
Al3+ (cmolc dm-3)
0,20
CTCefetiva (cmolc dm-3)
4,95
CTCpotencial (cmolc dm-3)
15,9
V (%)
24,0
Areia grossa (g kg-1)
629,0
Areia fina (g kg-1)
248,0
Silte, (g kg-1)
56,0
Argila, (g kg-1)
67,0
Classe textural
Arenosa
Densidade do solo (kg dm-3)
1,30
Densidade das partículas (kg dm-3)
2,65
Porosidade total (%)
50,9
CRA (m3 m-3)
0,43
área experimental, na profundidade de 0-20 cm,
antes da instalação do experimento
MO: matéria orgânica; SB: soma de base (Ca2+ + Mg2+ + K+ + Na+); A área experimental apresenta relevo do tipo suave
ondulado a ondulado e solo classificado como Argissolo
Vermelho-Amarelo (Embrapa, 2006) de textura
arenosa e de baixa fertilidade natural, o qual vem
sendo destinado, nos últimos 10 anos, para a produção
de abacaxi de sequeiro e pecuária de corte. A camada
de 0-20 cm do solo da área experimental foi amostrada
antes da instalação do experimento, sendo
determinadas suas características físicas (Embrapa,
1997) e químicas (Tedesco et al., 1995) (Quadro 1). O experimento foi instalado em pomar de
abacaxizeiros ‘Pérola’ de três meses de idade, plantado
em leirão de fileiras duplas no espaçamento de 0,80 ×
0,30 × 0,30 m. Adotou-se o delineamento experimental
de blocos casualizados, com os tratamentos arranjados
em esquema fatorial 3 × 2 × 6, com três repetições,
totalizando 108 unidades experimentais. DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... 1539 resíduos orgânicos de origem animal têm se
intensificado nos últimos anos (Hartz et al., 2000; Esse
et al., 2001; Souto et al., 2005; Silva & Menezes, 2007;
Santos et al., 2008; Azeez & van Averbeke, 2010;
Vanega Chacón et al., 2011; Freitas et al., 2012; Pitta
et al., 2012). No entanto, trabalhos que avaliem de
forma integrada a dinâmica de decomposição e
liberação de nutrientes desses resíduos nas condições
edafoclimáticas do nordeste brasileiro ainda são
incipentes (Menezes & Salcedo, 2007; Freitas et al.,
2012). Ademais, na literatura pertinente, inexistem
informações sobre a dinâmica desses processos
envolvendo a mistura desses resíduos. resíduos orgânicos de origem animal têm se
intensificado nos últimos anos (Hartz et al., 2000; Esse
et al., 2001; Souto et al., 2005; Silva & Menezes, 2007;
Santos et al., 2008; Azeez & van Averbeke, 2010;
Vanega Chacón et al., 2011; Freitas et al., 2012; Pitta
et al., 2012). No entanto, trabalhos que avaliem de
forma integrada a dinâmica de decomposição e
liberação de nutrientes desses resíduos nas condições
edafoclimáticas do nordeste brasileiro ainda são
incipentes (Menezes & Salcedo, 2007; Freitas et al.,
2012). Ademais, na literatura pertinente, inexistem
informações sobre a dinâmica desses processos
envolvendo a mistura desses resíduos. desses dois resíduos, denominado de esterco misto
(EM); duas camadas de incorporação (0-10 e 10-20 cm);
e seis períodos de avaliação (0, 30, 90, 150, 210 e 270
dias após a aplicação dos resíduos orgânicos - DAA). As amostras de esterco bovino e cama de frango
foram coletadas em pilhas dispostas a céu aberto,
conforme procedimentos descritos em Kiehl (1985). O
esterco bovino foi proveniente de estábulo destinado à 0
5
10
15
20
25
30
35
0
20
40
60
80
100
120
mar abr mai jun
jul
ago
set
out nov dez
Temperatura, C
o
Precipitação pluvial, mm
Mês
Precipitação
TMIN
TMAX
Figura 1. Precipitação pluvial e temperaturas
mínima e máxima do ar registrados durante o
experimento. DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... 0
5
10
15
20
25
30
35
0
20
40
60
80
100
120
mar abr mai jun
jul
ago
set
out nov dez
Temperatura, C
o
Precipitação pluvial, mm
Mês
Precipitação
TMIN
TMAX Pelo exposto, e partindo-se do pressuposto que a
mistura de esterco bovino e cama de frango em
proporções equivalentes resulta em decomposição e
liberação mais gradual em relação a desses resíduos
isolados, este trabalho objetivou avaliar a dinâmica
de decomposição e liberação de N, P e K dos resíduos
orgânicos esterco bovino e cama de frango isolados ou
misturados, em Argissolo Vermelho-Amarelo de baixa
fertilidade da região de Tabuleiros Costeiros
paraibanos. Figura 1. Precipitação pluvial e temperaturas
mínima e máxima do ar registrados durante o
experimento. MATERIAL E MÉTODOS Os fatores
em estudo constaram de três resíduos orgânicos:
esterco bovino (EB), cama de frango (CF) e a mistura INTRODUÇÃO Decomposição e mineralização são processos
biocatalíticos, complexos, dinâmicos e multifásicos que
envolvem quebra e conversão de monômeros
específicos (celulose, hemicelulose, lignina) por células
microbianas em formas inorgânicas passíveis de serem
absorvidas pela microbiota decompositora e pelas
plantas (Paul & Clark, 1996; Moreira & Siqueira,
2006). Tais processos são influenciados pela qualidade
dos resíduos (teores e relações entre C, N, P, S, lignina,
celulose e polifenóis), pela atividade da biota e pelas
condições edafoclimáticas (pH, textura, umidade,
temperatura e aeração do solo) (Eghball et al., 2002;
Moreira & Siqueira, 2006; Nascimento et al., 2011;
Vanega Chacón et al., 2011). O potencial de utilização desses resíduos na
adubação das culturas depende da capacidade deles
em disponibilizar nutrientes no momento adequado
(Esse et al., 2001; Menezes & Salcedo, 2007; Azeez &
van Averbeke, 2010; Pitta et al., 2012). Isso implica
na necessidade de se conhecer a dinâmica de
decomposição e liberação dos nutrientes neles contidos
(Hartz et al., 2000; Eghball et al., 2002), visando
estabelecer estratégias que permitam sincronizar a
liberação de nutrientes com a época de maior demanda
pelas culturas, evitando, assim, a imobilização e, ou,
a rápida mineralização desses (Esse et al., 2001;
Menezes & Salcedo, 2007; Pitta et al., 2012). Esterco bovino e cama de frango são resíduos
orgânicos bastante empregados na agricultura, por
causa da riqueza deles em C e nutrientes, bem como
seus efeitos benéficos nos atributos físicos, no aumento
do teor de matéria orgânica do solo e no fornecimento
de nutrientes às plantas (Cassol et al., 2001; Fiorezze
& Ceretta, 2006; Menezes & Salcedo, 2007; Silva & Pesquisas relacionadas com a dinâmica de
decomposição e liberação de nutrientes a partir de R. Bras. Ci. Solo, 38:1537-1546, 2014 MO: matéria orgânica; SB: soma de base (Ca2+ + Mg2+ + K+ + Na+);
CTCefetiva = SB + Al3+; CTCpotencial = SB + (H+Al); V: saturação
por bases = (SB/CTC) × 100; e CRA: capacidade de retenção de
água. CRA (m3 m-3) MO: matéria orgânica; SB: soma de base (Ca2+ + Mg2+ + K+ + Na+);
CTCefetiva = SB + Al3+; CTCpotencial = SB + (H+Al); V: saturação
por bases = (SB/CTC) × 100; e CRA: capacidade de retenção de
água. R. Bras. Ci. Solo, 38:1537-1546, 2014 Valéria Borges da Silva et al. 1540 produção de vacas leiteiras alimentadas com silagem
de milho e cana, enquanto a cama de frango tinha
como material absorvente a mistura de bagaço de cana
e palha+sabugo de milho. O esterco misto resultou da
mistura de esterco bovino e cama de frango na
proporção de 1:1 (m/m). Após coletadas e determinada
a umidade, as amostras foram secas ao ar e nelas
quantificados os teores de C, N, P, K (Tedesco et al.,
1995), celulose, hemicelulose e lignina (van Soest et
al., 1991) (Quadro 2). modelo não linear exponencial simples (Thomas &
Asakawa, 1993), do tipo y = yoe-kt, em que y é a
porcentagem de massa ou de nutriente remanescente
de cada resíduo, após um período de tempo t, em dias;
yo é a quantidade inicial de massa ou de nutrientes do
resíduo; e k corresponde à constante de decomposição
ou de liberação de nutrientes de cada resíduo. Com
base nos valores da constante k, calculou-se o tempo
de meia-vida (t½ = 0,693/k), que expressa o período de
tempo necessário para que metade da quantidade
inicial do resíduo seja decomposta ou para que metade
da quantidade de nutrientes seja liberada (Paul &
Clark, 1989). produção de vacas leiteiras alimentadas com silagem
de milho e cana, enquanto a cama de frango tinha
como material absorvente a mistura de bagaço de cana
e palha+sabugo de milho. O esterco misto resultou da
mistura de esterco bovino e cama de frango na
proporção de 1:1 (m/m). Após coletadas e determinada
a umidade, as amostras foram secas ao ar e nelas
quantificados os teores de C, N, P, K (Tedesco et al.,
1995), celulose, hemicelulose e lignina (van Soest et
al., 1991) (Quadro 2). A decomposição e liberação de nutrientes dos
resíduos orgânicos foram avaliadas pelo método das
sacolas de decomposição ou método litterbag (Wieder
& Lang, 1982). Após a caracterização química, os
resíduos foram secos em estufa (±65 oC) até peso
constante, passados em malha de 2 mm e acondicionados
em sacolas de náilon de 20 × 20 cm, com abertura de
malha de 1,0 mm2. RESULTADOS E DISCUSSÃO Em cada período de avaliação, coletou-se uma
sacola de cada resíduo, em cada camada avaliada,
totalizando 18 sacolas por período. Após coletadas, as
sacolas foram acondicionadas em caixas térmicas e
enviadas para laboratório. No laboratório, as sacolas
foram abertas, o resíduo orgânico nelas contido foi
limpo e seco em estufa (±65 oC) até peso constante;
determinou-se a massa, avaliando-se, dessa forma, a
decomposição dos materiais em cada período de
avaliação. Em seguida, foram retiradas subamostras
para quantificar os teores de N, P e K (Tedesco et al.,
1995). O N foi determinado pelo método Kjeldahl, após
digestão sulfúrica. Os teores de P foram determinados
colorimetricamente pelo método do azul de molibdênio,
enquanto os de K, por fotometria de chama, ambos do
extrato de digestão nítrico-perclórica (Tedesco et al.,
1995). CRA (m3 m-3) Cada sacola recebeu 20 g de um
dos resíduos avaliados; em seguida, cada uma foi
disposta horizontalmente no cume do leirão, em duas
diferentes camadas (0-10 e 10-20 cm). Os dados foram submetidos às análises de variância
e de regressão, utilizando-se o programa SAEG versão
9.0 (SAEG, 2007), adotando o nível de significância de
10 %. Valéria Borges da Silva et al. DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... 1541 decomposição (celulose, lignina, gorduras etc) (Pitta
et al., 2012; Shah et al., 2013). elevados de C e N e com menores valores das relações
C/N, lignina/N e polifenóis/N são decompostos mais
rapidamente (Moreira & Siqueira, 2006; Vanega
Chacón et al., 2011; Nascimento et al., 2011). Observou-se que a decomposição da CF foi mais
rápida na camada de 10-20 cm (k = 0,0040) e que a
profundidade de incorporação não influenciou a
velocidade de decomposição dos resíduos EB (k = 0,0010)
e EM (k = 0,0020) (Figura 2, Quadro 3). Para o EM, formado a partir da mistura de
proporções equivalentes de EB e CF, e que exibiu
comportamento intermediário entre CF e EB (Figura
2), é provável que tenha ocorrido maior ataque
microbiano às frações da CF, por apresentarem maior
proporção de formas de C facilmente biodegradáveis
de mais fácil e rápida decomposição (açúcares,
aminoácidos, proteínas etc) e menor proporção de
compostos orgânicos mais complexos e de mais lenta Em geral, a incorporação dos resíduos em
profundidade acelera a sua decomposição, em razão
do aumento da superfície de ataque microbiano, da
proliferação e da atividade decompositora promovida
pela melhoria das condições de temperatura e umidade
e pela diminuição dos ciclos de umedecimento e
secagem (Souto et al., 2005; Fatondji et al., 2009). Neste trabalho, a ausência de diferenças na velocidade
de decomposição dos resíduos EB e EM, em razão da
profundidade, pode ser explicada pela maior proporção
de compostos orgânicos mais complexos e de maior
hidrofobicidade (lignina, celulose e complexos lignina-
celulose) presente tanto no EB quanto no EM (Moreira
& Siqueira, 2006; Shah et al., 2013). 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 89,958e
-0,002x
2
R = 0,9304
ŷ(0-10 cm) = 87,439e
-0,002x
2
R = 0,841
ŷ(10-20 cm) = 79,797e
-0,004x
2
R = 0,8855
ŷ(0-10 cm) = 73,255e
-0,003x
2
R = 0,7695
ŷ(0-10 cm) = 100,04e
-0,001x
2
R = 0,9959
ŷ(10-20 cm) = 100,17e
-0,001x
2
R = 0,9887
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
Massa remanescente, %
Figura 2. Massa remanescente de esterco bovino,
cama de frango e esterco misto, nas camadas de
0-10 e 10-20 cm, em função dos períodos de
avaliação. DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... Quanto aos períodos de avaliação, constatou-se que
após 270 DAA foram decompostos, na média das duas
camadas, 27; 68; e 47 % da massa de EB, CF e EM,
respectivamente (Figura 2). Isso reflete, conforme
mencionado anteriormente, as diferenças na
composição química dos resíduos (Quadro 2) e o menor
ataque microbiano às substâncias mais recalcitrantes
(lignina e polifenóis), que se acumulam com o avançar
da decomposição (Esse et al., 2001; Souto et al., 2005;
Moreira & Siqueira, 2006; Pitta et al., 2012). Tais
valores são compatíveis com os reportados na
literatura (Esse et al., 2001; Souto et al., 2005; Pitta
et al., 2012), embora para EB o valor tenha sido
inferior aos 42 % reportados por Freitas et al. (2012). Ressalta-se, entretanto, que no trabalho de Freitas et
al. (2012) a decomposição foi avaliada sob condições
de fertirrigação, o que teria acelerado a decomposição
(Moreira & Siqueira, 2006). Cama de frango Massa remanescente, % Quanto aos valores de meia-vida, constatou-se que
esses foram elevados e maiores no EB (693 dias para
as duas camadas), seguido pelo EM (347 dias para
ambas as camadas) e CF (231 dias na camada de
0-10 cm e 173 dias na de 10-20 cm) (Quadro 3). Tais
resultados são explicados pelas diferenças na matriz
orgânica (Quadro 2) e na dinâmica de decomposição
desses (Figura 2). Os valores estimados para CF são
compatíveis com os 299 dias reportados por Pitta et
al. (2012) em Latossolos do Paraná, mas superiores
aos 140 dias reportados por Esse et al. (2001) para EB
em solos degradados da África. Nesse sentido, é
provável que o EB utilizado neste trabalho apresente
grau de humificação mais avançado (Melo et al., 2008)
e seja, portanto, entre os resíduos avaliados, o de menor
susceptibilidade à decomposição. Dia após a aplicação Figura 2. Massa remanescente de esterco bovino,
cama de frango e esterco misto, nas camadas de
0-10 e 10-20 cm, em função dos períodos de
avaliação. Figura 2. Massa remanescente de esterco bovino,
cama de frango e esterco misto, nas camadas de
0-10 e 10-20 cm, em função dos períodos de
avaliação. Decomposição dos resíduos Verificou-se padrão de decomposição diferenciado
entre os resíduos orgânicos avaliados (Figura 2). Para
a cama de frango (CF) e o esterco misto (EM), observou-
se uma fase de decomposição inicial mais rápida nos
primeiros 30 DAA, seguida de uma mais lenta nos
períodos seguintes. Para o esterco bovino (EB), houve
decomposição mais lenta nos primeiros 30 DAA e
estabilização nos períodos subsequentes (Figura 2). A decomposição mais rápida da CF em relação ao
EB corrobora os resultados de Azeez & van Averbeke
(2010) e pode ser explicada pelos maiores teores de C,
N e K, menores teores de lignina, celulose e
hemicelulose e menores valores das relações C/N e
lignina/N (Quadro 2). A qualidade dos resíduos
determina a velocidade de decomposição deles, de modo
que, em geral, resíduos orgânicos com teores mais Para descrever a dinâmica de decomposição e de
liberação de nutrientes dos resíduos, foi utilizado o Característica
Esterco bovino
Cama de frango
Esterco Misto
C (g kg-1)
210,9
349,0
279,9
N (g kg-1)
18,9
34,5
26,7
P (g kg-1)
1,75
1,32
1,56
K (g kg-1)
18,8
46,5
32,6
C/N
11,1
10,1
10,6
C/P
120,5
264,4
179,4
N/P
10,8
26,1
17,1
Lignina (g kg-1)
140,0
89,0
nd(1)
Celulose (g kg-1)
110,0
103,7
nd
Hemicelulose (g kg-1)
80,0
166,7
nd
Lignina/N
7,41
2,58
nd
Umidade (%)
23,6
13,6
nd
Quadro 2. Caracterização química dos resíduos orgânicos
(1) nd: não determinado. Quadro 2. Caracterização química dos resíduos orgânicos R. Bras. Ci. Solo, 38:1537-1546, 2014 Liberação de N Os resíduos diferiram quanto ao padrão de liberação
de N (Figura 3). Para CF e EM, houve uma fase inicial R. Bras. Ci. Solo, 38:1537-1546, 2014 1542 Valéria Borges da Silva et al. Resíduo
Camada
Parâmetro da equação
y0
k
t½
R2
%
g g-1
dia
Decomposição
Esterco bovino
0-10
100,04
0,0010 a
693
0,99
10-20
100,17
0,0010 a
693
0,98
Cama de frango
0-10
73,25
0,0030 b
231
0,76
10-20
79,79
0,0040 a
173
0,88
Esterco misto
0-10
87,43
0,0020 a
347
0,84
10-20
89,95
0,0020 a
347
0,93
N
Esterco bovino
0-10
104,82
0,0004 a
1733
0,67
10-20
99,20
0,0002 b
3466
0,89
Cama de frango
0-10
94,89
0,0003 b
2310
0,53
10-20
94,72
0,0005 a
1386
0,72
Esterco misto
0-10
95,44
0,0009 b
770
0,85
10-20
97,35
0,0013 a
533
0,88
P
Esterco bovino
0-10
45,47
0,0040 b
173
0,43
10-20
49,00
0,0041 a
169
0,48
Cama de frango
0-10
73,25
0,0033 b
210
0,76
10-20
79,79
0,0038 a
182
0,88
Esterco misto
0-10
87,44
0,0018 b
385
0,84
10-20
89,96
0,0024 a
289
0,93
K
Esterco bovino
0-10
91,41
0,0007 a
990
0,64
10-20
95,47
0,0007 a
990
0,75
Cama de frango
0-10
84,99
0,0025 a
277
0,84
10-20
78,91
0,0025 a
277
0,75
Esterco misto
0-10
89,44
0,0013 a
533
0,77
10-20
88,26
0,0013 a
533
0,71
Quadro 3. Parâmetros dos modelos de regressão ajustados (y = y0 e-kt) aos valores de decomposição e liberação
de N, P e K e tempo de meia-vida (t½) dos resíduos orgânicos avaliados, nas camadas de 0-10 e 10-20 cm
Médias entre camada, para um mesmo resíduo e variável, seguidas pela mesma letra não diferem entre si pelo teste F a 5 %. Quadro 3. Parâmetros dos modelos de regressão ajustados (y = y0 e-kt) aos valores de decomposição e liberação
de N, P e K e tempo de meia-vida (t½) dos resíduos orgânicos avaliados, nas camadas de 0-10 e 10-20 cm A liberação maior e mais rápida de N pelo EM
indica que a mistura de EB e CF, com composição
química e grau de maturação distinta, teve efeito
aditivo sobre a liberação de N (Figura 3). Liberação de P Os resíduos também diferiram quanto ao padrão
de liberação de P, havendo para o EB liberação mais
rápida nos primeiros 30 DAA (80 % do P liberado) e
tendência de estabilização nos períodos subsequentes
(Figura 4). Para CF, a liberação também foi alta nos
primeiros 30 DAA (40 % do P liberado), mas inferior a
do EB, havendo liberação gradual nos períodos
seguintes. Para o EM, a liberação de P aos 30 DAA
(apenas 20 %) foi comparativamente mais lenta do
que a dos EB e CF, seguida de uma fase de liberação
gradual nos períodos subsequentes (Figura 4). 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 97,348e
-0,0013x
2
R = 0,8808
ŷ(0-10 cm) = 95,448e
-0,0009x
2
R = 0,8571
ŷ(10-20 cm) = 94,724e
-0,005x
2
R = 0,7291
ŷ(0-10 cm) = 94,896e
-0,003x
2
R = 0,5346
ŷ(0-10 cm) = 104,82e
-0,004x
2
R = 0,6727
ŷ(10-20 cm) = 99,208e
-0,002x
2
R = 0,8979
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
N remanescente, %
Figura 3. Percentual remanescente de nitrogênio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. 0-10 cm
10-20 cm A liberação mais rápida de P pelo EB em relação à
CF deve-se ao maior teor de P, aos menores valores
da relação C/P e possivelmente ao predomínio de
fosfatos solúveis em água e fracamente ligados à matriz
sólida do EB (Cassol et al., 2001; Galvão & Salcedo,
2009), em comparação à prevalência de frações de P
orgânico (fosfatos monoésteres) de menor labilidade
na CF (Pitta et al., 2012; Souza et al., 2012). A
liberação mais lenta de P no EM pode ter sido
propiciada pela imobilização de P na biomassa
microbiana por causa do aumento do número de
microrganismos totais na mistura (Moreira &
Siqueira, 2006; Tiago et al., 2008). N remanescente, % A profundidade de incorporação influenciou a
liberação de P, constatando-se liberação mais rápida
para todos os resíduos na camada de 10-20 cm (Figura
3, Quadro 3). DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... 1543 velocidade de decomposição desse resíduo na camada
de 10-20 cm (Figura 2 e Quadro 3). Para EB e EM,
entretanto, houve maior liberação de N na camada de
0-10 cm em detrimento de não terem ocorrido
diferenças na decomposição desses resíduos entre as
camadas (Figura 2, Quadro 3). as estimativas dos valores de meia-vida foram
elevadas, principalmente para o EB (1.733 a 3.466
dias) e a CF (1.386 a 2.310 dias). Para o EM, os valores
foram menores, mas variaram de 533 a 770 dias nas
camadas de 0-10 e 10-20 cm, respectivamente (Quadro
3). Isso demonstra as limitações desses resíduos como
fonte de N em curto prazo, indicando a necessidade de
se aplicarem grandes quantidades deles para suprir a
demanda nutricional das culturas nesses solos. De forma geral, a liberação de N foi baixa,
permanecendo nas sacolas após 270 DAA,
aproximadamente 95,0; 85,0; e 72,0 % do teor inicial
de N nos resíduos EB, CF e EM, respectivamente
(Figura 3). Tais valores são inferiores aos obtidos por
Pitta et al. (2012) para CF (30 % após 270 DAA) e por
Freitas et al. (2012) para EB (46 % após 210 DAA),
embora sejam compatíveis com a faixa de amplitude
(82-95 %) reportada por Hartz et al. (2000). Ademais, Liberação de N Esse
comportamento provavelmente deve-se ao aumento do
número de microrganismos totais (fungos e bactérias)
na mistura (Tiago et al., 2008), os quais são
responsáveis pela transformação, ciclagem e
disponibilidade de nutrientes, particularmente o N,
na estrutura de resíduos orgânicos (Moreira &
Siqueira, 2006; Tiago et al., 2008). de liberação mais rápida seguida de uma mais lenta e
gradual nos períodos subsequentes. Para o EB,
entretanto, houve uma fase inicial de imobilização de
N até os 90 DAA, seguida de uma de baixa e lenta
liberação nos períodos subsequentes (Figura 3). A liberação maior e mais rápida de N pela CF em
relação ao EB e de imobilização de N no EB na fase
inicial corroborou os resultados de outros trabalhos
(Hartz et al., 2000; Eghball et al., 2002; Azeez & van
Averbeke, 2010; Pitta et al., 2012) e pode ser explicada
pelas diferenças na composição química (Quadro 2) e
na velocidade de decomposição dos resíduos (Figura
2). Ademais, na CF, predominam formas de N
orgânico (proteínas) de mais fácil e rápida liberação,
enquanto no EB, de maior recalcitrância, há maior
proporção de formas de N mais estáveis e de difícil e,
ou, mais lenta liberação (Eghball et al., 2002; Azeez
& van Averbeke, 2010). A profundidade de incorporação influenciou a
liberação de N pelos resíduos, havendo liberação
mais rápida de N para EB (0,0004 vs 0,0002) e EM
(0,0009 vs 0,0003) na camada de 0-10 cm e para CF
(0,0005 vs 0,0003), na camada de 10-20 cm (Figura 3,
Quadro 3). A liberação mais rápida de N pela CF na
camada de 10-20 cm está condizente com a maior R. Bras. Ci. Solo, 38:1537-1546, 2014 Liberação de K Diferentemente do constatado para N e P, a
liberação de K não foi influenciada pela profundidade
de incorporação (Figura 4, Quadro 3), corroborando
os resultados obtidos por Freitas et al. (2012). Tais
resultados podem ser explicados pela homogeneidade
das condições texturais e pela dinâmica simplificada
de liberação de K a partir de resíduos orgânicos (Braz
et al., 2002; Eghball et al., 2002; Moreira & Siqueira,
2006; Pitta et al., 2012). Os resíduos apresentaram padrão semelhante de
liberação de K, representado por uma fase de liberação
inicial mais rápida nos primeiros 30 DAA, seguida de
uma de liberação mais gradual (Figura 4). Contudo,
em ambas as fases, houve liberação mais rápida de K
na CF, seguida pelo EM e EB (Figura 4), indicando
que essa apresentou maior quantidade de frações de
K potencialmente mineralizáveis (Eghball et al., 2002;
Pitta et al., 2012). Segundo Zeviani et al. (2012), o
padrão de liberação de K pelos resíduos orgânicos de
origem animal ainda é desconhecido, o que limita o
aproveitamento dessas fontes como fonte de nutrientes. Verificou-se que após 270 DAA restavam nas
sacolas de decomposição aproximadamente 80,0; 47,0;
e 68,0 % do teor inicial de K nos resíduos EB, CF e
EM, respectivamente (Figura 4). Esses valores são 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 89,58e
-0,0024x
2
R = 0,9304
ŷ(0-10 cm) = 87,439e
-0,0018x
2
R = 0,841
ŷ(10-20 cm) = 79,797e
-0,0038x
2
R = 0,8855
ŷ(0-10 cm) = 73,255e
-0,0033x
2
R = 0,7695
ŷ(0-10 cm) = 49,007e
-0,004x
2
R = 0,4889
ŷ(10-20 cm) = 45,473e
-0,004x
2
R = 0,439
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
P remanescente, %
Figura 4. Percentual remanescente de fósforo em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. Liberação de P Isso indica que, apesar de não influenciar
a decomposição dos EB e EM, a incorporação dos
resíduos na camada de 10-20 cm intensificou a
liberação de P (Quadro 3), corroborando, assim, os
resultados encontrados por Freitas et al. (2012). É
possível que as condições de temperatura e umidade
do solo nessa camada tenham estimulado a atividade
microbiana e aumentado a mineralização do P contido
nos respectivos resíduos (Moreira & Siqueira, 2006). No que se refere aos períodos de avaliação, verificou-
se que após 270 DAA restavam nas sacolas cerca de
20,0; 32,0; e 53,0 % do teor inicial de P nos resíduos
EB, CF e EM, respectivamente (Figura 3), indicando
que a liberação de P pelos resíduos foi entre duas e
quatro vezes maior do que a de N (Figuras 2 e 3). Tais
valores são compatíveis com os obtidos por Freitas et
al. (2012) para EB (46 %, após 210 DAA) e Pitta et al. (2012) para CF (35 %, após 270 DAA). Apesar disso,
os valores de meia-vida foram elevados tanto na
camada de 0-10 (173-385 dias) quanto na de 10-20 cm
(169-289 dias) (Quadro 3). Figura 3. Percentual remanescente de nitrogênio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. Figura 3. Percentual remanescente de nitrogênio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. R. Bras. Ci. Solo, 38:1537-1546, 2014 1544 Valéria Borges da Silva et al. Valéria Borges da Silva et al. Liberação de K 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 88,268e
-0,0014x
2
R = 0,7116
ŷ(0-10 cm) = 89,441e
-0,001x
2
R = 0,7742
ŷ(10-20 cm) = 78,91e
-0,003x
2
R = 0,7527
ŷ(0-10 cm) = 84,992e
-0,002x
2
R = 0,8438
ŷ(0-10 cm) = 95,469e
-0,0007x
2
R = 0,7581
ŷ(10-20 cm) = 91,416e
-0,0007x
2
R = 0,6432
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
K remanescente, %
Figura 5. Percentual remanescente de potássio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 88,268e
-0,0014x
2
R = 0,7116
ŷ(0-10 cm) = 89,441e
-0,001x
2
R = 0,7742
ŷ(10-20 cm) = 78,91e
-0,003x
2
R = 0,7527
ŷ(0-10 cm) = 84,992e
-0,002x
2
R = 0,8438
ŷ(0-10 cm) = 95,469e
-0,0007x
2
R = 0,7581
ŷ(10-20 cm) = 91,416e
-0,0007x
2
R = 0,6432
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
K remanescente, %
Figura 5. Percentual remanescente de potássio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 88,268e
-0,0014x
2
R = 0,7116
ŷ(0-10 cm) = 89,441e
-0,001x
2
R = 0,7742
ŷ(10-20 cm) = 78,91e
-0,003x
2
R = 0,7527
ŷ(0-10 cm) = 84,992e
-0,002x
2
R = 0,8438
ŷ(0-10 cm) = 95,469e
-0,0007x
2
R = 0,7581
ŷ(10-20 cm) = 91,416e
-0,0007x
2
R = 0,6432
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
K remanescente, %
Figura 5. Quantidades de N, P e K liberadas Considerando os percentuais médios de N, P e K
liberados pelos resíduos, nas duas camadas, a
composição química desses (Quadro 2) e uma dose de
20 t ha-1 (base matéria seca), estimou-se que após
90 DAA seriam liberados para o solo: 5,0 kg ha-1
de N, 8,0 kg ha-1 de P e 20,0 kg ha-1 de K pelo EB;
70,0 kg ha-1 de N, 12,0 kg ha-1 de P e 335,0 kg ha-1 de
K pela CF; e 70,0 kg ha-1 de N, 7,0 kg ha-1 de P e 98,0 kg
ha-1 de K pelo EM. Após 270 DAA, as quantidades
aportadas seriam de: 19,0 kg ha-1 de N, 28,0 kg ha-1 de
P e 75,0 kg ha-1 de K pelo EB; 103,5 kg ha-1 de N; 18,0
kg ha-1 de P e 493,0 kg ha-1 de K pela CF; e 149,5 kg ha-1 de
N, 15,0 kg ha-1 de P e 209,0 kg ha-1 de K pelo EM. CASSOL. P.C.; GIANELLO, C. & COSTA, V.E.U. Frações de
fósforo em estrumes e sua eficiência como adubo
fosfatado. R. Bras. Ci. Solo, 25:635-644, 2001. EGHBALL, B.; WIENHOLD, B.J.; GILLEY, J.E. &
EIGENBERG, R.A. Mineralization of manure nutrients. J. Soil Water Conserv., 57:469-473, 2002. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA -
EMBRAPA. Centro Nacional de Pesquisa de Solos. Sistema brasileiro de classificação de solos. 2.ed. Rio de
Janeiro, 2006. 306p. Em síntese, tais resultados indicaram maior
capacidade de fornecimento de N pelo EM, de P pelo
EB e de K pela CF, tanto aos 90 quanto aos 270 DAA. Demonstraram também potencial limitado desses para
suprir a demanda nutricional, principalmente de N e
P, para culturas de ciclo curto nesses solos. Entretanto, para culturas de ciclo longo, a exemplo
do abacaxizeiro (540 dias), os resíduos CF e EM podem
ser alternativas para complementar a demanda
nutricional da cultura. Contudo, há a necessidade de
se avaliar o desempenho agronômico desses nessas
condições edafoclimáticas. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA -
EMBRAPA. Centro Nacional de Pesquisa de Solos. Manual de métodos de análise de solo. 2.ed. Rio de Janeiro,
1997. 212p. ESSE, P.C.; BUERKERT, A.; HIERNAUX, P. & ASSA, A. Decomposition and nutrient release from ruminant
manure on acid sandy soil in the Sahelian Zone of Niger,
West Africa. Agric. Ecosyst. Environ., 83:55-63, 2001. FATONDJI, D.; MARTIUS, C.; ZOUGMORE, R.; VLEK, P.L.G.;
BIELDERS, C.L. & KOALA, S. DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... DECOMPOSIÇÃO E LIBERAÇÃO DE N, P E K DE ESTERCO BOVINO E DE CAMA DE FRANGO... 1545 Quantidades de N, P e K liberadas Decomposition of organic
amendment and nutrient release under the Zai technique
in the Sahel. Nutr. Cycl. Agroecosyst., 85:225-239, 2009. LITERATURA CITADA superiores aos mencionados em outros trabalhos (Esse
et al., 2001; Eghball et al., 2002; Silva & Menezes,
2007; Pitta et al., 2012; Freitas et al., 2012) e se devem
possivelmente ao fato de terem sido utilizadas neste
trabalho sacolas com abertura de malha maior, o que
teria favorecido possivelmente a entrada de partículas
de solo contendo K no interior dessas sacolas (Wieder
& Lang, 1982; Esse et al., 2001). Em razão disso, os
valores de meia-vida foram elevados e corresponderam,
para ambas as camadas, a 990, 277 e 533 dias para
EB, CF e EM, respectivamente (Quadro 3). superiores aos mencionados em outros trabalhos (Esse
et al., 2001; Eghball et al., 2002; Silva & Menezes,
2007; Pitta et al., 2012; Freitas et al., 2012) e se devem
possivelmente ao fato de terem sido utilizadas neste
trabalho sacolas com abertura de malha maior, o que
teria favorecido possivelmente a entrada de partículas
de solo contendo K no interior dessas sacolas (Wieder
& Lang, 1982; Esse et al., 2001). Em razão disso, os
valores de meia-vida foram elevados e corresponderam,
para ambas as camadas, a 990, 277 e 533 dias para
EB, CF e EM, respectivamente (Quadro 3). AZEEZ, J.O.E. & AVERBEKE, W.V. Nitrogen mineralization
potential of three animal manures applied on a sandy
clay loam soil. Bioresou. Technol., 101:5645-5651, 2010. BRAZ, S.B.; NASCIMENTO JUNIOR, D.; CANTARUTTI, R.B.;
REGAZZI, A.J.; MARTINS, C.A. & FONSECA, D.M. Disponibilização dos nutrientes das fezes de bovinos em
pastejo para a forragem. R. Bras. Zootec., 31:1614-1623,
2002. BRASIL. Ministério da Agricultura. Escritório de Pesquisa e
Experimentação. Equipe de Pedologia e Fertilidade do
solo. I Levantamento exploratório-reconhecimento de
solos do Estado da Paraíba. II Interpretação para uso
agrícola dos solos do Estado da Paraíba. Rio de Janeiro,
1972. 638p. (Boletim Técnico, 15; SUDENE, Série
Pedológica, 8) Liberação de K Percentual remanescente de potássio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm, 0
20
40
60
80
100
120
140
0-10 cm
10-20 cm
0
20
40
60
80
100
120
140
Cama de frango
ŷ(10-20 cm) = 89,58e
-0,0024x
2
R = 0,9304
ŷ(0-10 cm) = 87,439e
-0,0018x
2
R = 0,841
ŷ(10-20 cm) = 79,797e
-0,0038x
2
R = 0,8855
ŷ(0-10 cm) = 73,255e
-0,0033x
2
R = 0,7695
ŷ(0-10 cm) = 49,007e
-0,004x
2
R = 0,4889
ŷ(10-20 cm) = 45,473e
-0,004x
2
R = 0,439
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
Esterco bovino
P remanescente, % 0-10 cm
10-20 cm Cama de frango Cama de frango K remanescente, % P remanescente, % 0
20
40
60
80
ŷ(10-20 cm) = 89,58e
-0,0024x
2
R = 0,9304
ŷ(0-10 cm) = 87,439e
-0,0018x
2
R = 0,841
ŷ(10-20 cm) = 79,797e
-0,0038x
2
R = 0,8855
,
0
20
40
60
80
100
120
140
0
30
60
90
120
150
180
210
240
270
Dia após a aplicação
Esterco misto
P remanescen Dia após a aplicação Dia após a aplicação Figura 5. Percentual remanescente de potássio em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. Figura 4. Percentual remanescente de fósforo em
amostras de esterco bovino, cama de frango e
esterco misto, nas camadas de 0-10 e 10-20 cm,
em função dos períodos de avaliação. R. Bras. Ci. Solo, 38:1537-1546, 2014 CONCLUSÕES 1. A decomposição dos resíduos orgânicos ocorre
de forma mais rápida na CF, mais lentamente no EB
e numa velocidade intermediária no EM; ademais, é
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EM independe da profundidade de incorporação. FIOREZZE, C. & CERETTA, C.A. Fontes orgânicas de
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in sandy soils amended with cattle manure for long
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liberam, em média, em relação aos teores iniciais,
respectivamente, 5,0; 15,0; e 28,0 % de N; 80,0; 68,0;
e 47,0 % de P; 20,0; 53,0; e 32,0 % de K. Em valores
absolutos, esses percentuais representam,
respectivamente, 19,0; 103,5; e 149,5 kg ha-1 de
N; 28,0; 18,0; e 15,0 kg ha-1 de P; e 75,0; 493,0; e
209,0 kg ha-1 de K. HARTZ, T.K.; MITCHELL, J.P. & GIANNINI, C. Nitrogen and
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CAMARGO, F.A.O., eds. Fundamentos da matéria
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matriz orgânicas de resíduos de origens diversificadas. R. Bras. Ci. Solo, 32:101-110, 2008. SILVA, T.O. & MENEZES, R.S.C. Adubação orgânica da batata
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após incorporação de adubos orgânicos em um Neossolo
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H. & VOLKWEISS, S.J. Análise de solo, planta e outros
materiais. Porto Alegre, Universidade Federal do Rio
Grande do Sul, 1995. 174p. PAUL, E.A. & CLARK, F.E. Soil microbiology and
biochemistry. Sand Diego, Academic Press, 1989. 275p. THOMAS, R.J. & ASAKAWA, N.M. Decomposition of leaf
litter from tropical forage grasses and legumes. Soil Biol. Biochem., 25:1351-1361, 1993. PAUL, E.A. & CLARK, F.E. Dynamics of residue
decomposition and soil organic matter turnover. In:
PAUL, E.A. & CLARK, F.E., eds. Soil microbiology and
biochemistry. 2.ed. Sand Diego, Academic Press, 1996. p.158-179. TIAGO, P.V.; MELZ, E.M. & SCHIEDECK, G. Comunidade de
bactérias e fungos de estercos antes e após
vermicompostagem e no substrato hortícola após uso de
vermicomposto. R. Ci. Agron., 39:187-192, 2008. PITTA, C.S.R.; ADAMI, P.F.; PELISSARI, A.; ASSAMANN,
T.S.; FRANCHIN, M.F.; CASSOL, L.C. & SARTOR, L.R. Year-round poultry litter decomposition and N, P, K
and Ca release. R. Bras. Ci. Solo, 36:1043-1053, 2012. VANEGA CHACÓN, E.A.; MENDONÇA, E.S.; SILVA, R.R.;
LIMA, P.C.; SILVA, I.R. & CANTARUTTI, R.B. Decomposição de fontes orgânicas e mineralização de
formas de nitrogênio e fósforo. R. Ceres, 58:373-383, 2011. SAEG. Sistema para Análises Estatísticas. Versão 9.0. Viçosa,
MG, Fundação Arthur Bernardes, 2007. van SOEST, P.J.; ROBERTSON, J.B. & LEWIS, B.A. Methods
for dietary fiber, neutral detergent fiber, and nonstarch
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MENEZES, R.S.C. Decomposição de materiais
celulósicos em solo preparado com e sem leirões, no
semiárido paraibano. Magistra, 20:371-378, 2008. R. Bras. Ci. Solo, 38:1537-1546, 2014 CONCLUSÕES ZEVIANI, W.M.; SILVA, C.A.; CARNEIRO, W.J.O. & MUNIZ,
J.A. Modelos não lineares para a liberação de K de estercos
animais em Latossolos. Ci. Rural, 42:1897-1796, 2012. SHAH, G.M.; RASHID, M.I.; SHAH, G.A.; GROOT, J.C.J. &
LANTINGA, E.A. Mineralization and herbage recovery
of animal manure nitrogen after application to various
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methods used in examining decomposition data obtained
from litter bags. Ecology, 63:1636-1642, 1982. R. Bras. Ci. Solo, 38:1537-1546, 2014
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Evaluation of Electronic Mental Health Implementation in Northern Territory Services Using the Integrated “Promoting Action on Research Implementation in Health Services” Framework: Qualitative Study (Preprint)
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Corresponding Author: Corresponding Author:
Buaphrao Raphiphatthana, BSc (Hons), PhD
Menzies School of Health Research
Charles Darwin University
Casuarina, 0810
Australia
Phone: 61 0889468478
Email: buaphrao raphiphatthana@menzies edu Phone: 61 0889468478
Email: buaphrao.raphiphatthana@menzies.edu.au Phone: 61 0889468478
Email: buaphrao.raphiphatthana@menzies.edu.au Abstract Background: Electronic mental health is a promising strategy to bridge the treatment gap in mental health care. Training
workshops have been delivered to service providers working with Aboriginal and Torres Strait Islander people at a primary health
care level to raise awareness and knowledge of electronic mental health approaches. Objective: This study aimed to understand service providers’perspectives and experiences of electronic mental health adoption. More specifically, it aimed to use the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS)
framework to further identify and understand how different factors facilitate or impede electronic mental health uptake within
primary health care settings providing services to Aboriginal and Torres Strait Islander people. Methods: Qualitative interviews were conducted with 57 service providers working with Aboriginal and Torres Strait Islander
people, who had undergone electronic mental health training workshops. Results: Several factors related to innovation (electronic mental health approach), recipients (service providers as an individual
and as a team), and context (local, organizational, and external contexts) were found to influence electronic mental health uptake. Particularly, organizational readiness, in terms of information technology resources and infrastructure, policies, workforce and
culture, and processes to mandate electronic mental health use, were found to be significant impediments to electronic mental
health utilization. These findings led to the development of a three-phase implementation strategy that aims to enhance electronic
mental health adoption by addressing organizational readiness before and post electronic mental health training. Conclusions: The i-PARIHS provides a useful determinant framework that deepens our understanding of how different factors
impede or facilitate electronic mental health adoption in this setting. This insight was used to develop a practical and comprehensive
implementation strategy to enhance the utilization of electronic mental health approaches within primary health care settings,
involving three phases: pretraining consultations, training workshops, and post-training follow-up support. (JMIR Ment Health 2020;7(5):e14835) doi: 10.2196/14835 (JMIR Ment Health 2020;7(5):e14835) doi: 10.2196/14835 Original Paper Original Paper Evaluation of Electronic Mental Health Implementation in Northern
Territory Services Using the Integrated “Promoting Action on
Research Implementation in Health Services” Framework:
Qualitative Study JMIR MENTAL HEALTH JMIR MENTAL HEALTH Raphiphatthana et al JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 1
(page number not for citation purposes) Evaluation of Electronic Mental Health Implementation in Northern
Territory Services Using the Integrated “Promoting Action on
Research Implementation in Health Services” Framework:
Qualitative Study Buaphrao Raphiphatthana1, BSc (Hons), PhD; Michelle Sweet1, BHealthSc, PhD; Stefanie Puszka1, BA, BIK (Hons),
GCYS; Megan Whitty2, BA (Hons), PhD; Kylie Dingwall1, BA (Hons), PhD; Tricia Nagel1, MBBS, FRANZCP, PhD 1Menzies School of Health Research, Casuarina, Australia
2Australian National University, Canberra, Australia 2Australian National University, Canberra, Australia Electronic Mental Health Within Australia’s First
Nations Context For First Nations, positive identities, extensive social networks,
connectivity with languages, and traditions can act as protective
factors for well-being; nevertheless, there is a much higher
prevalence of mental health concerns in comparison to other
Australian communities [22]. The multiple underlying factors
include consequences of colonization, marginalization,
intergenerational trauma, and enduring inequality and racism
[23,24]. Despite the high need for support, access to appropriate
services is poor, particularly for those who live in remote areas
[25]. With the rising presence of digital media in First Nations
communities, particularly for youth [26,27], an electronic mental
health approach has the potential to bridge the treatment gap
and overcome the barriers to services such as stigma and
geography. Evidence for the effectiveness of electronic mental health in
addressing mental health concerns has begun to emerge in the
last couple of decades. Several systematic reviews and
meta-analyses have shown that the approach is effective in
reducing symptoms for a range of mental illnesses, for example,
depression, anxiety, eating disorders, and substance misuse
[10-12]. Researchers and clinicians have, however, raised some concerns
about the approach. In particular, issues around cybersecurity,
confidentiality, data storage, and management have been
highlighted [13]. Other concerns include the level of responsivity
to crises, the lack of guidelines, and the limited number of
evidence-based tools and resources [14]. Nonetheless, efforts
are underway to address these concerns. Several professional
organizations have issued ethical guidelines for Web-based
counseling [15,16] as well as for risk management of crises
[17]. In Australia, electronic portals (ePortals) have been
developed to direct individuals to electronic mental health tools
and resources that have been filtered by experts, for example,
HeadtoHealth and Beacon [18]. The Australian government has
also launched a project to develop a certification framework to
ensure the quality of electronic mental health resources and
their safe application [19]. Research on the application of electronic mental health in
Australia First Nations contexts is still in its infancy; however,
the approach is perceived to be feasible, acceptable, and
appropriate [28]. From the service providers' perspective, the
approach is deemed useful for rapport building and engagement
with First Nations clients. Its applicability across a range of
clients, services, and settings is perceived to be broad and viable
[28]. Enthusiasm for electronic mental health is also expressed
by community members. Electronic Mental Health Background It is estimated that 450 million people worldwide are affected
by mental illness, that is, at least one in four people are burdened
by mental health conditions. This places mental illness as one
of the leading causes of ill health and disability worldwide [1]. Despite the high prevalence of mental illness, a significant
proportion of burdened individuals are not receiving treatment
[2-4]. The treatment gap is a global challenge involving a
complex interplay between multiple factors from policy to
individual elements and thus requires an innovative and feasible
solution. Menzies School of Health Research (Menzies) is a partner in
the eMHPrac project. In collaboration with the University Centre
for Rural Health (UCRH) and Queensland University of
Technology (QUT), we focus on raising awareness and
promoting electronic mental health resources along with
providing training and support to service providers working
with Aboriginal and Torres Strait Islander people (First Nations
hereafter) at the primary health care level, for example, nurse,
peer support worker, Indigenous health worker, psychologist,
and alcohol and other drug workers. In our use of the term First
Nations, we acknowledge the diversity of Australian Aboriginal
and Torres Strait Islander languages and traditions. The training
package aims to educate service providers on available
evidence-based electronic mental health resources and tools. The Menzies Aboriginal and Islander Mental Health Initiative
(AIMhi) Stay Strong app is one of the relatively few available
apps responsive to First Nations cultures. With the omnipresence of the internet and technology in today’s
society, many scholars recognize the potential of digital media
as a promising strategy to bridge the treatment gap in mental
health care. This surge of interest led to the emergence of the
electronic mental health field in 1999 [5]. An electronic mental
health approach has been defined as “the use of information
and communication technology... to support and improve mental
health conditions and mental health care” [6]. Typically, the
approach involves the use of a variety of digital platforms such
as telephone, mobile, computer, and Web-based apps. One of the attractions of the electronic mental health approach
is flexibility. Electronic mental health can be applied at different
levels of health care, such as promotion, prevention, early
intervention, active treatment, maintenance, and relapse
intervention. Its mode of utilization can also vary. Some tools
are designed to be self-directed and used without professional
support, whereas others are practitioner-guided [7,8]. Electronic Mental Health Background Other
strengths of electronic mental health include improved
accessibility, cost-efficiency, interactivity, and consumer
engagement [8,9]. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 2
(page number not for citation purposes) KEYWORDS eHealth; implementation science; health care delivery http://mental.jmir.org/2020/5/e14835/ http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 1
(page number not for citation purposes) XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al Introduction In line with global statistics, the disparity between demand and
supply of mental health services in Australia is significant [20]. To reduce the burden and treatment gap of mental illness in
Australia, the Australian government released the National
eMental Health Strategy, which aims to raise awareness and
utilization of electronic mental health approaches by all
Australians [21]. The initiative encompasses three components:
the head to health website (ePortal), Mindspot (electronic
clinic]), and nationwide electronic mental health training and
implementation support (electronic mental health in practice
[eMHPrac]). http://mental.jmir.org/2020/5/e14835/ Implementation of Electronic Mental Health It takes approximately 17 years for research to be translated
into clinical practice [30]. The time lag is referred to as the
translation gap, and it is a forefront issue in health research [31]. Minimizing this gap optimizes patient benefits and the use of
scarce resources. It is therefore imperative to gain an
understanding of the factors influencing translation to inform
robust implementation strategies. For the purpose of this study, innovation is defined as the
integration of external evidence (knowledge of research, clinical,
and patients’ experience relating to electronic mental health
approaches) with local priorities and practices to impact
electronic mental health uptake; recipients refers to the actors
involved in, affected by, and influencing implementation;
context is the setting in which the proposed change is to be
implemented and is considered across different levels from local
organizational to the external health system [34]. To date, little is known regarding effective strategies for
implementing electronic mental health resources in primary
health care services working with Australia’s First Nations. Findings from the very few studies in this field report common
themes essential to electronic mental health uptake relating to
three key domains: organizational factors, characteristics of the
adopters (ie, practitioners and clients), and the innovation itself. For example, factors that impose challenges to adoption are the
lack of organizational policy and procedural support for
electronic mental health utilization, and IT infrastructure and
resources. Poor IT literacy and knowledge of electronic mental
health also impedes the uptake of the electronic mental health
approach. Utilization is also dependent on the characteristics
of the innovation, that is, user-friendliness and cultural
responsiveness [32,33]. The specific objectives of this study were threefold. First, to
continue identifying important factors in the adoption of
electronic mental health from the service providers’perspective. Second, to further understand how they influence (ie, impede
or facilitate) electronic mental health adoption by service
providers working with First Nations, with reference to the
i-PARIHS framework. Third, to evaluate the usefulness of the
i-PARIHS framework in this specific context by determining
which elements are helpful as theoretical guides and how they
are operationalized in practice. From our findings, we aimed to
further the current understanding of electronic mental health
implementation and to provide practical guidance for future
implementation strategies for services and researchers working
with Australia’s First Nations. This Study This study aimed to further the current knowledge of electronic
mental health implementation in primary care services working
with Australia's First Nations. A conceptual framework, the
integrated Promoting Action on Research Implementation in
Health Services (i-PARIHS), was used to deepen the
understanding of factors influencing adoption. The framework
applied here is a revised version of the Promoting Action on
Research Implementation in Health Services (PARIHS) [34,35]. Electronic Mental Health Within Australia’s First
Nations Context Povey et al [29] conducted focus
groups exploring the response of First Nations community
members to their experience of two culturally adapted apps. Overall, the community members were optimistic about http://mental.jmir.org/2020/5/e14835/ XSL•FO
RenderX XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al and innovation were used to inform implementation efforts. This study, therefore, used the i-PARIHS to retrospectively
evaluate factors influencing electronic mental health adoption,
with a focus on the core constructs, context, innovation, and
recipients. By gaining a greater understanding of these three
constructs in relation to electronic mental health implementation
within primary health care services working with First Nations,
we will be able to develop a tailored facilitation strategy for
future electronic mental health implementation programs within
this context. electronic mental health and its potential for improving
well-being for First Nations. The three key factors identified as
influencing the acceptability of electronic mental health
approaches within indigenous settings were as follows: personal
factors (eg, motivation and information technology [IT] literacy),
environmental factors (eg, availability of resources), and app
characteristics (eg, graphics and content). Methods The Role of Menzies School of Health Research JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 3
(page number not for citation purposes) The Role of Menzies School of Health Research The eMHPrac project is a joint venture involving multiple
organizations across Australia to support electronic mental
health uptake using a number of dissemination and translation
strategies, including Web-based campaigning, promotions at
community activities and conferences, and provision of
electronic mental health training. Menzies have extensive
experience in developing and training service providers in
culturally grounded mental health interventions through the
AIMhi research program [36]. Therefore, our primary role in
the project is to provide electronic mental health training
workshops for service providers working with First Nations at
the primary care level. The
i-PARIHS
framework
conceptualizes
successful
implementation
as
“achievement
of
agreed
implementation/project goals, the uptake and embedding of the
innovation in practice, individuals, teams and stakeholders are
engaged, motivated, and ‘own’the innovation, variation related
to context is minimized across implementation settings” [34]. The framework posits that factors relating to innovation
(innovation, I), the individuals involved in the implementation
process (recipients, R), and the settings in which evidence-based
practice is to be implemented (context, C) are key considerations
for facilitation (Facn), which drives successful implementation. In other words, facilitation is an active ingredient that assesses
and draws knowledge from I, R, and C to inform strategies and
processes to enable successful implementation. The training aims to raise awareness of validated electronic
mental health tools and resources, specifically those that are
responsive to First Nations cultures as well as to build service
providers’ skills and confidence in applying them in their
practice. The training workshops, adapted from the AIMhi
training program Yarning about Indigenous Mental Health [37],
are delivered by Indigenous and non-Indigenous trainers. An The i-PARIHS is deemed useful as a determinant framework
for our analysis, as it resonates with findings from our earlier
work [33]. That is, factors related to the context, individuals, JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 3
(page number not for citation purposes) JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 3
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ JMIR MENTAL HEALTH Raphiphatthana et al in-depth description of the training workshop is reported
elsewhere [38]. Between June 2015 and November 2015, service providers who
had completed the training program were approached for a
follow-up interview via email and telephone. The aim was to
interview as many trainees as possible; however, the final
sample, 57, only represents a proportion of those trained. The Role of Menzies School of Health Research The
main reason for nonparticipation was staff movement into
different roles and organizations. The demographic information
of the trainees who participated in the interview, that is,
ethnicity, primary role, profession, and service type, is reported
in Table 1. Professions reported in the Other category included
support/administrative and managerial staff, nutritionists, and
dieticians. in-depth description of the training workshop is reported
elsewhere [38]. Service type (n=53) Aboriginal community−controlled health service Nongovernment community Government community epistemological position that sits between positivism and
constructionism. This epistemology recognizes that knowledge
is shaped by and acquires meaning in particular contexts, which
may be physical, cultural, or temporal. Through a contextualist
paradigm,
we
understand
electronic
mental
health
implementation as a process that is inalienable from local service
provision contexts [39]. A semistructured interview was selected epistemological position that sits between positivism and
constructionism. This epistemology recognizes that knowledge
is shaped by and acquires meaning in particular contexts, which
may be physical, cultural, or temporal. Through a contextualist
paradigm,
we
understand
electronic
mental
health
implementation as a process that is inalienable from local service
provision contexts [39]. A semistructured interview was selected Participants and Settings From October 2013 to December 2015, 261 participants received
training, and 21 training workshops were held in Darwin, Alice
Springs, and remote Northern Territory communities. The
training workshops were advertised through professional
networks, newsletters, and the Menzies website. Participants
either self-selected or were selected by their organizations to
attend the training. The participants were service providers
working with First Nations at the primary care level, such as
nurses,
support
workers,
Indigenous
health
workers,
psychologists, and alcohol and other drug workers. Table 1. Participants’ primary role, profession, and service type (N=57). Values, n (%)
Participants
Primary role (n=53)
2 (4)
Family well-being
15 (28)
Social and emotional well-being
7 (13)
Chronic disease
12 (23)
Youth
4 (8)
Alcohol and other drugs
1 (2)
Primary care
12 (23)
Other
Profession (n=54)
3 (6)
Aboriginal community worker
3 (6)
Alcohol and other drug worker
5 (9)
General nurse
1 (2)
Mental health nurse
2 (4)
Other nurse
3 (6)
Psychologist
11 (20)
Social worker
2 (4)
Peer support worker
1 (2)
Trainer/educator
6 (11)
Manager/coordinator/chief executive officer
17 (32)
Other
Service type (n=53)
8 (13)
Aboriginal community−controlled health service
30 (57)
Nongovernment community
13 (25)
Government community
2 (4)
Other
Research Design
A
li
i
h
b dd d i
li
di
epistemological position that sits between positivism and
constructionism. This epistemology recognizes that knowledge Table 1. Participants’ primary role, profession, and service type (N=57). Values, n (%) Primary role (n=53)
2 (4)
Family well-being
15 (28)
Social and emotional well-being
7 (13)
Chronic disease
12 (23)
Youth
4 (8)
Alcohol and other drugs
1 (2)
Primary care
12 (23)
Other
Profession (n=54)
3 (6)
Aboriginal community worker
3 (6)
Alcohol and other drug worker
5 (9)
General nurse
1 (2)
Mental health nurse
2 (4)
Other nurse
3 (6)
Psychologist
11 (20)
Social worker
2 (4)
Peer support worker
1 (2)
Trainer/educator
6 (11)
Manager/coordinator/chief executive officer
17 (32)
Other
Service type (n=53)
8 (13)
Aboriginal community−controlled health service
30 (57)
Nongovernment community
13 (25)
Government community
2 (4)
Other
Research Design
A qualitative approach, embedded in a contextualist paradigm,
was used to deepen our understanding of factors that influence,
that is, impede or facilitate, uptake of electronic mental health
approaches by primary care service working with First Nations
epistemological position that sits between positivism and
constructionism. Participants and Settings This epistemology recognizes that knowledge
is shaped by and acquires meaning in particular contexts, which
may be physical, cultural, or temporal. Through a contextualist
paradigm,
we
understand
electronic
mental
health
i
l
t ti
th t i i
li
bl f
l
l
i http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 4
(page number not for citation purposes) Aims of the Analysis The analysis aimed to identify factors and to form an
understanding of how they impede or facilitate electronic mental
health utilization. The i-PARIHS was used as a determinant
framework, that is, as a descriptive purpose for identifying
factors that influence implementation outcomes. Therefore, the
core elements and subelements that are supported by data are
presented according to the framework’s conceptualization. In
addition, as previously discussed, the facilitation construct is
not presented in the results, as it is considered to be the next
phase of the implementation process. The interviews took approximately 45 min to an hour to
complete. The interviews were recorded, and the audio was
transcribed verbatim by a professional transcription service and
then entered into the NVivo2 software program. The transcribed
data were not returned to participants for comments. Participants’ confidentiality was assured by the assignment of
ID codes. The extracts reported here are deidentified. All
participants provided written consent to interviews and audio
recordings, and ethics approval was granted by all relevant
ethics committees (reference number HREC 12-1881 and
CAHREC 12-100). The aim of the study was to explore the utilization of an
electronic mental health approach in general; however, many
of the responses were related to the use of the Stay Strong app. The app, developed as part of the AIMhi research program, is
a strength-based brief intervention designed to incorporate both
Indigenous and Western perspectives of mental health and
well-being [36]. The target audience for the training workshops
was service providers working with Indigenous Australians,
and so a portion of the training was focused on electronic mental
health apps that are culturally responsive to indigenous cultures. The Stay Strong app is one of the very few culturally adapted
tools, and thus attracted a lot of interest from the trainees. Data Collection The one-on-one, face-to-face, semistructured interviews
conducted in Alice Springs were carried out by MS; those in
Darwin were conducted by Katrina Kawaljenllo and SP. MS
and SP are female researchers who conducted the training
workshops and provided follow-up support. The interviews
were held at the researchers’or participants’workplaces, except
for three that were conducted over the phone as the participants
worked in remote communities, that is, Groote Eylandt and
Borroloola. The semistructured interview guide was developed
by the research team based on the team’s knowledge of the
implementation literature and experience researching and
working with service providers in electronic mental health
implementation. It was designed to explore participants’
attitudes and experiences with electronic mental health
approaches, and the perceived real or potential implementation
issues. Innovation Definition: The integration of external evidence (knowledge of
research, clinical, and patients’experience relating to electronic
mental health approaches) with local priorities and practice
impact on electronic mental health uptake. Research Design A qualitative approach, embedded in a contextualist paradigm,
was used to deepen our understanding of factors that influence,
that is, impede or facilitate, uptake of electronic mental health
approaches by primary care service working with First Nations. As explained by Braun and Clarke, contextualism is an JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 4
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ XSL•FO
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RenderX JMIR MENTAL HEALTH Raphiphatthana et al between the original PARIHS and the revised i-PARIHS
framework is the addition of the recipient construct, expansion
of the context element to encapsulate inner (local and
organizational context) and outer context (the wider health
system), and reconceptualization of facilitation to be an
overarching active factor that assesses and responds to the other
core constructs. The second phase of analysis involved
recategorization of the preidentified data extracts from the first
phase to the revised i-PARIHS framework elements and
subelements (BR). Therefore, the final themes were established
deductively based on the relevant principles of the i-PARIHS
framework. The remapping of data extracts to the i-PARIHS
elements and interpretation of the data were consulted with TN
and MS to ensure rigor and validity. Owing to the time lag
between the interviews and the final stages of analyses, the final
findings were not reviewed by the participants. However, our
findings will be disseminated through the newsletter, which
will be sent to participants on the distribution list. as the method to explore participants’ perceptions and
experiences of using electronic mental health resources. The research team was involved in the development of the Stay
Strong app (which is included in the training workshops),
delivery of the training workshops, follow-up support, and
conducting the interviews. This limitation is emphasized in the
Discussion section. Data Analysis A thematic analysis was used to analyze the data. The analysis
oscillated between deductive and inductive approaches and was
carried out in two phases. The first phase involved
familiarization with the data by multiple readings, and the
preliminary coding of the data was conducted independently
by 2 authors, MS and MW. At this stage, the codes were formed
inductively, and common perspectives were grouped together
with an assigned descriptive code name. The comparison
revealed consistency across categories and broad agreement on
codes and key themes. Data saturation was reached. Through
collaboration, 3 members of the research team (MS, MW, and
TN) reinterpreted participants’experiences by using the original
PARIHS framework to retrospectively evaluate key elements
influencing electronic mental health implementation. The coded
extracts were categorized into themes in reference to the
elements and subelements of the framework through extensive
discussion between the authors. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 5
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ Underlying Knowledge Sources The three key areas of knowledge relating to electronic mental
health approaches identified as essential in the decision-making
process to use a particular resource include the context in which
the resource was developed, the population it was developed
for, and whether it has been validated. Utilization was dependent
on whether the user was confident that the information
embedded in the resource aligned with the local settings and Post analysis, the i-PARIHS framework was discovered, and
thus another phase of analysis took place to ensure that the most
up-to-date conceptual framework was used. The key differences http://mental.jmir.org/2020/5/e14835/ XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al those who worked with youth were more receptive to the
innovation. target populations. Some of the participants expressed a lack
of knowledge and confusion regarding the context and the
population for which the innovation was developed and
validated. Therefore, they were uncertain about the suitability
of the resources for their clients: I work with young people between twelve and eighteen
and technology is their bible, you know the things
they go to most. So I find that the use of electronic
mental health is like a good resource that young
people feel comfortable using. [P205, youth and
family worker, nongovernment community] A lot of the time there are resources listed but then
you are not really sure where they were developed,
in what context, and then if they would be useful in
the Central Australian context. Sometimes it could
be more tailored towards Top End, or somewhere
else completely. [P81, social worker, nongovernment
community] Electronic mental health was also perceived to be potentially
beneficial and useful for First Nations communities: A lot of the work we do is based on community needs
and a very traditional way of doing things…but even
they want more different ways of doing things as well
and capturing information on film and photographs
and applications like this... So I think there is a real
need for communities to use applications and things
like this. [P81, social worker, nongovernment
community] e-health in general, some of them are not [developed
for First Nations clients] so we wouldn’t be using it.
[P89, psychologist, Aboriginal community−controlled
health service] No change to clinical practice... we just incorporate
into our framework of how we work with young
people. [P32,
social
worker,
nongovernment
community] Challenges to electronic mental health utilization in
communities, particularly those living in remote areas, were
limited availability and accessibility to resources such as IT and
the internet as well as low IT literacy levels: Uptake of electronic mental health approaches was dependent
upon the perceived fit of the innovation to the organization’s
priorities. Specific to the Stay Strong app, some participants
reported that conducting brief interventions was not an
immediate priority; hence, the app was not fully integrated into
practice. Utilization was also dependent on the perceived fit
with service providers’ roles and responsibilities. For example,
a participant commented that alcohol and other drug workers
perceived their primary responsibility to include the following
but not providing brief interventions: It depends on the situation or the context…a lot of
stuff can be difficult in remote communities because
of access to internet or things like that. [P98, social
worker, Aboriginal nongovernmental organization)] http://mental.jmir.org/2020/5/e14835/ Degree of Fit With Existing Practice and Values The degree of change required to incorporate electronic mental
health into current practice was perceived by a minority of the
participants to influence the speed of adoption. For example, a
participant said: It just required us all to change practice a bit slightly
too. So we dragged the ball a bit. [P129, peer support
worker, government community] It just required us all to change practice a bit slightly
too. So we dragged the ball a bit. [P129, peer support
worker, government community] However, utilization was dependent on the apps’ cultural
responsivity to First Nations cultures. This was expressed by 1
participant: This, however, appears to vary across organizations as another
participant reported that minimal change in practice was needed: e-health in general, some of them are not [developed
for First Nations clients] so we wouldn’t be using it. [P89, psychologist, Aboriginal community−controlled
health service] e-health in general, some of them are not [developed
for First Nations clients] so we wouldn’t be using it. [P89, psychologist, Aboriginal community−controlled
health service] Motivation In general, most participants expressed enthusiasm toward
electronic mental health approaches; however, some participants
commented that having the motivation to incorporate it into
practice was a challenge. The lack of motivation was partly due
to high workload: You can fill out all the information you need on an
app and then email your information straight across
so it makes life a little bit easier for us. [P124, social
worker, nongovernment community] People are already feeling overwhelmed by their work
and then all of a sudden, “Oh, I’ve got to do it this
way”, because you have to put more thought and
effort into it and that turns people off. [P133, other
nurse, government community] However, the technological requirements of electronic mental
health approaches were perceived to impede implementation
by most participants. Specifically, participants reported
difficulties accessing the appropriate technology, that is, iPads
or the internet as well as having trouble with various aspects of
the technology such as charging, passwords, and connectivity
requirements: Nevertheless, motivation was acknowledged to be intrinsic,
which could overcome the challenges of circumstance. As one
participant said: Your barrier is yourself... when you’ve got a hundred
other things that you’re focusing on with a young
person, adding another one in often seems like a
barrier but it isn’t. [P193, youth and family worker,
nongovernment community] We have challenges with our apps and our iPad
because the communities I go to don’t have WIFI
hotspots so I can’t access internet. [P108, general
nurse, government community] Values and Beliefs Moreover, for the participants who worked across multiple sites,
having to remember to take the technology with them was a
challenge: Electronic mental health approaches were perceived by the
majority of the participants to fit with the times and future
directions and to be particularly applicable for young people: We didn’t even think to grab the i-Pad when we were
heading out, or even ask for it. [P85, alcohol and other
drug worker, Aboriginal community−controlled health
service] More relevant in peoples' modern lives. It's using
technology these days, and if we can keep using paper
based and forms that are wordy, that sort of thing,
we are not going to be effective so yes, I think it's
good. It's simple language and it's digital which is
where we need to be. [P129, peer support worker,
government community] Recipient The definition of recipient is as follows: “Actors involved in,
affected by, and influencing implementation. Characteristics of
the individuals, eg, motivation, values, beliefs, skills and
knowledge, and factors relating to the team’s culture influence
individuals and teams in supporting or resisting an innovation.” Trialability Having a good understanding of how electronic mental health
resources are effectively used in a real-life context was identified
to be important in the training and implementation process. It was also considered to be a good and valuable option for
therapeutic practice. However, 1 participant was concerned that
the utilization of electronic mental health resources without
professional supervision would negatively impact the
face-to-face treatment process: So it’s all good and well to role play or simulate using
it, but until you have the client there – especially with
a young person whose vocabulary isn’t the same as
an adult, you know, that’s where more of the training
would happen. [P205, youth and family worker,
nongovernment community] So it’s all good and well to role play or simulate using
it, but until you have the client there – especially with
a young person whose vocabulary isn’t the same as
an adult, you know, that’s where more of the training
would happen. [P205, youth and family worker,
nongovernment community] Some of these e-Health resources adopt psychological
procedures [that may not be] appropriate for the
client, [so] the client’s attribution is, “Oh, it doesn’t
work”. So when they eventually are referred to see a
psychologist... it spoils their chances of benefitting
from psychological therapy because they come in with
a negative attitude already. [P84, psychologist,
organization not reported] One participant reported having undergone a trial of electronic
mental health resources with their clients to assess suitability: We trial them on a small group of people to see if it’s
suitable for our population and see where we go from
there. [P124,
social
worker,
nongovernment
community] We trial them on a small group of people to see if it’s
suitable for our population and see where we go from
there. [P124,
social
worker,
nongovernment
community] Successful implementation of electronic mental health was also
perceived to be dependent on the organization’s appraisal of
the approach. One participant expressed that integration of
electronic mental health into the general orientation is dependent
on whether the organization saw the value of the approach. This suggests that, at least for this organization, electronic
mental health approaches can be implemented in a step-by-step
process. JMIR MENTAL HEALTH JMIR MENTAL HEALTH Raphiphatthana et al Specific to the Stay Strong app, some participants expressed
that the app has relative advantages over a traditional paper
format in terms of data recording and management. They also
voiced that it helped streamline their approach and
communication between staff: Usability and Relative Advantages Overall, most participants perceived electronic mental health
resources to be more interactive, engaging, and useful for
bridging literacy gaps as well as enabling clients to exert
ownership over their well-being journey: It was far better than having a therapist really
because they could do it at their speed, and it was
often someone with anxiety and a bit of OCD and
things like that. They have to get things just right and
having a computer to deal with is really very, very
good for them. [P41, occupation, and organization
not reported] Thus, the Stay Strong app was not incorporated into their
practice. In addition, participants in a managerial role explained
the nonutilization of eMental Health due to its irrelevance to
the role (eg, acting manager). Characteristics of the target population (age, culture, and IT
literacy) were also important considerations. With regard to
age, some participants expressed concerns over electronic mental
health incompatibility with the older population: One participant also commented that electronic mental health
is flexible, easily accessible, and a useful resource: the older people don’t really relate to the animation
and to the touching of the screen. They don’t really
feel comfortable. [P58, general nurse, Aboriginal
community−controlled health service] I do like that you can call a phone number for mental
health and you can get advice whether you’re a
patient or whether you’re a professional. I like the
way you can go online and you can do little tests and
go, “Okay. I’m not suicidal” or “Okay. My child’s
not depressed.” [P108, general nurse, government
community] The majority of the participants perceived electronic mental
health to be applicable and suitable for young people, and so http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 6
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RenderX http://mental.jmir.org/2020/5/e14835/ comfortable about intervening in a more therapeutic
way.
[P89,
psychologist,
Aboriginal
community–controlled health service] comfortable about intervening in a more therapeutic
way. [P89,
psychologist,
Aboriginal
community–controlled health service] If the co-ordinator sat down and said, “Okay. We’re
going to apply this [the app]…”, then obviously we
would follow it through–that would be something that
we could focus on - but that was never forwarded to
us in any way. [P20, social support worker and
well-being, nongovernment community] Collaboration and Teamwork Some participants expressed the view that staff encouraging,
teaching, and holding each other accountable with regard to the
use of electronic mental health may facilitate adoption. For
instance, 1 participant commented that collaboration and
encouragement could prompt utilization. Finally, the lack of confidence and skills to conduct therapeutic
interventions was perceived to impede service providers’ use
of electronic mental health resources. For example, specific to
the Stay Strong app, 1 participant reported that alcohol and other
drug workers were reluctant to use the app as they were not: And a lack of IT infrastructure within communities: [P94,
manager/coordinator/chief
executive
officer
nongovernment community] The staff, they are not getting enough support, or they
might need more training or support, or workshops
internally
to
use
it
adequately. [P94,
manager/coordinator/chief
executive
officer
nongovernment community] … so many of the patients we work with don’t use
technology in that way. A lot of people use mobile
phones … not necessarily Smart Phones. Some might
enjoy the i-Pad and being able to play games on it,
but … accessing computers and the internet within
the mob that we work with is not so much. [P91,
administration
support,
Aboriginal
community−controlled health service] … so many of the patients we work with don’t use
technology in that way. A lot of people use mobile
phones … not necessarily Smart Phones. Some might
enjoy the i-Pad and being able to play games on it,
but … accessing computers and the internet within
the mob that we work with is not so much. [P91,
administration
support,
Aboriginal
community−controlled health service] Goals Specific to the Stay Strong app, some participants reported
having trouble remembering using it in their day-to-day practice. An explicit goal setting of where, when, and with whom the
electronic mental health resource will be used was suggested http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 7
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RenderX JMIR MENTAL HEALTH Raphiphatthana et al You need to have will coming from the top…It starts
there. Why is there no will? Partly because that
person may have so many things… so many battles
from the front that something like this falls behind. [P84, psychologist, organization not reported] by 1 participant as a potentially useful technique to combat this
issue: I had these big ideas. I was like, “Oh, I’m going to
use it. It’s going to be great. I’m going to get all these
really good results” [but]when I do home visits,
I…forget to take it with me … – you’ve almost got to
allocate a scheduled time of who you want to do it
with and say, “Okay. So I’m going to do it on this
time.”
[P95, mental health support worker,
nongovernment community] The lack of IT resources prevented both service providers and
community members from using an electronic mental health
approach. Many participants reported limited resources such as
iPads within their workplace: I tried to get our iPad – a single iPad that we have
here – and it was lost or someone had it and it was
just difficult [P122, general nurse, government
community] http://mental.jmir.org/2020/5/e14835/ Skills and Knowledge The lack of knowledge around electronic mental health
approaches poses a barrier to utilization. This includes
uncertainty around the effectiveness, accessibility, and
applicability of the tools and resources. Having the ability to
efficiently sort out information and select the right tools and
resources for target clients was perceived by 1 participant to be
an important skill in electronic mental health adoption: And a lack of IT infrastructure within communities: And a lack of IT infrastructure within communities: And a lack of IT infrastructure within communities: Places that young people and older people can go
and get online, that is actually going to make a bit of
a difference. [P60, Aboriginal community worker,
Aboriginal community−controlled health service] Lack of supervision due to skilled staff shortage served as a
barrier for incorporating electronic mental health into the general
practice: There is some great resources out there but I need to
know where to access them, how to access them, and
what to do with them once I have them... Because
there is so much out there as well. It's a bit
overwhelming at the same time. So to try and pick the
right resource for the right setting, for the right people
and finding that online, can be a daunting experience
as a practitioner in trying to work out where to start
with that. [P81, social worker, nongovernment
community] It was a time when we had very few therapists and so
the AOD workers were mostly left to their own devices
to interact with clients as they saw fit and we weren’t
able to provide closer supervision and so while we
did roll out the app, it was seeds not going into fertile
ground at that time. [P89, psychologist, Aboriginal
community−controlled health service] It was a time when we had very few therapists and so
the AOD workers were mostly left to their own devices
to interact with clients as they saw fit and we weren’t
able to provide closer supervision and so while we
did roll out the app, it was seeds not going into fertile
ground at that time. [P89, psychologist, Aboriginal
community−controlled health service] Low levels of IT literacy pose a challenge to electronic mental
health adoption. Unfamiliarity with different ways of using
technologies impedes the utilization of the approach by both
service providers and community members. Poor IT literacy
within communities was attributed to limited access to
technology: Insufficient support and training within the organization were
also perceived to hinder utility and negatively impact fidelity
of use: The staff, they are not getting enough support, or they
might need more training or support, or workshops
internally
to
use
it
adequately. [P94,
manager/coordinator/chief
executive
officer
nongovernment community] The staff, they are not getting enough support, or they
might need more training or support, or workshops
internally
to
use
it
adequately. Power and Authority Staff in an authoritative role were considered important in
driving successful implementation. This was highlighted by 1
participant who attributed nonutilization of electronic mental
health to the absence of clear directions from those in power: Time, Resources, and Support Finding the time to explore and learn more about electronic
mental health resources was a challenge for many participants. High workload was reported to also delay senior staff, who was
perceived to be an important driver, to action implementation: JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 8
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RenderX JMIR MENTAL HEALTH Raphiphatthana et al receptive to innovations and more inclined to invest effort into
the implementation process. Structure and Systems “Have you used your Stay Strong yet?” [P121,
occupation, and organization not reported] Have you used your Stay Strong yet? [P121,
occupation, and organization not reported] Two main aspects of the organizational structure and systems
were identified as influential in electronic mental health
adoption. One aspect relates to the adequacy of the existing (or
the lack thereof) structure and systems to support electronic
mental health utilization, and the other involves the effectiveness
of the integration process in the existing system. Organizational Priorities Delays in the implementation process were reported to be partly
due to resource constraints, that is, technology, workforce, and
structures to support electronic mental health approaches. Prioritization of financial and training investment was also
perceived to impact implementation. Decisions on which aspects
within the organization are worth investing in may render
insufficient resources for some programs. Whether our program has the financial resources to
invest in that, I’m not sure. And also whether we are
going to have sort of an iPad available for all
programs to use which would make it more accessible
for my program. And that is still undecided yet. [P149,
trainer/educator, government community] What’s happening is that we are living in an economic
environment where we get funding for six months or
twelve months and we’re not attracting staff to stay
here or people who can help with the training and
the knowledge base of the team…there are a lot of
indigenous organisations who are constrained by
those very factors. [P89, psychologist, Aboriginal
community−controlled health service] Strategic investment in training may potentially assist
organizations in sustaining implementation efforts in the long
term. For example, one participant reported: We thought training the local Aboriginal staff was a
better option... they are the more appropriate people
to talk to their own community... and they are going
to be there longer. So in that way, we tried to
overcome some of the turnover and issues. [P135,
manager/coordinator/chief
executive
officer,
government community] Policy was another structural issue identified to influence the
utilization of the electronic mental health approach. Inadequate
policies regarding the use and access of the technology and the
client’s
confidentiality
were
reported
to
delay
the
implementation process: So the policy and procedure needed to be created to
get around our usage and downloading apps; and
how the staff could access it; and who they need to
go to if they found an app that they would like to use. [P149, trainer/educator, nongovernment community] Evaluation and Feedback Processes As illustrated above, evaluation of the effectiveness of electronic
mental health approaches may incentivize practitioners to
continue using the approach and support fidelity. The three key areas identified to be lacking in the organizational
structure and systems were resources, policies, and procedures
to support the use of the electronic mental health approach. Insufficient IT resources were highlighted as an impediment to
utilization. Many participants expressed difficulties in obtaining
iPads in a timely manner and gaining access to the internet,
particularly in remote settings. In addition, shortage of skilled
staff and high staff turnover meant difficulties retaining
knowledge and expertise within the workplace, and thus limited
support and supervision for the general staff. One participant
attributed these issues to inadequate funding, particularly for
First Nations organizations: Local Context Formal and Informal Leadership Support Formal and Informal Leadership Support We’re quite keen on the AOD workers up-skilling in
their abilities to at least do brief interventions. So
when it comes time to concentrate on that, that will
be where the app comes into its own and having had
it introduced into the team and then some follow-up
recently, it will make implementation quite easy. [P89,
psychologist, Aboriginal community-controlled health
service] Having leaders within the organization showing interest and
providing direct support was perceived to facilitate uptake. It
created incentives and provided opportunities for service
providers to reflect and evaluate the utility of the electronic
mental health approach. The manager was supportive of the app… she would
ask how we were going with the application and we
would sit down and show her that it is really working
and
purchasing
some
results. [P224,
manager/coordinator/chief
executive
officer,
Aboriginal community−controlled health service] The manager was supportive of the app… she would
ask how we were going with the application and we
would sit down and show her that it is really working
and
purchasing
some
results. [P224,
manager/coordinator/chief
executive
officer,
Aboriginal community−controlled health service] A culture of accountability was expressed by one participant to
be potentially useful for enhancing adoption: We have very little accountability generally in our
workplace which I would like more of... accountability
from management would be good... And perhaps even
the peer accountability between the dietitians. [P157,
nutritionist, government community] However, 1 participant reported that too much advocacy could
create pressure instead of being supportive: Definitely supported to use it... There was a lot of –
I don’t know. I would say a lot of pressure. Like,
“Have you used your Stay Strong yet?” [P121,
occupation, and organization not reported] Definitely supported to use it... There was a lot of –
I don’t know. I would say a lot of pressure. Like,
“Have you used your Stay Strong yet?” [P121,
occupation, and organization not reported] Definitely supported to use it... There was a lot of –
I don’t know. I would say a lot of pressure. Like, http://mental.jmir.org/2020/5/e14835/ Interorganizational Networks and Relationships Communication across services working with the same client
may help to ensure nonoverlapping of interventions and
resources. The issue of repetition posing a negative impact on
the clients’ experience of electronic mental health approaches
was expressed by 1 participant: We’ve just been held up by, you know … at the
Director level … somewhere in the corporate
communications area…it’s getting approval for these
things. [P134, public health nutritionist, government
community] Everybody is coming on board with these apps, and
often we all service the same clients…How often do
these clients get asked to do this because the clients
that we work with often… it’s passive resistance…I
wonder if it’s [us] asking them to do the same thing. [P106 occupation and organization not reported] Many participants reported difficulties obtaining iPads in a
timely manner due to reasons such as unavailability and empty
battery as well as remembering to use it as part of their practice. To address some of these difficulties, one participant suggested
developing on the ground processes to systemize the utilization
of IT resources: f
[
]
g
g
[P106, occupation and organization not reported] Culture A work culture that values on-going staff development was
identified as important for successful implementation. The
commitment to upskilling staff may render organizations more http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 9
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RenderX JMIR MENTAL HEALTH Raphiphatthana et al accreditation process of resources and tools, investment in
technology in remote areas, and funding to First Nations
organizations. Organizational processes to streamline the integration of
electronic mental health into the existing system were identified
to be essential for successful implementation. Inadequate
integration may increase workload or render the innovation lost
within the system, and thus impede staff from incorporating the
approach into their practice. In addition, insufficient processes
at the higher management level was reported to delay the
implementation process and constrained utilization: The Integrated Promoting Action on Research
Implementation in Health Services as a Determinant
Framework [the] process could be streamlined…say, “Here –
here’s an iPad”– or whatever the process is. [P122,
general nurse, government community] [the] process could be streamlined…say, “Here –
here’s an iPad”– or whatever the process is. [P122,
general nurse, government community] To further understand the conditions for successful
implementation of electronic mental health approaches, this
study aimed to provide insights into factors that impede or
facilitate electronic mental health uptake within primary health
care services for Australia’s First Nations in the Northern
Territory. The i-PARIHS was used as a determinant framework
to deepen our understanding and interpretation of service
providers’ experiences and perspectives on electronic mental
health adoption. Finally, having a procedure to train new staff and disseminate
new resources among existing staff was perceived to be
important for effective use of electronic mental health in the
long term. I think there should be a process for it so if something
new comes out, you tend to go around and show
people how they use it and practise it. So you’ll teach
someone your bad habits... I think better processes
and having some evaluation of it. “We use this
because it’s been shown to …” you know, “do this
or do that” – not just “Isn’t this pretty and here’s
another thing to use”. [P138, general nurse,
government community] The framework had been previously used in a similar
manner—as a retrospective analysis by Laycock et al [40], who
found it to enhance their understanding of factors that influenced
the interactive dissemination process. Similarly, we found that
the framework helped us to understand service providers’
experiences systematically. It also deepened our understanding
of how the innovation, recipient, and context, relating to
electronic mental health adoption, operationalized in a real-life
context. However, we also found that some of the i-PARIHS
elements could not be analyzed as separate constructs due to
their interdependencies. This is due to both the complex nature
of implementation, that is, the inter-relations and flow-on effects
among factors in determining implementation outcomes as well
as the difficulty in teasing out conceptual differences among
some of the i-PARIHS subelements. History of Innovation and Change The history of introducing technology to the workplace may
ease the integration of the electronic mental health approach
into the system. For example, 1 participant reported: IT policies are pretty up to date with regards to using
iPads and stuff like that, which all happened when
we did start getting the iPads in to do – ‘cause we
used them for Survey Monkey to do feedback. [P188,
case manager, nongovernment community] For example, the lack of human resources and IT infrastructure
at the organizational level (context) impacted the availability
of supervision and technological devices for individual
practitioners (recipients). Therefore, when discussing the
negative impact of the lack of resources and support on
electronic mental health uptake, it is difficult to categorize if
its related to context or recipients when there is an
interdependency, that is, the lack in the organizational structure
has a flow-on effect on the individual practitioner. http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 10
(page number not for citation purposes) Policy Drivers and Priorities The importance of the external health system in the successful
implementation of electronic mental health approaches was
foreshadowed throughout. As described in the previous
elements, participants expressed the importance of electronic
mental health approaches to be validated for the First Nations,
IT infrastructure within community and organization, and human
resources in electronic mental health uptake. This suggests a
perceived need for policies to cover a range of areas, that is, the Another example is the issue of knowledge related to electronic
mental health approaches. Participants expressed a lack of JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 10
(page number not for citation purposes) XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al proficiency in electronic mental health adoption was highlighted
in previous studies in primary health care for First Nations. This
suggests the potential usefulness of basic IT skills training for
service providers [32,33]. In addition, service providers had
limited knowledge of electronic mental health approaches,
particularly of the context in which the resources were
developed, the population they were developed for, and whether
they have been validated. This resonates with the general lack
of electronic mental health awareness and knowledge exhibited
by the wider population [24,46,47]. knowledge and confusion regarding the context and the
population for which electronic mental health approaches were
developed and validated. Thus, they were uncertain about the
suitability of the resources for their clients. This important issue
is related to recipients as it highlights that, practitioners
themselves lack the knowledge; however, it is also related to
innovation as it demonstrates the importance of information
from research, clinical, patient, and local experience in electronic
mental health uptake. Moreover, there were no definitions for
the subelements within each of the core elements, and thus we
had to rely on our understanding. This lack of knowledge impeded utilization, as confidence that
the resources are appropriate for First Nations and the local
settings was essential to uptake. The need for electronic mental
health resources to be culturally responsive to First Nations has
been previously highlighted [29,33]. As part of the electronic
mental health training program provided by Menzies, culturally
responsive tools and resources such as the Stay Strong app are
promoted. The training program also promotes directories, such
as Head to Health and Beacon, which provide links to other
relevant resources. Principal Findings Similar to previous studies, service providers showed optimism
and enthusiasm for electronic mental health approaches and
viewed them as valuable and potentially useful resources,
particularly for young people [32,33]. In addition, the approach
was perceived to be suitable and useful for First Nations
communities, resonating with previous findings from other
health organizations and community members [29,33]. With
the omnipresence of information and communication technology
in today’s society, digital media such as smartphones and
computers are inevitably becoming part of and shaping First
Nations’lives [26,27]. Although still in its infancy, the literature
suggests promising potential for electronic mental health to
enhance First Nations well-being and culture [41,42]. Similar to the systematic reviews’ findings, we found that
organizational factors played a major role in electronic mental
health uptake [44,45]. In particular, the lack of IT resources and
infrastructure posed a significant barrier to utilization. Insufficient workforce due to staff shortage and high staff
turnover was an impediment as it meant high workload and
difficulties retaining knowledge and expertise within the
workplace. Uptake was hindered by inadequate policies to guide
the appropriate use of technology and confidentiality, and
insufficient processes to streamline the integration of electronic
mental health approaches into existing systems. Direction and
support from leaders in the organization were also important
for providing incentives and may enhance fidelity in electronic
mental health use. These barriers were previously noted by
Bennet et al [32]. However, limited availability and access to IT resources in some
areas impeded electronic mental health utilization and
contributed to community members’ low levels of IT literacy. The lack of resources, particularly in remote areas, is recognized
by the Australian Government. Efforts are being made under
the Mobile Black Spot Program and the National Broadband
Network as part of the government Closing the Gap 2016
initiative to ensure the availability of IT across Australia [43]. In line with findings from systematic reviews on factors essential
to the successful implementation of electronic mental health
approaches [44,45], the present findings demonstrate the
importance of service providers’ knowledge, skills, and
perception in electronic mental health adoption. Particularly,
service providers’ low levels of IT literacy pose a challenge to
electronic mental health utilization. Policy Drivers and Priorities Nonetheless, future adaptation of the training
program will develop service providers’ skills in obtaining and
evaluating relevant information on electronic mental health
approaches to facilitate independent selection of suitable tools
for their clients. With regard to the conceptualization of facilitation (an active
ingredient that assesses and aligns innovation, recipients, and
context), we found it to provide greater depth to our
implementation strategy. We viewed ourselves as an external
facilitator aiming to empower internal facilitators and service
providers to incorporate electronic mental health as part of their
practice. We intend to use our findings relating to the innovation
(electronic mental health approach), recipients (service providers
as an individual and as a team), and context (local,
organizational, and external contexts) to inform future
facilitation strategies and catalyze electronic mental health
uptake. The key findings, as discussed below, were therefore used to
develop a structured approach or framework for implementing
electronic mental health approaches within First Nations primary
health care services. The adoption of electronic mental health was also dependent
on the perceived fit of the approach to the organizational ethos
as well as on the practitioners’ role and responsibilities, which
is in accordance with previous findings [32,33,44,45]. More
specifically, service providers who held managerial roles, and
those who did not work with clients in a therapeutic way did
not use the approach. However, it was recognized that leaders
within the organization, who may not work with clients directly,
can have a positive impact by championing the approach and
influencing others to use it as part of their practice. This
resonates with the literature that identifies internal facilitators
as important figures in successful implementation [48,49]. http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 11
(page number not for citation purposes) Pretraining Consultations Organizational readiness is conceptualized to comprise both
individual difference and structural factors, reflecting the extent
to which the organization and its members are inclined to
accept, embrace and adopt a particular plan to purposefully
alter the status quo. Individual difference refers to the
characteristics of those being asked to change (eg, skills,
knowledge, attitude, perception), and structural factors refer to
the circumstances under which the change is occurring (eg,
infrastructure, policies, resources availability) [51]. Our findings demonstrate the importance of organizational
readiness in successful implementation. Organizational factors
highlighted as essential to uptake are IT resources and
infrastructure; policies around the use of technology and
confidentiality; human resources; processes to streamline
integration of electronic mental health into the existing system;
and leadership/internal facilitators to advocate, action
implementation, train, and provide support and supervision for
on-going electronic mental health utilization within the
organization. Thus, before training, it is pertinent for
organizations to assess their level of readiness across the
highlighted areas to identify aspects that require improvements. Key considerations are listed in Textbox 1. To ensure that
organizations have thoroughly reflected on their readiness and
improve the identified areas, we propose at least two
consultation sessions. The objective of the first session is to aid
organizations to assess their level of electronic mental health
readiness and develop an action plan to address areas in which
adaptation is required. The second session’s aim is to provide
an opportunity for further identification and refinement of
strategies to address the areas of improvement. The importance of organizational readiness in successful
implementation is well recognized in the literature. Multifaceted
approaches to implementation are considered an optimal strategy
for influencing change [52]. In the literature, implementation
frameworks such as the four-stage implementation model
developed by the National Implementation Research Network
[53] and the Quality Implementation Framework [54], propose
multiple stages to implement with the initial stages, focusing
on need analysis and development of strategies addressing
organizational readiness. Reflecting on our electronic mental health implementation
strategy, we recognize the limitations of one-off trainings and
the need for a more comprehensive facilitation strategy that
addresses organizational readiness. Taken together, the present
findings and the literature, we propose a multistaged
implementation strategy aimed at enhancing electronic mental
health uptake by addressing the different aspects of
organizational readiness relating to electronic mental health
implementation at both the individual and structural levels. Principal Findings The importance of IT A scoping review found that organizational drivers (decision
support, administration, and system intervention) and leadership
drivers received significantly less attention than competency
drivers (staff and user selection, training, and supervision) in
the electronic mental health implementation literature [50]. The
importance of organizational structure, systems, and leadership XSL•FO
RenderX Raphiphatthana et al JMIR MENTAL HEALTH Implementation
Research
Network
and
The
Quality
Implementation Framework [53,54], we propose a three-phase
implementation strategy for electronic mental health
implementation in health care, involving: (1) pretraining
consultations to address organizational readiness, (2) training
workshops to increase awareness, knowledge, and skills relevant
to electronic mental health utilization, and to train internal
facilitators to supervise, support, and conduct trainings within
the organization, and (3) follow-up support to boost the trainees’
motivation and the organizations’ efforts to incorporate
electronic mental health into practice and the existing system,
and to provide support for internal facilitators in supervising
and training other staff. support in electronic mental health adoption highlighted in the
present findings as well as in the previous findings [32,33]
suggest that much more attention to these elements is needed. It is important to note that Puszka et al [33] reported on chief
executive officers’, mangers’, and directors’ perspectives on
potential barriers and enablers of electronic mental health
implementation, whereas this study provided the experiences
of service providers who have undergone electronic mental
health training and had some time to apply what they have
learned into their practice. Nonetheless, similar issues around
organizational readiness have been raised by both cohorts, thus
emphasizing the need for organizational readiness to be
addressed in implementation strategies. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 12
(page number not for citation purposes) Training The training should be contextualized and involve information
and activities relevant to the trainees. That is, it should include
resources that are applicable to the trainees’ local settings and
target populations, that is, resources that are culturally relevant
to local indigenous populations as well as incorporating IT
training to ensure adequate levels of IT literacy for electronic
mental health utilization. The training should also provide
trainees with the skills to find, assess, and evaluate information
to select the right electronic mental health resources for their
specific clients. In addition, a time should be set aside for
implementation planning, so trainees can plan where, when,
and with whom they would use an electronic mental health
approach. Pretraining Consultations The assessment should function as both a diagnostic and evaluative tool to track the trajector
of organizational readiness over the course of the intervention
•
Comparison of the frequency of electronic mental health use before and after the intervention
•
Frequency and quality of trainings conducted within the organization by internal trainers
•
Service providers’ skills, knowledge, and perception of electronic mental health approaches before, during, and postintervention
•
Records of discussions during pretraining consultations and follow-up support to illuminate the progression and type of support needed throughou
the intervention
T
i i
offered. Specifically, it should provide internal facilitators wit offered. Specifically, it should provide internal facilitators with
the knowledge and skills to support, supervise, and conduct
electronic mental health training with the organization. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 13
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ Pretraining Consultations This stage should also instigate reflections on who within the
organization should attend training and who would be
appropriate to carry the role of an internal facilitator. Moreover,
information regarding organizations’ local settings and target
populations, as well as the service providers’ levels of IT skills
and literacy, should be noted. This information would be useful
in informing the training sessions so that the resources and
information are contextualized and more relevant and useful
for trainees. Given the importance of preparatory stages as outlined in other
existing implementation frameworks, for example, the National JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 12
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 12
(page number not for citation purposes) XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al Textbox 1. The three-phase facilitation process for electronic mental health implementation. Phase 1: Pretraining consultations
Consultations assess the following aspects of organizational readiness:
•
Information technology (IT) resources and infrastructure, that is, computers, iPads, and Wi-Fi connectivity
•
Policies and procedures to guide collection, confidentiality, and security of data
•
Current conducive work culture and climate (eg, accountability, on-going learning, staff turnover, and priorities)
•
Staff skills and literacy in IT and electronic mental health approaches
•
Suitability of client population and cultural context
•
Internal facilitators to champion, train, and support on-going e utilization
Phase 2: Training
Workshops occur when suitable organizational readiness is achieved through mutual agreement and incorporate the following elements:
•
IT skills training if needed (determined through consultation)
•
Presentation of electronic mental health resources relevant to organizations’ local and cultural needs
•
Strategies to find and evaluate electronic mental health resources
•
Information regarding electronic portals hosting resources that are filtered by experts
•
Planning when, where, and with whom, electronic mental health resources will be used
•
Training for internal facilitators to supervise and support on-going electronic mental health use
Phase 3: Follow-up support
•
Face-to-face, email, and telephone follow-up to support on-going use
•
Face-to-face, email, and telephone follow-up for internal facilitators to support their training, support, and supervision
•
Review progress of organizational readiness and support implementation
•
Guidelines for evaluating the implementation strategy
•
Assessment of organizational readiness before, during, and postintervention. Pretraining Consultations The assessment should cover individual characteristics as well as
structural factors, as discussed in the literature [51]. The assessment should function as both a diagnostic and evaluative tool to track the trajectory
of organizational readiness over the course of the intervention
•
Comparison of the frequency of electronic mental health use before and after the intervention
•
Frequency and quality of trainings conducted within the organization by internal trainers
•
Service providers’ skills, knowledge, and perception of electronic mental health approaches before, during, and postintervention
•
Records of discussions during pretraining consultations and follow-up support to illuminate the progression and type of support needed throughout
the intervention
Training
The training should be contextualized and involve information
offered. Specifically, it should provide internal facilitators with
the knowledge and skills to support, supervise, and conduct
l
i
l h
l h
i i
i h h
i
i Textbox 1. The three-phase facilitation process for electronic mental health implementation. Phase 1: Pretraining consultations
Consultations assess the following aspects of organizational readiness:
•
Information technology (IT) resources and infrastructure, that is, computers, iPads, and Wi-Fi connectivity
•
Policies and procedures to guide collection, confidentiality, and security of data
•
Current conducive work culture and climate (eg, accountability, on-going learning, staff turnover, and priorities)
•
Staff skills and literacy in IT and electronic mental health approaches
•
Suitability of client population and cultural context
•
Internal facilitators to champion, train, and support on-going e utilization
Phase 2: Training
Workshops occur when suitable organizational readiness is achieved through mutual agreement and incorporate the following elements:
•
IT skills training if needed (determined through consultation)
•
Presentation of electronic mental health resources relevant to organizations’ local and cultural needs
•
Strategies to find and evaluate electronic mental health resources
•
Information regarding electronic portals hosting resources that are filtered by experts
•
Planning when, where, and with whom, electronic mental health resources will be used
•
Training for internal facilitators to supervise and support on-going electronic mental health use
Phase 3: Follow-up support
•
Face-to-face, email, and telephone follow-up to support on-going use
•
Face-to-face, email, and telephone follow-up for internal facilitators to support their training, support, and supervision
•
Review progress of organizational readiness and support implementation
•
Guidelines for evaluating the implementation strategy
•
Assessment of organizational readiness before, during, and postintervention. Pretraining Consultations The assessment should cover individual characteristics as well a
structural factors, as discussed in the literature [51]. The assessment should function as both a diagnostic and evaluative tool to track the trajector
of organizational readiness over the course of the intervention
•
Comparison of the frequency of electronic mental health use before and after the intervention
•
Frequency and quality of trainings conducted within the organization by internal trainers
•
Service providers’ skills, knowledge, and perception of electronic mental health approaches before, during, and postintervention
•
Records of discussions during pretraining consultations and follow-up support to illuminate the progression and type of support needed throughou
the intervention Textbox 1. The three-phase facilitation process for electronic mental health implementation. Phase 1: Pretraining consultations
Consultations assess the following aspects of organizational readiness:
•
Information technology (IT) resources and infrastructure, that is, computers, iPads, and Wi-Fi connectivity
•
Policies and procedures to guide collection, confidentiality, and security of data
•
Current conducive work culture and climate (eg, accountability, on-going learning, staff turnover, and priorities)
•
Staff skills and literacy in IT and electronic mental health approaches
•
Suitability of client population and cultural context
•
Internal facilitators to champion, train, and support on-going e utilization
Phase 2: Training
Workshops occur when suitable organizational readiness is achieved through mutual agreement and incorporate the following elements:
•
IT skills training if needed (determined through consultation)
•
Presentation of electronic mental health resources relevant to organizations’ local and cultural needs
•
Strategies to find and evaluate electronic mental health resources
•
Information regarding electronic portals hosting resources that are filtered by experts
•
Planning when, where, and with whom, electronic mental health resources will be used
•
Training for internal facilitators to supervise and support on-going electronic mental health use
Phase 3: Follow-up support
•
Face-to-face, email, and telephone follow-up to support on-going use
•
Face-to-face, email, and telephone follow-up for internal facilitators to support their training, support, and supervision
•
Review progress of organizational readiness and support implementation
•
Guidelines for evaluating the implementation strategy
•
Assessment of organizational readiness before, during, and postintervention. The assessment should cover individual characteristics as well a
structural factors, as discussed in the literature [51]. Acknowledgments This research was supported by Menzies and funded by the eMHPrac project under the National eMental Health Strategy. The
authors would like to acknowledge their eMHPrac partners, QUT, and UCRH, as collaborators in the development of the AIMhi
Stay Strong app, and training workshops. The authors would also like to thank the participants for their time and insights. Follow-Up Support The literature highlights the importance of follow-up supervision
post one-off workshops in influencing therapist behavior and
patient outcomes [9,32]. Specific to electronic mental health
implementation, Bennett-Levy et al [55] reported the positive
impact of post-training consultations regarding the enhancement
of skills and exploration of electronic mental health resources. Therefore, follow-up support should be provided to trainees
and organizations following training workshops. It should
include occasional face-to-face meetings and regular emails and
phone calls to motivate on-going use and to address any issues
regarding electronic mental health utilization, such as skills and
confidence, technical setups, and troubleshooting. In addition,
separate support should be provided to the internal facilitators A separate training for the selected individuals who would take
on the internal facilitator role within the organization should be XSL•FO
RenderX JMIR MENTAL HEALTH Raphiphatthana et al the data extracts, the initial codes, and themes identified as
relevant to electronic mental health implementation were not
influenced by either the i-PARIHS or PARIHS framework. As
this initial stage is strongly informed by data, it is unlikely that
the sequence of applying the framework to the data would have
majorly impacted the results. to ensure the fidelity of electronic mental health training and
implementation within the organization. Finally, organizational
readiness should be reevaluated to identify any further gaps and
to ensure the effectiveness of the ongoing progress in the
integration of the electronic mental health approach into the
wider system. Limitations The present findings are consistent with those in the literature
regarding factors essential for successful implementation of
electronic mental health approaches in health care settings. However, it is important to acknowledge the context of our
participants, who are service providers working with Australia’s
First Nations in the Northern Territory. Certain factors, such as
limited resources, IT literacy, and high staff turnover, may be
more salient to this context relative to other services in urban
areas working with nonindigenous populations. Our position as an eMHPrac partner as well as developers of
the AIMhi Stay Strong app, which is included in the training
workshop, may have introduced a positive bias and influenced
data collection and the analysis process. However, due to our
position, we have established rapport with the participants before
the interviews, which may have rendered participants more
comfortable in sharing their experiences. In addition, through
the delivery of our training program, we have developed an
understanding of local service delivery contexts. Nonetheless,
we were cognizant of the way in which our subjectivity may
have influenced and shaped the different stages in the research
process. Conclusions Through the lens of the i-PARIHS framework, this study
provides a practical understanding of how different factors
impede or facilitate electronic mental health implementation
within primary health care servicing Indigenous Australians. This insight laid a foundation for a comprehensive facilitation
strategy, involving three stages: pretraining consultations,
training workshops, and post-training follow-up support, aiming
to enhance the success of electronic mental health
implementation. The analysis was carried out in two stages upon the discovery
of the revised i-PARIHS framework. It is possible that starting
the analysis with the i-PARIHS framework may have led to
some nuances in the results. However, the data were initially
analyzed inductively, that is, codes and themes were identified
without the guidance of any particular framework. Therefore, Conflicts of Interest Conflicts of Interest None declared. Authors' Contributions BR designed the study, analyzed the data, and wrote the manuscript. MW analyzed the data and edited the manuscript. MS, SP,
KD, and TN designed the study and data collection tools, developed and conducted training, collected data, analyzed data, and
edited the manuscript. TN also supervised all activities. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 14
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RenderX XSL•FO
RenderX Please cite as:
Raphiphatthana B, Sweet M, Puszka S, Whitty M, Dingwall K, Nagel T
Evaluation of Electronic Mental Health Implementation in Northern Territory Services Using the Integrated “Promoting Action on
Research Implementation in Health Services” Framework: Qualitative Study
JMIR Ment Health 2020;7(5):e14835
URL: http://mental.jmir.org/2020/5/e14835/
doi: 10.2196/14835
PMID: 32452812 Abbreviations AIMhi: Aboriginal and Islander Mental Health Initiative
eMHPrac: electronic mental health in practice
ePortal: electronic portal
i-PARIHS: integrated Promoting Action on Research Implementation in Health Services
IT: information technology
Menzies: Menzies School of Health Research
PARIHS: Promoting Action on Research Implementation in Health Services
QUT: Queensland University of Technology
UCRH: University Centre for Rural Health Please cite as:
Raphiphatthana B, Sweet M, Puszka S, Whitty M, Dingwall K, Nagel T
Evaluation of Electronic Mental Health Implementation in Northern Territory Services Using the Integrated “Promoting Action on
Research Implementation in Health Services” Framework: Qualitative Study
JMIR Ment Health 2020;7(5):e14835
URL: http://mental.jmir.org/2020/5/e14835/
doi: 10.2196/14835
PMID: 32452812 ©Buaphrao Raphiphatthana, Michelle Sweet, Stefanie Puszka, Megan Whitty, Kylie Dingwall, Tricia Nagel. Originally published
in JMIR Mental Health (http://mental.jmir.org), 26.05.2020. This is an open-access article distributed under the terms of the
Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work, first published in JMIR Mental Health, is properly cited. The
complete bibliographic information, a link to the original publication on http://mental.jmir.org/, as well as this copyright and
license information must be included. JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 17
(page number not for citation purposes) http://mental.jmir.org/2020/5/e14835/ http://mental.jmir.org/2020/5/e14835/ JMIR Ment Health 2020 | vol. 7 | iss. 5 | e14835 | p. 17
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https://jmedicalcasereports.biomedcentral.com/track/pdf/10.1186/1752-1947-5-331
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English
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Bilateral simultaneous rupture of the quadriceps tendon in a patient with psoriasis: a case report and review of the literature
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Journal of medical case reports
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cc-by
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CASE REPORT Open Access Open Access © 2011 Senevirathna et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Abstract Introduction: Bilateral quadriceps tendon rupture is not common in the absence of systemic disease. Patients with
chronic systemic diseases such as uremia and systemic lupus erythematosus and patients who are being treated
with systemic steroids or local steroid injections are more prone to tendon rupture. The tendon can rupture
spontaneously or as a result of trauma. We report an unusual case of simultaneous bilateral traumatic quadriceps
tendon rupture in a patient with psoriasis who was being treated with topical steroid preparations. Case presentation: A 57-year-old Caucasian man with a known history of psoriasis, for which he was being
treated with topical steroid preparations, presented to our hospital with clinical signs of bilateral quadriceps
tendon rupture after he fell while walking down stairs. The diagnosis was confirmed by bilateral ultrasound scans
of the thighs. The patient underwent surgery to repair both quadriceps tendons. Post-operatively, the patient was
immobilized first in bilateral cylinder casts for six weeks, then in knee braces for the next four weeks. His knees
were actively mobilized during physiotherapy. Conclusion: Bilateral quadriceps tendon rupture is a rare occurrence in patients with psoriasis who are being
treated with topical steroids. Introduction been unable to bear weight on his legs since then and
presented to our Accident and Emergency Department
with painful swelling over both knees. Bilateral quadriceps tendon rupture is extremely rare in
the absence of systemic disease. The co-existence of sys-
temic and local disease is taken into consideration in
the pathogenesis of these ruptures. The pre-disposing
factors for spontaneous tendon rupture include chronic
systemic disease, treatment with systemic steroids or
local steroid injections, or trauma [1-4]. In the present
report, we describe a rare case of simultaneous bilateral
traumatic quadriceps tendon rupture in a patient with
psoriasis who was being treated only with topical steroid
preparations and was not taking systemic steroids. His physical examination revealed that both knees
were very tender to touch over the suprapatellar region
and had massive suprapatellar swelling. He was unable
to perform a straight leg raise on both sides, although
active quadriceps contraction was seen. On palpation, a
defect in the continuity of both quadriceps tendons was
found. Plain radiographs of both knees revealed joint effusion,
patella baja, and disruption of soft tissues superior to
the patella. An avulsion fracture of the patella on the
left side was suspected (Figure 1). Bilateral ultrasound
scans of the thighs confirmed the diagnosis of bilateral
quadriceps tendon rupture at the osseotendinous junc-
tions and a calcified fragment indicating a possible avul-
sion fracture within the detached end of the left
quadriceps tendon. Bilateral simultaneous rupture of the quadriceps
tendon in a patient with psoriasis: a case report
and review of the literature Shanaka Senevirathna*, Sarkell Radha and Aysha Rajeev Shanaka Senevirathna*, Sarkell Radha and Aysha Rajeev Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 JOURNAL OF MEDICAL
CASE REPORTS JOURNAL OF MEDICAL
CASE REPORTS * Correspondence: shanaka_s2004@yahoo.com
Department of Trauma & Orthopaedics, Queen Elizabeth Hospital,
Gateshead, NE9 6SX, UK Case report A 57-year-old Caucasian man with a history of psoriasis,
for which he was taking topical steroid preparations, fell
while walking down stairs. Initially, his left leg gave way,
and he landed on his hyperflexed right knee. He had * Correspondence: shanaka_s2004@yahoo.com
Department of Trauma & Orthopaedics, Queen Elizabeth Hospital,
Gateshead, NE9 6SX, UK The patient underwent bilateral surgical exploration of
the knees with longitudinal incisions. The peri-operative Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 Page 2 of 4 Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 Figure 3 Image obtained three months after surgery showing
full, straight leg raise using both knees. Figure 1 Radiograph of the left knee showing quadriceps
rupture. Figure 1 Radiograph of the left knee showing quadriceps
rupture. Figure 3 Image obtained three months after surgery showing
full, straight leg raise using both knees. findings were complete rupture of the tendons bilater-
ally from the superior pole of the patella (Figure 2). Bilateral quadriceps surgical repairs were performed
using mid-line incisions over the knees, and the rup-
tured ends of the quadriceps tendons were identified. The ruptured ends were freshened and repaired using
Vicryl 2-0 sutures through drill holes in the patella to
the tendons. Medial and lateral retinacular repair was
performed using Vicryl 2-0 and 1-0 sutures. findings were complete rupture of the tendons bilater-
ally from the superior pole of the patella (Figure 2). Bilateral quadriceps surgical repairs were performed
using mid-line incisions over the knees, and the rup-
tured ends of the quadriceps tendons were identified. The ruptured ends were freshened and repaired using
Vicryl 2-0 sutures through drill holes in the patella to
the tendons. Medial and lateral retinacular repair was
performed using Vicryl 2-0 and 1-0 sutures. physiotherapy. He was discharged from the clinic after
six months and returned to work. Discussion
F Few reports of quadriceps tendon rupture exist in the
literature. An older adult patient may present with an
inability to walk, and a diagnosis of proximal myopathy
usually precedes the true diagnosis of spontaneous rup-
ture. Often the presentation causes diagnostic confusion
because of bilateral involvement and the absence of
trauma. Post-operatively, the patient was immobilized first in
bilateral cylinder casts for six weeks, then in knee braces
for the next four weeks. His knees were actively mobi-
lized during physiotherapy. The physiotherapy protocol
was initially active knee range of motion exercises,
which were followed by passive assisted and polymeric
exercises. The patient had an uneventful post-operative
recovery and was able to perform straight leg raises
without a lag by the time of his three-month follow-up
examination (Figure 3). His final knee range of motion
was 0° to 125° in both legs after a full course of Lewis et al. [5] reported a case of bilateral quadriceps
tendon rupture in a bodybuilder that was attributed to
anabolic steroid misuse. Many cases of bilateral quadri-
ceps tendon rupture have been reported in patients with
chronic renal failure [1-3]. Bhole et al. [4] discussed the mechanisms, variability at
the rupture site, pathogenesis, and histopathological
changes of quadriceps tendon rupture in patients with
uremia. The various systemic diseases that pre-dispose
people to quadriceps tendon rupture include rheumatoid
arthritis, arteriosclerosis, diabetes mellitus, systemic lupus
erythematosus (SLE), primary and secondary hyperpar-
athyroidism, gout, tuberculosis, vasculitis, and steroid
injections to the tendons [3,4]. Few cases of simultaneous
quadriceps tendon and contralateral patellar tendon rup-
ture have been described in the literature [1,3,6,7]. Figure 2 Intra-operative image showing quadriceps rupture. In their case report, Muratli et al. [1] took into con-
sideration mechanical factors and co-existing systemic
and local factors associated with quadriceps tendon rup-
ture. The most important factor seems to be the blood
supply to the tendon, which comes from the arterioles
of the nearby muscles and connective tissue. After
micro-trauma,
the
blood
supply
to
the
tendon
diminishes because of the infiltration of mononuclear
cells and thrombosis of the micro-circulation, and thus
the tendon becomes more susceptible to rupture [8]. Figure 2 Intra-operative image showing quadriceps rupture. Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
http://www.jmedicalcasereports.com/content/5/1/331 Page 3 of 4 Page 3 of 4 Anzel et al. [9] stated that athletes and laborers are
more susceptible to ruptures. Discussion
F Endothelial swelling with
peri-vascular lymphocytic exudate has been described in
patients with arthritis, and peri-vascular mononuclear
cell infiltrate in the peri-vascular area was observed in a
patient with SLE. In some patients with SLE who are
being treated with corticosteroids, tendon rupture has
been observed without any inflammatory reactions [8]. In patients with rheumatoid arthritis, increased levels of
collagenase may play a role in the development of ten-
don degeneration and subsequent rupture [10]. Bilateral
quadriceps tendon rupture has also been reported in
patients with amyloidosis [11]. who was being treated for enthesopathy of the quadriceps
tendons on both sides. They believe that chronic entheso-
pathy of the superior pole of patella made their patient’s
quadriceps tendons susceptible to complete rupture due
to eccentric loading. McMaster [13] showed that normal
tendons would not normally break, even if half-severed,
until the loading profile reached about 10 to 15 kPa/mm2,
a level at which the belly of the muscle, its osseotendinous
insertion, and even the femur would fail. Lighthart et al. [16] compared the biomechanical
strength between bone tunnel repair and suture anchors. They found no statistical difference in mean initial displa-
cement after 10 cycles between suture anchor and bone
tunnel repairs on the lateral or medial side. They also
observed no difference in displacement between the two
types of repairs with the patient in a resting position (no
load) or in leg extension with load after 1000 cycles. Rasul and Fischer [12] reported that isolated quadri-
ceps tendon rupture usually occurs after trauma in the
sixth or seventh decade of life. The sites of rupture are
classified as the musculotendinous, mid-tendinous, and
osseotendinous junctions [13]. Rupture through the sub-
stance of the tendon, which is extremely rare, has been
reported in a case of glomerulonephritis [14]. Tendon
ruptures in patients with chronic renal failure tend to
occur at a low activity level and may give the initial
impression of being trivial [4]. The rehabilitation protocol following quadriceps ten-
don repair is more or less standardized. After surgery,
the knees are immobilized in extension for six weeks,
followed by gradual weight bearing and gait training
with the patient in knee braces. The patient is then
weaned off the knee braces, and the patient’s range of
motion is then increased to strengthen the knees [17]. Most reported case series have described good func-
tional outcomes. Conclusion
Si
l Simultaneous bilateral quadriceps tendon rupture is a
rare occurrence in patients with psoriasis. Our patient
was not being treated with systemic steroids or any
other medications that would have weakened the quad-
riceps tendons. Herein we report one of the causes of
bilateral quadriceps tendon rupture. Rogers et al. [6] reported a case of quadriceps tendon
rupture with contralateral patellar tendon rupture in a
47-year-old healthy man and emphasized the impor-
tance of the position of the limb and the degree of knee
flexion at the time of injury. Discussion
F Approximately two-thirds of patients
recovered to the same or better peak torque/body
weight ratio, average power, maximum average peak tor-
que, and total work/body weight ratio in affected and
unaffected limbs [18]. Most patients who undergo bilat-
eral simultaneous or unilateral tendon repair can expect
a good recovery of range of motion and can return to
their previous occupation, but many have persistent
weakness and difficulty returning to higher-level sport-
ing activities [18,19]. On the basis of his series of 55 cases of simultaneous
bilateral quadriceps tendon ruptures, Shah [2] stated
that falls are the main cause (76%) and that the com-
monest site of rupture is the osseotendinous junction
(60%). The patients in his study were almost always
treated surgically (96%). According to his report, the
patient’s gender, mechanism of injury, and tear location,
as well as the time to diagnosis and repair, were not
related to outcome, whereas the patient’s age, multiple
risk factors, renal or endocrine disease, or diabetes were
related to outcome. Quadriceps tendon rupture after trauma occurs by
direct injury or by the sudden, violent contraction of the
muscle against the body weight with the knees in a
semi-flexed position in an effort to prevent a fall or to
lift something or simply in descending stairs [3,14]. SS was the main author who wrote the manuscript. SR helped with taking
the photographs. AR was the senior author and managed the patient’s
surgery. Consent In people younger than 50 to 60 years of age, the
patellar tendon is the weakest link in the quadriceps
mechanism, fracturing 50 to 60 times more frequently
than in other ruptures. Indirect trauma accounts for
more ruptures of the quadriceps tendon than direct
trauma, and the site of rupture is suprapatellar in two-
thirds of patients and infrapatellar in one-third of
patients [3]. Written informed consent was obtained from the patient
for publication of this case report and any accompany-
ing images. A copy of the written consent is available
for review by the Editor-in-Chief of this journal. Authors’ contributions
SS was the main author who wrote the manuscript. SR helped with taking
the photographs. AR was the senior author and managed the patient’s
surgery. Competing interests
h
h
d
l
h Received: 13 January 2011 Accepted: 29 July 2011
Published: 29 July 2011 Received: 13 January 2011 Accepted: 29 July 2011
Published: 29 July 2011 Authors’ contributions
SS
h
h Arumilli et al. [15] reported a case of bilateral simulta-
neous complete quadriceps tendon rupture in a patient Page 4 of 4 Page 4 of 4 Senevirathna et al. Journal of Medical Case Reports 2011, 5:331
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contralateral quadriceps tendon in a healthy individual. Orthopedics 2003,
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quadriceps rupture following chronic symptomatic tendinopathy: a case
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16. Lighthart WA, Cohen DA, Levine RG, Parks BG, Boucher HR: Suture anchor
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versus suture through tunnel fixation for quadriceps tendon rupture: a
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Outcomes following repair of quadriceps tendon ruptures. J Orthop
Trauma 1998, 12:273-279. 19. Konrath GA, Chen D, Lock T, Goitz HT, Watson JT, Moed BR, D’Ambrosio G:
Outcomes following repair of quadriceps tendon ruptures. J Orthop
Trauma 1998, 12:273-279. Submit your next manuscript to BioMed Central
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Cite this article as: Senevirathna et al.: Bilateral simultaneous rupture of
the quadriceps tendon in a patient with psoriasis: a case report and
review of the literature. Journal of Medical Case Reports 2011 5:331. doi:10.1186/1752-1947-5-331
Cite this article as: Senevirathna et al.: Bilateral simultaneous rupture of
the quadriceps tendon in a patient with psoriasis: a case report and
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English
| null |
A population-level invasion by transposable elements triggers genome expansion in a fungal pathogen
|
eLife
| 2,021
|
cc-by
| 19,105
|
A population-level invasion by
transposable elements triggers
genome expansion in a fungal
pathogen A Publication date:
2021-09-16 Permanent link:
https://doi.org/10.3929/ethz-b-000509333 ETH Library ETH Library A population-level invasion by
transposable elements triggers
genome expansion in a fungal
pathogen Author(s): Author(s):
Oggenfuss, Ursula; Badet, Thomas; Wicker, Thomas; Hartmann, Fanny E.; Singh, Nikhil Kumar; Abraham, Leen; Karisto, Petteri
; Vonlanthen, Tiziana; Mundt, Christopher; McDonald, Bruce A.; Croll, Daniel A population-level invasion by
transposable elements triggers genome
expansion in a fungal pathogen A population-level invasion by
transposable elements triggers genome
expansion in a fungal pathogen Ursula Oggenfuss1, Thomas Badet1, Thomas Wicker2, Fanny E Hartmann3,4,
Nikhil Kumar Singh1, Leen Abraham1, Petteri Karisto4†, Tiziana Vonlanthen4,
Christopher Mundt5, Bruce A McDonald4, Daniel Croll1* 1Laboratory of Evolutionary Genetics, Institute of Biology, University of Neuchâtel,
Neuchatel, Switzerland; 2Institute for Plant and Microbial Biology, University of
Zurich, Zurich, Switzerland; 3Ecologie Systématique Evolution, Bâtiment 360,
Univ. Paris-Sud, AgroParisTech, CNRS, Université Paris-Saclay, Orsay, France;
4Plant Pathology, Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland;
5Department of Botany and Plant Pathology, Oregon State University, Corvallis,
United States Abstract Genome evolution is driven by the activity of transposable elements (TEs). The spread
of TEs can have deleterious effects including the destabilization of genome integrity and expansions. However, the precise triggers of genome expansions remain poorly understood because genome
size evolution is typically investigated only among deeply divergent lineages. Here, we use a large
population genomics dataset of 284 individuals from populations across the globe of Zymoseptoria
tritici, a major fungal wheat pathogen. We built a robust map of genome-wide TE insertions and
deletions to track a total of 2456 polymorphic loci within the species. We show that purifying selec-
tion substantially depressed TE frequencies in most populations, but some rare TEs have recently
risen in frequency and likely confer benefits. We found that specific TE families have undergone a
substantial genome-wide expansion from the pathogen’s center of origin to more recently founded
populations. The most dramatic increase in TE insertions occurred between a pair of North American
populations collected in the same field at an interval of 25 years. We find that both genome-wide
counts of TE insertions and genome size have increased with colonization bottlenecks. Hence, the
demographic history likely played a major role in shaping genome evolution within the species. We
show that both the activation of specific TEs and relaxed purifying selection underpin this incipient
expansion of the genome. Our study establishes a model to recapitulate TE-driven genome evolu-
tion over deeper evolutionary timescales. Competing interest: The authors
declare that no competing
interests exist. Funding: See page 19
Preprinted: 12 February 2020
Received: 09 April 2021
Accepted: 28 August 2021
Published: 16 September 2021 Reviewing Editor: Detlef
Weigel, Max Planck Institute
for Developmental Biology,
Germany Reviewing Editor: Detlef
Weigel, Max Planck Institute
for Developmental Biology,
Germany Originally published in: g
y p
eLife 10, https://doi.org/10.7554/eLife.69249 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. RESEARCH ARTICLE Introduction Bursts of insertions of different retrotransposon families were observed
across eukaryotic lineages including Homo sapiens, Zea mays, Oryza sativa, and Blumeria graminis
(Shen et al., 1991; SanMiguel et al., 1998; Eichler and Sankoff, 2003; Piegu et al., 2006; Lu et al.,
2017; Frantzeskakis et al., 2018). Prolonged bursts without effective counterselection are thought
to underpin genome expansions. In the symbiotic fungus Cenococcum geophilum, the burst of TEs
resulted in a dramatically expanded genome compared to closely related species (Peter et al., 2016). Similarly, a burst of a TE family in brown hydras led to an approximately threefold increase of the
genome size compared to related hydras (Wong et al., 2019). Across the tree of life, genome sizes
vary by orders of magnitude and enlarged genomes invariably show hallmarks of historic TE invasions
(Kidwell, 2002). Population size variation is among the few correlates of genome size across major
groups, suggesting that the efficacy of selection plays an important role in controlling TE activity
(Lynch, 2007). Reduced selection efficacy against deleterious TE insertions is expected to lead to a
ratchet-like increase in genome size. In fungi, TE-rich genomes often show an isochore structure alter-
nating gene-rich and TE-rich compartments (Rouxel et al., 2011). TE-rich compartments often harbor
rapidly evolving genes such as effector genes in pathogens or resistance genes in plants (Raffaele
and Kamoun, 2012; Jiao and Schneeberger, 2019). Taken together, incipient genome expansions
are likely driven by population-level TE insertion dynamics. The fungal wheat pathogen, Zymoseptoria tritici, is one of the most important pathogens on
crops, causing high yield losses in many years (Torriani et al., 2015). Z. tritici emerged during the
domestication of wheat in the Fertile Crescent where the species retained high levels of genetic
variation (Zhan et al., 2005; Stukenbrock et al., 2011). The pathogen migrated to all temperate
zones where wheat is currently grown and underwent multiple migration bottlenecks, in particular
when colonizing Oceania and North America (Zhan et al., 2005; Estep et al., 2015). The genome is
completely assembled and shows size variation between individuals sampled across the global distri-
bution range (Feurtey et al., 2020; Badet et al., 2020; Goodwin et al., 2011). The TE content of the
genome shows a striking variation of 17–24% variation among individuals (Badet et al., 2020). Z. Introduction Transposable elements (TEs) are mobile repetitive DNA sequences with the ability to independently
insert into new regions of the genome. TEs are major drivers of genome instability and epigen-
etic change (Eichler and Sankoff, 2003). Insertion of TEs can disrupt coding sequences, trigger
chromosomal rearrangements, or alter expression profiles of adjacent genes (Lim, 1988; Petrov
et al., 2003; Slotkin and Martienssen, 2007; Hollister and Gaut, 2009; Oliver et al., 2013). Hence, TE activity can have phenotypic consequences and impact host fitness. While TE insertion
dynamics are driven by the selfish interest for proliferation, the impact on the host can range
from beneficial to highly deleterious. The most dramatic examples of TE insertions underpinned
rapid adaptation of populations or species (Feschotte, 2008; Chuong et al., 2017), particularly Copyright Oggenfuss et al. This article is distributed under
the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that
the original author and source
are credited. 1 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 1 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics following environmental change or colonization events. Beneficial TE insertions are expected to
experience strong positive selection and rapid fixation in populations. However, most TE inser-
tions have neutral or deleterious effects upon insertions. Purifying selection is expected to rapidly
eliminate deleterious insertions from populations unless constrained by genetic drift (Walser
et al., 2006; Baucom et al., 2009; Cridland et al., 2013; Stuart et al., 2016; Lai et al., 2017;
Stritt et al., 2018). Additionally, genomic defense mechanisms can disable transposition activity. Across eukaryotes, epigenetic silencing is a shared defense mechanism against TEs (Slotkin and
Martienssen, 2007). Fungi evolved an additional and highly specific defense system introducing
repeat-induced point (RIP) mutations into any nearly identical set of sequences. The relative impor-
tance of demography, selection, and genomic defenses determining the fate of TEs in populations
remain poorly understood. A crucial property predicting the invasion success of TEs in a genome is the transposition rate. TEs tend to expand through family-specific bursts of transposition followed by prolonged phases
of transposition inactivity. A dynamic TE landscape shaped by strong purifying selection A dynamic TE landscape shaped by strong purifying selection
We detected 4753 TE copies, grouped into 30 families with highly variable copy numbers in the refer-
ence genome IPO323 (Figure 2—source data 1 and Figure 2—figure supplement 1A). To establish
a comprehensive picture of within-species TE dynamics, we analyzed 295 genomes from a worldwide
set of six populations spanning the distribution range of the wheat pathogen Z. tritici. To ascertain
the presence or absence of TEs across the genome, we developed a robust pipeline (Figure 1A). In
summary, we called TE insertions by identifying reads mapping to both a TE sequence and a specific
location in the reference genome. Then, we assessed the minimum sequencing coverage to reliably
recover TE insertions and removed 11 genomes with an average read depth below 15× (Figure 1B). We tested for evidence of TEs using read depth at target site duplications (Figure 1C) and scanned
the genome for mapped reads indicating gaps at TE loci (Figure 1D). We found robust evidence for
a total of 18,864 TE insertions grouping into 2465 individual loci. Of these loci, 35.5 % (n = 876) have
singleton TEs (i.e., this locus is only present in one isolate: Figure 2A, Figure 2—source data 3). An
overwhelming proportion of loci (2345 loci or 95.1%) have a TE frequency below 1 %. Singleton TE
insertions in particular can be the product of spurious Illumina read mapping errors (Nakamura et al.,
2011). To assess the reliability of the detected singletons, we focused on seven isolates for which
PacBio long-read data was available (Badet et al., 2020). Aligned PacBio reads confirmed the exact
location of 71 % (22 of 31 singleton insertions among seven isolates; see Materials and methods for
further details). We found no significant difference in read coverage between confirmed and uncon-
firmed singleton insertions (Figure 2—figure supplement 1B,C and Figure 2—source data 2). The abundance of singleton TE insertions strongly supports the idea that TEs actively copy into new
locations but also indicates that strong purifying selection maintains nearly all TEs at low frequency
(Figure 2A). The density of TE loci on accessory chromosomes, which are not shared among all
isolates of the species, is almost twice the density found on core chromosomes (102 vs 58 TEs per Mb;
Figure 2B, Figure 2—figure supplement 2A). This suggests relaxed selection against TE insertion on
the functionally dispensable and gene-poor accessory chromosomes. Introduction tritici
recently gained major TE-mediated adaptations to colonize host plants and tolerate environmental
stress (Omrane et al., 2015; Omrane et al., 2017; Krishnan et al., 2018; Meile et al., 2018). Clusters
of TEs are often associated with genes encoding important pathogenicity functions (i.e. effectors),
recent gene gains or losses (Hartmann and Croll, 2017), and major chromosomal rearrangements
(Croll et al., 2013; Plissonneau et al., 2016). Transposition activity of TEs also had a genome-wide
impact on gene expression profiles during infection (Fouché et al., 2019). The well-characterized
demographic history of the pathogen and evidence for recent TE-mediated adaptations make Z. tritici
an ideal model to recapitulate the process of TE insertion dynamics, adaptive evolution, and changes
in genome size at the population level. Here, we retrace the population-level context of TE insertion dynamics and genome size changes
across the species range by analyzing populations sampled on four continents for a total of 284
genomes. We developed a robust pipeline to detect newly inserted TEs using short read sequencing
datasets. Combining analyses of selection and knowledge of the colonization history of the pathogen,
we tested whether population bottlenecks were associated with substantial changes in the TE content
and the size of genomes. Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 2 of 25 Research article Research article Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics A dynamic TE landscape shaped by strong purifying selection We found no difference in TE
allele frequency distribution between recombination hotspots and the rest of the genome (Figure 2—
figure supplement 2B). Similarly, the TE density and the number of insertions did not vary between
recombination hotspots and the genomic background (Figure 2—figure supplement 2C). TEs grouped into 23 families and 11 superfamilies, with 88.2 % of all copies belonging to class I/
retrotransposons (n = 2175; Figure 2C, Figure 2—figure supplement 3A,B). RLG/Gypsy (n = 1483)
and RLC/Copia (n = 623) elements constitute the largest long terminal repeats (LTR) superfamilies. Class II/DNA transposons are dominated by DHH/Helitron (n = 249). As expected, TE families shared
among fewer isolates tend to show also lower global copy numbers (i.e., all isolates combined), while
TE families that are present in all isolates generally have high global copy numbers (Figure 2D). We detected 153 loci with TEs inserted into genes with most of the insertions being singletons
(44.7 %; n = 68) or of very low frequency (Figure 2E). Overall, TE insertions into exonic sequences
were less frequent than expected compared to insertions into up- and downstream regions, which
is consistent with effective purifying selection (Figure 2F). Insertions into introns were also strongly
under-represented, likely due to the small size of most fungal introns (~50–100 bp) and the high prob-
ability of disrupting splicing or adjacent coding sequences. We also found that insertions 800–1000 bp
away from coding sequences of a focal gene were under-represented. Given the high gene density,
with an average spacing between genes of 1.744 kb, TE insertions within 800–1000 bp of a coding
gene tend to be near adjacent genes already. Taken together, TEs in the species show a high degree
of transposition activity and are subject to strong purifying selection. Detection of candidate TE loci underlying recent adaptation We thus expect reads to
map to the TE surrounding region and the target site duplication but not the TE itself. At the target site, a local duplication of read depth is expected. (ii) We selected all reads in an interval of 100 bp up- and downstream including the target site duplication to detect deviations in the number of
reads terminating near the target site duplication. (D) Validation of insertions present in the reference genome. (i) Analyses read coverage at target
site duplications. (ii) Decision map if a TE should be kept as a true insertion or rejected as a false positive. Only predicted TE insertions that overlap
Figure 1 continued on next page Number of detected insertions
Australia 2001
Europe 1999
Europe 2016
Middle East 1992
North America 1990
North America 2015
0
20
40
60
20
40
60
80
B
15
Coverage (x) Raw data processing
TE detection
Downsampling
analysis
TE position correction
TE position
correction
Overlap with
predicted TEs
Outlier test
Convertion to hint file
Convertion to bed
Mapping short reads
Detection spliced
junction reads
A A B TS
TS
S
TSD
SD
TSD
TSD
S
TS
number
stop
points
leftmost
-100 bp
i
ii
C
TS
rightmost
+100 bp
illumina
reads
reference genome
isolate
Illumina reads
TE C ii Final call presence/absence variation
Singleton validation Final call presence/absence variation
Singleton validation reference
genome
isolate
spliced junction
reads
spliced junction
reads
isolate
i
ii
accept as
absence
Unassigned
confirmed
presence
reject
presence
D
TE
predicted TE in
ngs_te_mapper structural variation locus
reference genome
PacBio
sequence
TS
isolate
TE
TS
reference genome
PacBio
sequence
TE consensus sequence
TS
500bp
500bp
i
ii
E D i
D E ii ii TE consensus sequence Figure 1. Robust discovery and validation of transposable element (TE) insertions: (A) General analysis pipeline. (B) Read depth downsampling analysis
for one isolate per population with an average coverage of the population. The vertical black line indicates the coverage at which on average 90 %
of the maximally detectable variants were recovered. Dashed black lines indicate the standard error. The threshold for a minimal mean coverage was
set at 15 × (red line). (C) Validation of insertions absent in the reference genome. Detection of candidate TE loci underlying recent adaptation Detection of candidate TE loci underlying recent adaptation
The TE transposition activity can generate adaptive genetic variation. To identify the most likely candi-
date loci, we analyzed insertion frequency variation among populations as an indicator for recent
selection. Across all populations, the insertion frequencies differed only weakly with a strong skew
toward extremely low FST values (mean = 0.0163; Figure 3A,B, Figure 3—figure supplement 1). To further analyze evidence for TE-mediated adaptive evolution, we screened a genome-wide SNP
dataset for evidence of selective sweeps using selection scans. We found 16.5 % of all TE loci located
in regions of selective sweep. Given our population sampling of two population pairs, we tested for Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 3 of 25 Research article Evolutionary Biology | Genetics and Genomics Number of detected insertions
Australia 2001
Europe 1999
Europe 2016
Middle East 1992
North America 1990
North America 2015
0
20
40
60
20
40
60
80
TS
TS
S
TSD
SD
TSD
TSD
S
TS
number
stop
points
leftmost
reference
genome
isolate
B
spliced junction
reads
spliced junction
reads
isolate
15
-100 bp
i
ii
i
ii
Coverage (x)
C
accept as
absence
Unassigned
confirmed
presence
reject
presence
D
TS
rightmost
+100 bp
illumina
reads
reference genome
isolate
Illumina reads
TE
TE
Raw data processing
TE detection
Downsampling
analysis
TE position correction
Final call presence/absence variation
TE position
correction
Overlap with
predicted TEs
Outlier test
Convertion to hint file
Convertion to bed
Mapping short reads
Detection spliced
junction reads
A
predicted TE in
ngs_te_mapper
Singleton validation
structural variation locus
reference genome
PacBio
sequence
TS
isolate
TE
TS
reference genome
PacBio
sequence
TE consensus sequence
TS
500bp
500bp
i
ii
E
Figure 1. Robust discovery and validation of transposable element (TE) insertions: (A) General analysis pipeline. (B) Read depth downsampling analysis
for one isolate per population with an average coverage of the population. The vertical black line indicates the coverage at which on average 90 %
of the maximally detectable variants were recovered. Dashed black lines indicate the standard error. The threshold for a minimal mean coverage was
set at 15 × (red line). (C) Validation of insertions absent in the reference genome. (i) TE insertions that are not present in the reference genome show a
duplication of the target site and the part of the reads that covers the TE will not be mapped against the reference genome. Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics Figure 1 continued evidence of split reads were kept as TE insertions in downstream analyses. (E) Singleton validation using long-read PacBio sequencing. (i) Analysis if TE
insertions overlap with a detected insertion/deletion locus (Badet et al., 2021). (ii) Homology search of the TE insertion flanking sequences based on
the reference genome against PacBio reads. In addition, the consensus sequence of the inserted TE was used for matches between the flanks.ii The online version of this article includes the following figure supplement(s) for figure 1: Source data 1. TE insertion validations for non-reference copies. Source data 2. TE consensus sequences. Source data 2. TE consensus sequences. Figure supplement 1. Validation of transposable element (TE) insertion predictions. Figure supplement 2. Establishment of transposable element (TE) loci with differing start and end positions in t Figure supplement 3. Bias for reads with a GC content lower than 30 % per population. Figure supplement 3. Bias for reads with a GC content lower than 30 % per population. adaptive TE insertions in selective sweep regions either in the North American or European popula-
tion pairs. Hence, we selected loci having low TE insertion frequencies (<5%) in all populations except
either the recent North American or European population (>20%) (Figure 3B). Based on these criteria,
we obtained seven candidate loci possibly underlying local adaptation (six in North America, one
in Europe; Figure 4A, Figure 4—source data 1). All loci carry inserted retrotransposons with four
RLG_Luna, one RLG_Mercurius, and one RLG_Deimos. adaptive TE insertions in selective sweep regions either in the North American or European popula-
tion pairs. Hence, we selected loci having low TE insertion frequencies (<5%) in all populations except
either the recent North American or European population (>20%) (Figure 3B). Based on these criteria,
we obtained seven candidate loci possibly underlying local adaptation (six in North America, one
in Europe; Figure 4A, Figure 4—source data 1). All loci carry inserted retrotransposons with four
RLG_Luna, one RLG_Mercurius, and one RLG_Deimos. One TE insertion is 3815 bp downstream of a gene encoding an RTA1-like protein, which can func-
tion as transporters with a transmembrane domain and have been associated with resistance against
several antifungal compounds (Soustre et al., 1996). The insertion is also 5785 bp upstream of a
gene encoding a protein kinase domain (Figure 4B). Evolutionary Biology | Genetics and Genomics The TE insertion was not detected in the Middle
East or the two European populations and was at low frequencies in the Australian (3.7%) and North
American 1990 (1.7%) populations, but increased to 53 % of all isolates in the North American 2015
population (fixation index FST = 0.42; Figure 4—source data 1). Isolates that carry the insertion show
a significantly higher resistance to azole antifungal compounds (Figure 4C). The TE is in the subtelo-
meric region of chromosome 12, with a moderate GC content, a low TE, and a high gene density
(Figure 4D). The TE belongs to the family RLG_Luna, which shows a substantial burst across different
chromosomes within the species (Figure 4E,F). We found no association between the phylogenetic
relationships among isolates based on the two closest genes and the presence or absence of the TE
insertion (Figure 4G). A second candidate adaptive TE insertion belongs to the RLG_Mercurius family
and is located between two genes of unknown function (Figure 4—figure supplement 1). A third
potentially adaptive TE insertion of a RLC_Deimos is 229 bp upstream of a gene encoding a SNARE
domain protein and 286 bp upstream of a gene encoding a flavin amine oxidoreductase. Furthermore,
the TE is inserted in a selective sweep region (Figure 4—figure supplement 1). SNARE domains
play a role in vesicular transport and membrane fusion (Bonifacino and Glick, 2004). An additional
four candidates for adaptive TE insertions belong to RLG_Luna and were located distantly to genes
(Figure 4—figure supplement 1). We experimentally tested whether the TE insertions in proximity to
genes were associated with higher levels of fungicide resistance. For this, we measured growth rates
of the fungal isolates in the presence or absence of an azole fungicide widely deployed against the
pathogen. We found that the insertion of TEs at two loci was positively associated with higher levels of
fungicide resistance, suggesting that the adaptation was mediated by the TE (Figure 4C, Figure 4—
figure supplement 1). Detection of candidate TE loci underlying recent adaptation (i) TE insertions that are not present in the reference genome show a
duplication of the target site and the part of the reads that covers the TE will not be mapped against the reference genome. We thus expect reads to
map to the TE surrounding region and the target site duplication but not the TE itself. At the target site, a local duplication of read depth is expected. (ii) We selected all reads in an interval of 100 bp up- and downstream including the target site duplication to detect deviations in the number of
reads terminating near the target site duplication. (D) Validation of insertions present in the reference genome. (i) Analyses read coverage at target
site duplications. (ii) Decision map if a TE should be kept as a true insertion or rejected as a false positive. Only predicted TE insertions that overlap
Figure 1 continued on next page Figure 1 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 4 of 25 Research article Research article Population-level expansions in TE content p
p
If TE insertion dynamics are largely neutral across populations, TE frequencies across loci should reflect
neutral population structure. To test this, we performed a principal component analysis based on a
set of six populations on four continents that represent the global genetic diversity of the pathogen
(Figure 5A) and 900,193 genome-wide SNPs (Figure 5B). The population structure reflected the
demographic history of the pathogen with clear continental differentiation and only minor within-site
differentiation. To account for the lower number of TE loci, we performed an additional principal
component analysis using a random SNP set of similar size to the number of TE loci. The reduced
SNP set retained the geographic signal of the broader set of SNPs (Figure 5C). In stark contrast,
TE frequencies across loci showed only weak clustering by geographic origin with the Australian Oggenfuss et al. eLife 2021;0:e69249. Population-level expansions in TE content DOI: https://doi.org/10.7554/eLife.69249 5 of 25 Research article Evolutionary Biology | Genetics and Genomics B
C
F
0
50
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Chromosomes
Insertions per Mb
A
D
0
100
200
0
1000
2000
3000
4000
E
0
1000
2000
3000
4000
RLG_Sol
RLC_Deimos
RLG_Venus
RLG_Luna
RIX_Lucy
DHH_Ada
RXX_Wasp
RLG_Mercurius
RXX_Kepler
RLG_Namaka
RLG_Aurora
RLX_Cancri
XXX_Maki
DTX_MITES_Satyr
DTX_Kolyma
RLC_Io
RLC_Leda
RLG_Uranus
RLG_Jupiter
RLX_LARD_Gridr
RLG_Neptunus
RLX_TRIM_Jari
DTX_MITES_Antaios
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
0
10
1
2
3
4
5
6
7
8
9
10
Allele count
11-100
100-284
−0.75
−0.25
0.25
0.75
1.25
20
30
mean copy
number per
isolate
core
accessory
upstream
downstream
exon
intron
Number of isolates carrying TE family
Number
erved/expected [log2]
Fraction [%]
Global number of copies per TE family
2.5
10
7.5
5
0.5
1.0
1.5
2.0
2.5
density
exon
intron B
0
50
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Chromosomes
Insertions per Mb
A
0
10
1
2
3
4
5
6
7
8
9
10
Allele count
11-100
100-284
20
30
core
accessory
Fraction [%] B A C
F
0
50
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Chromosomes
Insertions per Mb
−1000
−800
−600
−400
−200
n
o
rt
ni
exon
200
400
600
800
1000
Position [bp]
D
0
100
200
0
1000
2000
3000
4000
E
0
1000
2000
3000
4000
RLG_Sol
RLC_Deimos
RLG_Venus
RLG_Luna
RIX_Lucy
DHH_Ada
RXX_Wasp
RLG_Mercurius
RXX_Kepler
RLG_Namaka
RLG_Aurora
RLX_Cancri
XXX_Maki
DTX_MITES_Satyr
DTX_Kolyma
RLC_Io
RLC_Leda
RLG_Uranus
RLG_Jupiter
RLX_LARD_Gridr
RLG_Neptunus
RLX_TRIM_Jari
DTX_MITES_Antaios
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
0
10
1
2
3
4
5
6
7
8
9
10
Allele count
11-100
100-284
−1.25
−0.75
−0.25
0.25
0.75
1.25
20
mean copy
number per
isolate
upstream
downstream
exon
intron
Number of isolates carrying TE family
Number
Observed/expected [log2]
Fraction [%]
Global number of copies per TE family
2.5
10
7.5
5
0.0
0.5
1.0
1.5
2.0
2.5
1
10
100
Allele count [log10]
density
exon
intron
Figure 2. Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics Figure 2 continued Figure 2 continued Allele frequency distribution of TE insertions into introns and exons. (F) Number of TE insertions within 1 kb up- and downstream of genes on core
chromosomes including introns and exons (100 bp windows). The blue arrow indicates a gene schematic with exons and an intron, the green triangles
indicate TE insertions. The dotted blue line indicates no deviation from the expected value (i.e., mean number of TEs per window). The online version of this article includes the following figure supplement(s) for figure 2: Source data 1. TEs in reference. Source data 2. Presence absence matrix TE loci. Source data 2. Presence absence matrix TE loci. Source data 3. Singletons. Source data 3. Singletons. Figure supplement 1. Validation of singleton insertions detected by mapped Illumina reads using PacBio read alignments for confirmation. Figure supplement 2 TE insertion loci characteristics ure supplement 1. Validation of singleton insertions detected by mapped Illumina reads using PacBio read align Figure supplement 2. TE insertion loci characteristics. Figure supplement 3. Hierarchy superfamilies. Figure supplement 3. Hierarchy superfamilies. population being the most distinct (Figure 5D). We found a surprisingly strong differentiation of the
two North American populations sampled at a 25 -year interval in the same field in Oregon. population being the most distinct (Figure 5D). We found a surprisingly strong differentiation of the
two North American populations sampled at a 25 -year interval in the same field in Oregon. p p
p
yi
g
Unusual patterns in population differentiation at TE loci suggests that TE activity may substan-
tially vary across populations (Figure 6, Figure 4—source data 1). To analyze this, we first identified
the total TE content across all loci per isolate. We found generally lower TE numbers in the Middle
Eastern population from Israel (Figure 6A–C, Figure 6—figure supplement 1), which is close to the
pathogen’s center of origin (Stukenbrock et al., 2007). Populations that underwent at least one
migration bottleneck showed a substantial burst of TEs across all major superfamilies. These popula-
tions included the two populations from Europe collected in 1999 and 2016 and the North American
population from 1990, as well as the Australian population. We found a second stark increase in TE
content in the North American population sampled in 2015 at the same site as the population from
1990. Evolutionary Biology | Genetics and Genomics Strikingly, the isolate with the lowest number of analyzed TEs collected in 2015 was comparable
to the isolate with the highest number of TEs at the same site in 1990. We tested whether sequencing
coverage could explain variation in the detected TEs across isolates, but we found no meaningful
association (Figure 2—figure supplement 3C). We analyzed whether the population-specific expan-
sions were correlated with shifts in the frequency spectrum of TEs in the populations (Figure 6D). We
found that the first step of expansions observed in Europe compared to the Middle East (Israel) was
associated with an upwards shift in allele frequencies. This is consistent with transposition activity
creating new copies in the genomes and stronger purifying selection in the Middle East. Similarly, the
North American populations showed also signatures consistent with relaxation of selection against TEs
(i.e., fewer low-frequency TEs). We found a significant difference (two-sample Kolmogorov–Smirnov
test, two-sided) in the curve shapes between the population from the Middle East and North America
2015 (Figure 6—source data 1). We analyzed variation in TE copy numbers across families and found
that the expansions were mostly driven by RLG elements including the families Luna, Sol, and Venus,
the RLC family Deimos, and the LINE family Lucy (Figure 6E, Figure 6—figure supplement 2). We
also found a North American–specific burst in DHH elements of the family Ada (increase from 4.6 to
6.1 copies on average per isolate), an increase specific to Swiss populations in LINE elements, and
an increase in RLC elements in the Australian and the two North American populations. Analyses of
complete Z. tritici reference-quality genomes that include isolates from the Israel, Australia, Switzer-
land (1999), and North American (1990) population revealed high TE contents in Australia and North
America (Oregon 1990) (Badet et al., 2020). The reference-quality genomes confirmed also that the
increase in TEs was driven by LINE, RLG, and RLC families in Australia and DHH, RLG, and RLC families
in North America (Badet et al., 2020). Population-level expansions in TE content Transposable element (TE) landscape across populations. (A) Allele frequencies of the TE insertions across all isolates. (B) TE insertions
per Mb on core chromosomes (dark) and accessory chromosomes (light). Dashed lines represent mean values. Blue: global mean of 75.65 insertion
Mb, dark: core chromosome mean of 58 TEs/Mb, light: accessory chromosome mean of 102.24 insertions/Mb. (C) Number of TE insertions per fam
(D) TE frequencies among isolates and copy numbers across the genome. The blue line indicates the maximum number of isolates (n = 284). (E)
Figure 2 continued on next page Allele count C
0
1000
2000
3000
4000
RLG_Sol
RLC_Deimos
RLG_Venus
RLG_Luna
RIX_Lucy
DHH_Ada
RXX_Wasp
RLG_Mercurius
RXX_Kepler
RLG_Namaka
RLG_Aurora
RLX_Cancri
XXX_Maki
DTX_MITES_Satyr
DTX_Kolyma
RLC_Io
RLC_Leda
RLG_Uranus
RLG_Jupiter
RLX_LARD_Gridr
RLG_Neptunus
RLX_TRIM_Jari
DTX_MITES_Antaios
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
Allele count
10
Number D
0
100
200
0
1000
2000
3000
4000
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
mean copy
number per
isolate
Number of isolates carrying TE family
Global number of copies per TE family
2.5
10
7.5
5 C Number Global number of copies per TE family E
0.0
0.5
1.0
1.5
2.0
2.5
1
10
100
Allele count [log10]
density
exon
intron F
−1000
−800
−600
−400
−200
n
o
rt
ni
exon
200
400
600
800
1000
Position [bp]
−1.25
−0.75
−0.25
0.25
0.75
1.25
upstream
downstream
exon
intron
Observed/expected [log2] F E Figure 2. Transposable element (TE) landscape across populations. (A) Allele frequencies of the TE insertions across all isolates. (B) TE insertions
per Mb on core chromosomes (dark) and accessory chromosomes (light). Dashed lines represent mean values. Blue: global mean of 75.65 insertions/
Mb, dark: core chromosome mean of 58 TEs/Mb, light: accessory chromosome mean of 102.24 insertions/Mb. (C) Number of TE insertions per family. (D) TE frequencies among isolates and copy numbers across the genome. The blue line indicates the maximum number of isolates (n = 284). (E)
Figure 2 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 6 of 25 Research article Research article Research article Evolutionary Biology | Genetics and Genomics Figure 3 continued the gene density in windows of 100 kb with darker blue representing higher gene density. Red bars indicate the TE density in windows of 100 kb with a
darker red representing higher TE density. Green triangles indicate positions of TE insertions with among population FST value shown on the y-axis. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1. Global pairwise FST distributions shown separately for the 21 chromosomes. Figure supplement 1. Global pairwise FST distributions shown separately for the 21 chromosomes. Given the limitations of short-read assemblies, we implemented corrective measures to compensate
for potential variation in assembly qualities. We corrected for variation in the GC content of different
sequencing datasets by downsampling reads to generate balanced sequencing read sets prior to
assembly (see Materials and methods). We also excluded all reads mapping to accessory chromo-
somes because different isolates are known to differ in the number of these chromosomes. Genome
assemblies were checked for completeness by retrieving the phylogenetically conserved BUSCO
genes (Figure 7A). Genome assemblies across different populations carry generally >99% complete
BUSCO gene sets, matching the completeness of reference-quality genomes of the same species
(Badet et al., 2020). The completeness of the assemblies showed no correlation with either TE or GC
content of the genomes. GC content was inversely correlated with genome size consistent with the
expansion of repetitive regions having generally low GC content (Figure 7B). We found that the core
genome size varied substantially among populations with the Middle East, Australia, as well as the two
older European and North American populations having the smallest core genome sizes (Figure 7C). We found a notable increase in core genome size in both the more recent European and North Amer-
ican populations. The increase in core genome size is positively correlated with the count and cumula-
tive length of all inserted TEs (Figure 7D, E and G) and negatively correlated with the genome-wide
GC content (Figure 7F,G, Benjamini and Speed, 2012). Hence, core genome size shows substantial
variation within the species matching the recent expansion in TEs across continents. We found the
most variable genome sizes in the more recent North American population (Figure 7—figure supple-
ment 1B). Finally, we contrasted variation in genome size with the detected TE insertion dynamics. Discussion TEs play a crucial role in generating adaptive genetic variation within species but are also drivers of
deleterious genome expansions. We analyzed the interplay of TEs with selective and neutral processes
including population differentiation and incipient genome expansions. TEs have substantial transposi-
tion activity in the genome but are strongly counterselected and are maintained at low frequency. TE
dynamics showed distinct trajectories across populations with more recently established populations
having higher TE content and a concurrent expansion of the genome. For
this, we assessed the variable genome segment as the difference between the smallest and largest
analyzed core genome. To reflect TE dynamics, we calculated the cumulative length of all detected
TE insertions in any given genome. We found that the cumulative length of inserted TEs represents
between 4.8% and 184 % of the variable genome segment defined for the species or 0.2–2.6% of the
estimated genome size per isolate (Figure 7—figure supplement 1C,D). TE-mediated genome size expansions The combined effects of actively copying TE families and relaxed purifying selection lead to an accu-
mulation of new TE insertions in populations. Consequently, mean genome sizes in populations
should increase over generations. We estimated the cumulative length of TE insertions based on
the length of the corresponding TE consensus sequences and found a strong increase in the total TE
length in populations outside the Middle East center of origin and a second increase between the
two North American populations (Figure 7—figure supplement 1A). To test for incipient genome
expansions within the species, we first assembled genomes of all 284 isolates included in the study. Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 7 of 25 Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics Figure 3. Differentiation in transposable element insertion frequencies across the genome. (A) Global pairwise FST distribution
chromosomes. The red horizontal line indicates the mean FST (=0.0163). TEs with a strong local short-term frequency difference
highlighted (blue: increase in Europe; green: increase in North America). (B) Allele frequency changes between the population Figure 3. Differentiation in transposable element insertion frequencies across the genome. (A) Global pairwise FST distributions shown across the 21
chromosomes. The red horizontal line indicates the mean FST (=0.0163). TEs with a strong local short-term frequency difference among populations are
highlighted (blue: increase in Europe; green: increase in North America). (B) Allele frequency changes between the populations. The same TE loci as in
(A) are highlighted. (C) Circos plot describing from the outside to the inside: The black line indicates chromosomal position in Mb. Blue bars indicate
Figure 3 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 8 of 25 Recent selection acting on TE insertions TE frequencies in the species show a strong skew toward singleton insertions across populations. However, our short read based analyses are possibly skewed toward over-counting singletons as indi-
cated by independent long-read mapping evaluations. Nevertheless, the skew toward low-frequency
TE insertions indicates both that TEs are undergoing transposition and that purifying selection main-
tains frequencies at a low level. Similar effects of selection on active TEs were observed across plants
and animals, including Drosophila melanogaster and Brachypodium distachyon (Cridland et al., 2013;
Stritt et al., 2018; Luo et al., 2020). TE insertions were under-represented in or near coding regions,
showing a stronger purifying selection against TEs inserting into genes. Coding sequences in the Z. tritici genome are densely packed with an average distance of only ~1 kb (Goodwin et al., 2011). Consistent with this high gene density, TE insertions were most frequent at a distance of 200–400 bp
away from coding sequences. A rapid decay in linkage disequilibrium in the Z. tritici populations (Croll
et al., 2015; Hartmann et al., 2018) likely contributed to the efficiency of removing deleterious inser-
tions. Some TE superfamilies have preferred insertion sites in coding regions and transcription start Oggenfuss et al. eLife 2021;0:e69249. Recent selection acting on TE insertions DOI: https://doi.org/10.7554/eLife.69249 9 of 25 Research article Evolutionary Biology | Genetics and Genomics Research article
Evolutionary Biology | Genetics and Geno
A
0.00
0.03
0.06
0.09
0.12
0
5
10
15
20
25
*
0
25
0
25
50
75
Chromosome
Middle East,
Australia, Europe
North America
Isolates containing copies
of TE family [%]
1x
2x
3x
number of copies
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
75
100
50
100
5x
4x
number of TEs per isolate
0
10
20
30
40
Europe
1999
Europe
2016
North
America
1990
North
America
2015
Middle
East
1992
Australia
2001
Protein kinase
RTA1
3815 bp
5785 bp
RLG_Luna
density
EC50 value after 4 days
absent
present
*
significant with
Bonferroni correction
0
25
50
75
40
45
50
55
40
90
140
position [kb]
Gene and TE density [%]
GC content [%]
100
60
190
240
290
340
390
440
490
540
0.0
0.1
0.2
0.3
0.4
FST value
B
C
D
E
F
G RTA1 like protein
Protein kinase domain
no TE
with TE
−4
−2
0
2
RLC_Deimos_chr1
RLG_Luna_chr9
RLG_Luna_chr12
RLG_Mercurius_chr2
RLG_Luna_chr2
RLG_Luna_chr14
RLG_Luna_chr12
−1
−3
1
3
4
Distance to gene [kb]
RLG
RLC
DHH
Figure 4. Candidate adaptive transposable element (TE) insertions. (A) Distribution of all extremely differentiated TEs and their distance to the clos
gene. Color indicates the superfamily. The stars indicate TE insertions not found in the reference genome. (B) Location of the RLG_Luna TE insertion
chromosome 12 corresponding to its two closest genes. Recent selection acting on TE insertions (C) Resistance against azole fungicides among isolates as a function of TE presence or abse
(D) Genomic niche of the RLG_Luna TE insertion on chromosome 12: FST values for each TE insertion, gene content (blue), TE content (green) and G
Figure 4 continued on next page y
gy |
A
0.00
0.03
0.06
0.09
0.12
0
5
10
15
20
25
*
number of TEs per isolate
0
10
20
30
40
Europe
1999
Europe
2016
North
America
1990
North
America
2015
Middle
East
1992
Australia
2001
Protein kinase
RTA1
3815 bp
5785 bp
RLG_Luna
density
EC50 value after 4 days
absent
present
*
significant with
Bonferroni correction
0
25
50
75
40
45
50
55
40
90
140
position [kb]
Gene and TE density [%]
GC content [%]
100
60
190
240
290
340
390
440
490
540
0.0
0.1
0.2
0.3
0.4
FST value
B
C
D
E
G RTA1 like protein
−4
−2
0
2
RLC_Deimos_chr1
RLG_Luna_chr9
RLG_Luna_chr12
RLG_Mercurius_chr2
RLG_Luna_chr2
RLG_Luna_chr14
RLG_Luna_chr12
−1
−3
1
3
4
Distance to gene [kb]
RLG
RLC
DHH A B C 0.00
0.03
0.06
0.09
0.12
0
5
number of TEs per isolate
0
10
20
30
40
Europe
1999
Europe
2016
North
America
1990
North
America
2015
Middle
East
1992
Australia
2001
3815 bp
5785 bp
de
EC50 value after 4 days
0
25
50
75
40
45
50
55
40
90
140
position [kb]
Gene and TE density [%]
GC content [%]
100
60
190
240
290
340
390
440
490
540
0.0
0.1
0.2
0.3
0.4
FST value
D
E
G RTA1 like protein
RLG
RLC
DHH D number of TEs per isolate
0
10
20
30
40
Europe
1999
Europe
2016
North
America
1990
North
America
2015
Middle
East
1992
Australia
2001
E E G RTA1 like protein
Protein kinase domain
no TE
with TE G RTA1 like protein 0
25
0
25
50
75
Chromosome
Middle East,
Australia, Europe
North America
Isolates containing copies
of TE family [%]
1x
2x
3x
number of copies
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
75
100
50
100
5x
4x
F F Protein kinase domain Figure 4. Candidate adaptive transposable element (TE) insertions. (A) Distribution of all extremely differentiated TEs and their distance to the closest
gene. Color indicates the superfamily. Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics Figure 4 continued content (yellow). The gray section highlights the insertion site. (E) Number of RLG_Luna copies per isolate and population. (F) Frequency changes of
RLG_Luna between the two North American populations compared to the other populations. Colors indicate the number of copies per chromosome. (G) Phylogenetic trees of the coding sequences of either the gene encoding the RTA1-like protein or the protein kinase domain. Isolates of the two
North American populations and an additional 11 isolates from other populations not carrying the insertion are shown. Blue color indicates TE presence,
yellow indicates TE absence. The online version of this article includes the following figure supplement(s) for figure 4: The online version of this article includes the following figure supplement(s) for figure 4:
Source data 1. Top loci information. The online version of this article includes the following figure supplement(s) for figure 4:
Source data 1. Top loci information. Source data 1. Top loci information. Figure supplement 1. Additional top loci. D
PC 1 (8.00 %)
PC 2 (5.20 %)
-0.10
-0.05
0.00
0.05
0.10
PC 2 (1.57 %)
PC 1 (2.39 %)
-0.15
-0.10
-0.05
0.00
0.05
B
C
−0.10
−0.05
0.00
0.05
0.10
0.15
−0.15 −0.10
−0.05
0.00
0.05
PC 1 (10.40 %)
PC 2 (6.10 %)
-0.05
0.10
0.05
0.00
-0.10
0.15
-0.10
-0.05
0.00
0.05
Transposable element loci
Genome-wide SNPs (subset)
Genome-wide SNPs
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
A
n=97
n=53
n=56
n=33
n=30
n=27
Australia 2001
Figure 5. Population differentiation at transposable element (TE) and genome-wide SNP loci. (A) Sampling locations of the six populations. Middle
East represents the region of origin of the pathogen. In North America, the two populations were collected at an interval of 25 years in the same field
in Oregon. In Europe, two populations were collected at an interval of 17 years from two fields in Switzerland <20 km apart. Dark arrows indicate the
historic colonization routes of the pathogen. (B) Principal component analysis (PCA) of 284 Zymoseptoria tritici isolates, based on 900,193 genome-wide
SNPs. (C) PCA of a reduced SNP data set with randomly selected 203 SNPs matching approximately the number of analyzed TE loci. (D) PCA based on
193 TE insertion loci. Loci with allele frequency <5 % are excluded. Evolutionary Biology | Genetics and Genomics The online version of this article includes the following figure supplement(s) for figure 5:
Source data 1. Isolates. Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
A
n=97
n=53
n=56
n=33
n=30
n=27
Australia 2001 A D
PC 2 (1.57 %)
C
−0.10
−0.05
0.00
0.05
0.10
0.15
−0.15 −0.10
−0.05
0.00
0.05
PC 2 (6.10 %)
-0.05
0.10
0.05
0.00
-0.10
0.15
-0.05
0.00
0.05
Transposable eleme
Genome-wide SNPs (subset) PC 1 (8.00 %)
PC 2 (5.20 %)
-0.10
-0.05
0.00
0.05
0.10
-0.15
-0.10
-0.05
0.00
0.05
B
Genome-wide SNPs D B D
PC 2 (1.57 %)
PC 1 (2.39 %)
-0.05
0.10
0.05
0.00
-0.10
0.15
-0.10
-0.05
0.00
0.05
Transposable element loci
) C
−0.10
−0.05
0.00
0.05
0.10
0.15
−0.15 −0.10
−0.05
0.00
0.05
PC 1 (10.40 %)
PC 2 (6.10 %)
Genome-wide SNPs (subset C Figure 5. Population differentiation at transposable element (TE) and genome-wide SNP loci. (A) Sampling locations of the six populations. Middle
East represents the region of origin of the pathogen. In North America, the two populations were collected at an interval of 25 years in the same field
in Oregon. In Europe, two populations were collected at an interval of 17 years from two fields in Switzerland <20 km apart. Dark arrows indicate the
historic colonization routes of the pathogen. (B) Principal component analysis (PCA) of 284 Zymoseptoria tritici isolates, based on 900,193 genome-wide
SNPs. (C) PCA of a reduced SNP data set with randomly selected 203 SNPs matching approximately the number of analyzed TE loci. (D) PCA based on
193 TE insertion loci. Loci with allele frequency <5 % are excluded. The online version of this article includes the following figure supplement(s) for figure 5:
Source data 1. Isolates. Figure 5. Population differentiation at transposable element (TE) and genome-wide SNP loci. (A) Sampling locations of the six populations. Middle
East represents the region of origin of the pathogen. In North America, the two populations were collected at an interval of 25 years in the same field
in Oregon. In Europe, two populations were collected at an interval of 17 years from two fields in Switzerland <20 km apart. Dark arrows indicate the
historic colonization routes of the pathogen. (B) Principal component analysis (PCA) of 284 Zymoseptoria tritici isolates, based on 900,193 genome-wide
SNPs. Research article Research article Evolutionary Biology | Genetics and Genomics (C) PCA of a reduced SNP data set with randomly selected 203 SNPs matching approximately the number of analyzed TE loci. (D) PCA based on
193 TE insertion loci. Loci with allele frequency <5 % are excluded. The online version of this article includes the following figure supplement(s) for figure 5:
Source data 1. Isolates. Source data 1. Isolates. Recent selection acting on TE insertions The stars indicate TE insertions not found in the reference genome. (B) Location of the RLG_Luna TE insertion on
chromosome 12 corresponding to its two closest genes. (C) Resistance against azole fungicides among isolates as a function of TE presence or absence. (D) Genomic niche of the RLG_Luna TE insertion on chromosome 12: FST values for each TE insertion, gene content (blue), TE content (green) and GC
Figure 4 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 10 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 11 of 25 Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics A
0
30
60
90
120
Number of TEs per isolate
RLG_Sol
RLC_Deimos
RLG_Venus
RLG_Luna
RIX_Lucy
DHH_ Ada
RXX_Wasp
RLG_Mercurius
RXX_Kepler
RLG_Namaka
RLG_Aurora
RLX_Cancri
XXX_Maki
DTX-MITES_Satyr
DTX_Kolyma
RLC_Io
RLC_Leda
RLG_Uranus
RLG_Jupiter
RLX_LARD_Gridr
RLG_Neptunus
RLX_TRIM_Jari
DTX-MITES_Antaios
0:0
1:1
2:2−5
3:6−15
4:16−25
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
B
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
C
DHH
DTX
RIX
RLC
RLG
RLX
RXX
XXX
0.0
12.5
0
5
0
15
0
30
0
60
0
2
0
7.5
0
1
Number of TEs per superfamily
Europe
1999
Europe
2016
North America 1990
North America
2015
Australia
2001
Middle East
1992
DHH_Ada
DTX_Kolyma
DTX−MITES_Antaios
DTX−MITES_Satyr
RIX_Lucy
RLC_Deimos
RLC_Io
RLC_Leda
RLG_Aurora
RLG_Jupiter
RLG_Luna
RLG_Mercurius
RLG_Namaka
RLG_Neptunus
RLG_Sol
RLG_Uranus
RLG_Venus
RLX_Cancri
RLX−LARD_Gridr
RLX−TRIM_Jari
RXX_Kepler
RXX_Wasp
XXX_Maki
0
12.5
0
4
0
1
0
1
0
15
0
30
0
3
0
3
0
4
0
1
0
20
0
6
0
3
0
1
0
40
0
1
0
15
0
1
0
1
0
1
0
2
0
6
0
1
Number of TEs per family
Europe
1999
Europe
2016
North America 1990
North America
2015
Australia
2001
Middle East
1992
D
E
frequency per population
proportion
0.00
0.25
0.50
0.75
0.1
0.2
1.0
0.15
0.25
Australia 2001
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
Figure 6. Global population structure of transposable element (TE) insertion polymorphism. (A) Total TE copies per isolat
superfamilies. (B) TE copies per family and (C) superfamily. (D) TE insertion frequency spectrum per population. The curve
self-starting Nls asymptomatic regression model (E). TE family copy numbers per isolate. Research article Research article Evolutionary Biology | Genetics and Genomics The online version of this article includes the following figure supplement(s) for figure 6:
Source data 1. Kolmogorof–Smirnov. Figure supplement 1. Population changes additional. Figure supplement 2. Heatmap loci. Figure 6 continued The online version of this article includes the following figure supplement(s) for figure 6
Source data 1. Kolmogorof–Smirnov. Figure supplement 1. Population changes additional. Figure supplement 2. Heatmap loci. Figure 6 continued Source data 1. Kolmogorof Smirnov. Figure supplement 1. Population changes additional. Figure supplement 2. Heatmap loci. sites (Miyao et al., 2003; Fu et al., 2013; Gilly et al., 2014; Quadrana et al., 2016). Hence, some
heterogeneity in the observed insertion site distribution across the genome is likely due to insertion
preferences of individual TEs. We also found evidence for positive selection acting on TEs with the
strongest candidate locus being a TE insertion on chromosome 12. This locus showed a frequency
increase only in the more recent North American population, which experienced the first systematic
fungicide applications and subsequent emergence of fungicide resistance in the decade prior to the
last sampling (Estep et al., 2015). The nearest gene encodes a RTA1-like protein, a transmembrane
exporter that is associated with resistance toward different stressors, including antifungal compounds,
and shows strong copy number variation in several fungi (Soustre et al., 1996; Rogers and Barker,
2003; Sirisattha et al., 2004; Ali et al., 2013; Yew et al., 2016; Liang et al., 2018). Hence, the TE
insertion may have positively modulated RTA1 expression to resist antifungals. Transposition activity in a genome and counteracting purifying selection are expected to establish
an equilibrium over evolutionary time (Charlesworth and Charlesworth, 2009). However, temporal
bursts of TE families and changes in population size due to bottlenecks or founder events are likely
to shift the equilibrium. Despite purifying selection, we were able to detect signatures of positive
selection by scanning for short-term population frequency shifts. Population genomic datasets can
be used to identify the most likely candidate loci underlying recent adaptation. The shallow genome-
wide differentiation of Z. tritici populations provides a powerful background to test for outlier loci
(Hartmann et al., 2018). We found the same TE families to have experienced genome-wide copy
number expansions, suggesting that the availability of adaptive TE insertions may be a by-product of
TE bursts in individual populations. Population-level TE invasions and relaxed selection Across the surveyed populations from four continents, we identified substantial variation in TE counts
per genome. The increase in TEs matches the global colonization history of the pathogen with an
increase in TE copies in more recently established populations (Zhan et al., 2003; Stukenbrock et al.,
2007). Compared to the Israeli population located nearest the center of origin in the Middle East,
the European populations showed a threefold increase in TE counts. The Australian and North Amer-
ican populations established from European descendants retained high TE counts. We identified a
second increase at the North American site where TE counts nearly doubled again over a 25 year
period. Compared to the broader increase in TEs from the Middle East, the second expansion at the
North American site was driven by a small subset of TE families alone. Analyses of completely assem-
bled reference-quality genomes from the same populations confirmed that genome expansions were
primarily driven by the same TE families belonging to the RLG, RLC, and DHH superfamilies (Badet
et al., 2020). Consistent with the contributions from individual TEs, we found that the first expansion
in Europe led to an increase in low-frequency variants, suggesting higher transposition activity of
many TEs in conjunction with strong purifying selection. The second expansion at the North American
site shifted TE frequencies upwards, suggesting relaxed selection against TEs. The population-level
context of TEs in Z. tritici shows how heterogeneity in TE control interacts with demography to deter-
mine extant levels of TE content and, ultimately, genome size. Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Figure 6 continued on next page A A
0
30
60
90
120
Number of TEs per isolate
Retrotransposons
RLG Gypsy
RLC Copia
RIX (LINE unclassified)
DTX (TIR unclassified)
DHH Helitron
Unclassified
DNA transposons
RLX (unclassified)
RXX (unclassified)
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992 A C
DHH
DTX
RIX
RLC
RLG
RLX
RXX
XXX
0.0
12.5
0
5
0
15
0
30
0
60
0
2
0
7.5
0
1
Number of TEs per superfamily
Europe
1999
Europe
2016
North America 1990
North America
2015
Australia
2001
Middle East
1992 B
DHH_Ada
DTX_Kolyma
DTX−MITES_Antaios
DTX−MITES_Satyr
RIX_Lucy
RLC_Deimos
RLC_Io
RLC_Leda
RLG_Aurora
RLG_Jupiter
RLG_Luna
RLG_Mercurius
RLG_Namaka
RLG_Neptunus
RLG_Sol
RLG_Uranus
RLG_Venus
RLX_Cancri
RLX−LARD_Gridr
RLX−TRIM_Jari
RXX_Kepler
RXX_Wasp
XXX_Maki
0
12.5
0
4
0
1
0
1
0
15
0
30
0
3
0
3
0
4
0
1
0
20
0
6
0
3
0
1
0
40
0
1
0
15
0
1
0
1
0
1
0
2
0
6
0
1
Number of TEs per family
Europe
1999
Europe
2016
North America 1990
North America
2015
Australia
2001
Middle East
1992 C
y B D D
frequency per population
proportion
0.00
0.25
0.50
0.75
0.1
0.2
1.0
0.15
0.25
Australia 2001
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990 RLG_Sol
RLC_Deimos
RLG_Venus
RLG_Luna
RIX_Lucy
DHH_ Ada
RXX_Wasp
RLG_Mercurius
RXX_Kepler
RLG_Namaka
RLG_Aurora
RLX_Cancri
XXX_Maki
DTX-MITES_Satyr
DTX_Kolyma
RLC_Io
RLC_Leda
RLG_Uranus
RLG_Jupiter
RLX_LARD_Gridr
RLG_Neptunus
RLX_TRIM_Jari
DTX-MITES_Antaios
0:0
1:1
2:2−5
3:6−15
4:16−25
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
E E E Figure 6. Global population structure of transposable element (TE) insertion polymorphism. (A) Total TE copies per isolate. Colors identify TE
superfamilies. (B) TE copies per family and (C) superfamily. (D) TE insertion frequency spectrum per population. The curve fitting was performed with a
self-starting Nls asymptomatic regression model (E). TE family copy numbers per isolate. Figure 6 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 12 of 25 TE invasion dynamics underpins genome size expansions (C) Core genome size variation amo
the isolates of the populations (excluding accessory chromosomes) (D) Correlation of core genome size and number of detected TEs (E) Correlatio Australia 2001
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
B
53.0
53.5
54.0
GC content [%] BUSCO
completeness [%]
A
98.7
99.0
99.3
99.6 Australia 2001
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
BUSCO
completeness [%]
A
B
98.7
99.0
99.3
99.6
53.0
53.5
54.0
GC content [%] B A C
Core genome
size [Mb]
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
30
32
34
28 C
Core genome
size [Mb]
Europe
1999
Europe
2016
Australia
2001
Middle East
1992
30
32
34
28 C Core genome
size [Mb]
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
30
32
28
D
30
31
32
33
34
30
60
90
120
F
TEs per isolate
−1.0
−0.5
0.0
0.5
1.0
Spearman
Correlation
E
30
31
32
33
34
53.0
53.5
54.0
Genome−wide GC [%]
Core genome
size [Mb]
Core genome
size [Mb]
0.2
0.4
0.6
0.8
cumulative TE length [Mb]
30
31
32
33
34
Core genome
size [Mb]
0.18
−0.13
0.14
0.17
−0.95
0.77
0.79
−0.67
−0.68
0.99
BUSCO score
Core Genome size
GC content
TEs per isolate
Core Genome size
GC content
TEs per isolate
TE length per isolate
G
R = 0.76, p < 2.2e-16
R = 0.78, p < 2.2e-16
R = 0.95, p < 2.2e-16
Figure 7. Core genome size and transposable element (TE) evolution across populations. (A) BUSCO completeness variation among genome
assemblies. Black lines indicate the mean genome size per population. (B) Genome-wide GC content variation. (C) Core genome size variation among
the isolates of the populations (excluding accessory chromosomes). (D) Correlation of core genome size and number of detected TEs. (E) Correlation
of core genome size and the cumulative length of all TEs detected as inserted. (F) Correlation of core genome size and genome-wide GC content. TE invasion dynamics underpins genome size expansions TE invasion dynamics underpins genome size expansions
The number of detected TEs was closely correlated with core genome size; hence, genome size
expansions were at least partly caused by the very recent proliferation of TEs. Genome assemblies
of large eukaryotic genomes based on short-read sequencing are often fragmented and contain
chimeric sequences (Nagarajan and Pop, 2013). Focusing on the less repetitive core chromosomes in
the genome of Z. tritici reduces such artifacts substantially. Because genome assemblies are the least
complete in the most repetitive regions, any under-represented sequences may rather underestimate 13 of 25 13 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Research article Evolutionary Biology | Genetics and Genomics Australia 2001
Europe 2016
Europe 1999
Middle East 1992
North America 2015
North America 1990
BUSCO
completeness [%]
A
B
C
Core genome
size [Mb]
Europe
1999
Europe
2016
North America
1990
North America
2015
Australia
2001
Middle East
1992
30
32
34
28
D
30
31
32
33
34
30
60
90
120
F
TEs per isolate
−1.0
−0.5
0.0
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Correlation
E
98.7
99.0
99.3
99.6
53.0
53.5
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30
31
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34
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53.5
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size [Mb]
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size [Mb]
0.2
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size [Mb]
0.18
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−0.67
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GC content
TEs per isolate
Core Genome size
GC content
TEs per isolate
TE length per isolate
G
R = 0.76, p < 2.2e-16
R = 0.78, p < 2.2e-16
R = 0.95, p < 2.2e-16
Figure 7. Core genome size and transposable element (TE) evolution across populations. (A) BUSCO completeness variation among genome
assemblies. Black lines indicate the mean genome size per population. (B) Genome-wide GC content variation. Research article Research article Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics Spearman correlation matrix of BUSCO completeness, core genome size, number of detected TEs, and genome-wide GC content. The online version of this article includes the following figure supplement(s) for figure 7:
Figure supplement 1. Genome size expansion. Figure 7 continued Spearman correlation matrix of BUSCO completeness, core genome size, number of detected TEs, and genome-wide GC content. The online version of this article includes the following figure supplement(s) for figure 7:
Figure supplement 1. Genome size expansion. Figure 7 continued Spearman correlation matrix of BUSCO completeness, core genome size, number of detected TEs, and genome-wide GC content. The online version of this article includes the following figure supplement(s) for figure 7:
Figure 7 continued than overestimate within-species variation in genome size. Hence, we consider the assembly sizes to
be a robust correlate of total genome size. The core genome size differences observed across the
species range match genome size variation typically observed among closely related species. Among
primates, genome size varies by ~70 % with ~10 % between humans and chimpanzees (Rogers and
Gibbs, 2014; Miga et al., 2020). In fungi, genome size varies by several orders of magnitude within
phyla but is often highly similar among closely related species (Raffaele and Kamoun, 2012). Inter-
estingly, drastic changes in genome size have been observed in the Blumeria and Pseudocercospora
genera where genome size changed by 35–130% between the closest known species (González-
Sayer et al., 2021; Frantzeskakis et al., 2018). Beyond analyses of TE content variation correlating
with genome size evolution, proximate mechanisms driving genome expansions are poorly under-
stood. By establishing large population genetic datasets, such as those possible for crop pathogens,
analyses of genome size evolution become tractable at the population level. TEs might not only contribute to genome expansion directly by adding length through additional
copies, but also by increasing the rate of chromosomal rearrangements and ectopic recombination
(Bourque et al., 2018; Blommaert, 2020). However, TEs are not the only repetitive elements that can
lead to a genome size expansion. In Arabidopsis thaliana genomes, the 45 S rDNA has been shown
to have the strongest impact on genome size variation, followed by 5 S rDNA variation, and contribu-
tions by centromeric repeats and TEs (Long et al., 2013). Fungal isolate collection and sequencing We analyzed 295 Z. tritici isolates covering six populations originating from four geographic locations
and four continents (Figure 1—source data 1), including: Middle East 1992 (n = 30 isolates, Nahal
Oz, Israel), Australia 2001 (n = 27, Wagga Wagga), Europe 1999 (n = 33, Berg am Irchel, Switzerland),
Europe 2016 (n = 52, Eschikon, ca. 15 km from Berg am Irchel, Switzerland), and North America 1990
and 2015 (n = 56 and n = 97, Willamette Valley, Oregon; McDonald et al., 1996; Linde et al., 2002;
Zhan et al., 2002; Zhan et al., 2003; Zhan et al., 2005). Illumina short-read data from the Middle
Eastern, Australian, European 1999, and North American 1990 populations were obtained from the
NCBI Sequence Read Archive (SRA) under the BioProject PRJNA327615 (Hartmann et al., 2017). For
the Switzerland 2016 and Oregon 2015 populations, asexual spores were harvested from infected
wheat leaves from naturally infected fields and grown in YSB liquid media including 50 mgL–1 kana-
mycin and stored in silica gel at −80 °C. High-quality genomic DNA was extracted from liquid cultures
using the DNeasy Plant Mini Kit from Qiagen (Venlo, The Netherlands). The isolates were sequenced
on an Illumina HiSeq in paired-end mode and raw reads were deposited at the NCBI SRA under the
BioProject PRJNA596434. TE invasion dynamics underpins genome size expansions (G)
Figure 7 continued on next page D
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G F
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53.5
54.0
Genome−wide GC [%]
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size [Mb]
R = 0.95, p < 2.2e-16 G F Figure 7. Core genome size and transposable element (TE) evolution across populations. (A) BUSCO completeness variation among genome
assemblies. Black lines indicate the mean genome size per population. (B) Genome-wide GC content variation. (C) Core genome size variation among
the isolates of the populations (excluding accessory chromosomes). (D) Correlation of core genome size and number of detected TEs. (E) Correlation
of core genome size and the cumulative length of all TEs detected as inserted. (F) Correlation of core genome size and genome-wide GC content. (G)
Figure 7 continued on next page Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 14 of 25 Research article In conjunction, recent work demonstrates
how repetitive sequences are drivers of genome size evolution over short evolutionary timescales. The activity of TEs is controlled by complex selection regimes within species. Actively transposing
elements may accelerate genome evolution and underpin expansions. Hence, genomic defenses
should evolve to efficiently target recently active TEs. Here, we show that TE activity and counter-
acting genomic defenses have established a tenuous equilibrium across the species range. We show
that population subdivisions are at the origin of highly differentiated TE content within a species
matching genome size changes emerging over the span of only decades and centuries. In conclusion,
population-level analyses of genome size can recapitulate genome expansions typically observed
across much deeper time scales providing fundamentally new insights into genome evolution. Downsampling analysis We performed a downsampling analysis to estimate the sensitivity of the TE detection with ngs_
te_mapper based on variation in read depth. We selected one isolate per population matching the
average coverage of the population. We extracted the per-base pair read depth with the genomecov
function of bedtools version 2.27.1 and calculated the genome-wide mean read depth (Quinlan and
Hall, 2010). The number of reads in the original fastq file was reduced in steps of 10 % to simu-
late the impact of reduced coverage. We analyzed each of the obtained reduced read subsets with
ngs_te_mapper using the same parameters as described above. The correlation between the number
of detected insertions and the read depth was visualized using the function nls with model SSlogis
in R and visualized with ggplot2 (Wickham, 2016). The number of detected TEs increased with the
number of reads until reaching a plateau indicating saturation (Figure 1B). Saturation was reached
at a coverage of approximately 15 ×; hence, we retained only isolates with an average read depth
above 15 × for further analyses. We thus excluded one isolate from the Oregon 2015 population and
10 isolates from the Switzerland 2016 population. TE insertion detection The quality of Illumina short reads was determined with FastQC version 0.11.5 (https://www.bioin-
formatics.babraham.ac.uk/projects/fastqc/) (Figure 1A). To remove spuriously sequenced Illumina 15 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 15 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics adaptors and low-quality reads, we trimmed the sequences with Trimmomatic version 0.36, using
the following filter parameters: illuminaclip:TruSeq3-PE-2.fa:2:30:10 leading:10 trailing:10 sliding-
window:5:10 minlen:50 (Bolger et al., 2014). We created repeat consensus sequences for TE families
(sequences are available on https://github.com/crolllab/datasets (copy archived at swh:1:rev:364745
6a2f7ed986b690501d690f09d02d3f586e); Laboratory of Evolutionary Genetics @ UNINE, 2021;
Figure 1—source data 2) in the complete reference genome IPO323 (Goodwin et al., 2011) with
RepeatModeler version open-4.0.7 (http://www.repeatmasker.org/RepeatModeler/) based on the
RepBase Sequence Database and de novo (Bao et al., 2015). TE classification into superfamilies and
families was based on an approach combining detection of conserved protein sequences and tools to
detect non-autonomous TEs (Badet et al., 2020). To detect TE insertions, we used the R-based tool
ngs_te_mapper version 79ef861f1d52cdd08eb2d51f145223fad0b2363c integrated into the McClin-
tock pipeline version 20cb912497394fabddcdaa175402adacf5130bd1, using bwa version 0.7.4-r385
to map Illumina short reads, samtools version 0.1.19 to convert alignment file formats and R version
3.2.3 (Li and Durbin, 2009; Li et al., 2009; Linheiro and Bergman, 2012; R Development Core
Team, 2017; Nelson et al., 2017). Validation procedure for predicted TE insertions ngs_te_mapper detects the presence but not the absence of a TE at any given locus. We devised
additional validation steps to ascertain both the presence and the absence of a TE across all loci
in all individuals. TEs absent in the reference genome were validated by re-analyzing mapped Illu-
mina reads. Reads spanning both parts of a TE sequence and an adjacent chromosomal sequence
should only map to the reference genome sequence and cover the target site duplication of the
TE (Figure 1C). We used bowtie2 version 2.3.0 with the parameter --very-sensitive-local to
map Illumina short reads of each isolate on the reference genome IPO323 (Langmead and Salzberg,
2012). Mapped Illumina short reads were then sorted and indexed with samtools, and the resulting
bam file was converted to a bed file with the function bamtobed in bedtools. We extracted all mapped
reads with an end point located within 100 bp of the target site duplication (Figure 1C). We tested
whether the number of reads with a mapped end around the target site duplication significantly devi-
ated if the mapping ended exactly at the boundary. A mapped read ending exactly at the target site
duplication boundary is indicative of a split read mapping to a TE sequence absent in the reference
genome. To test for the deviation in the number of read mappings around the target site duplication,
we used a Poisson distribution and the ppois function in R version 3.5.1 (Figure 1C). We identified
a TE as present in an isolate if tests on either side of the target site duplication had a p-value<0.001
(Figure 5—source data 1; Figure 1—figure supplement 1B and Figure 1—source data 1). For TEs present in the reference genome, we analyzed evidence for spliced junction reads span-
ning the region containing the TE. Spliced reads are indicative of a discontinuous sequence and,
hence, absence of the TE in a particular isolate (Figure 1D). We used STAR version 2.5.3a to detect
spliced junction reads with the following set of parameters: --runThreadN 1 --outFilterMul-
timapNmax 100 --winAnchorMultimapNmax 200 --outSAMmultNmax 100 --outSAMtype BAM
Unsorted --outFilterMismatchNmax 5 --alignIntronMin 150 --alignIntronMax 15,000 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 16 of 25 16 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics Evolutionary Biology | Genetics and Genomics (Dobin et al., 2013). Clustering of TE insertions into loci g
We identified insertions across isolates as being the same locus if all detected TEs belonged to the
same TE family and insertion sites differed by ≤100 bp (Figure 1—figure supplement 2). We used the
R package GenomicRanges version 1.28.6 with the functions makeGRangesFromDataFrame and find-
Overlaps and the R package devtools version 1.13.4 (Lawrence et al., 2013; Wickham and Chang,
2016). We used the R package dplyr version 0.7.4 to summarize datasets (https://dplyr.tidyverse.
org/). Population-specific frequencies of insertions were calculated with the function allele.count in
the R package hierfstat version 0.4.22 (Goudet, 2005). We conducted a principal component analysis
for TE insertion frequencies filtering for a minor allele frequency ≥5 %. We also performed a prin-
cipal component analysis for genome-wide single nucleotide polymorphism (SNP) data obtained from
Hartmann et al., 2017 and Singh et al., 2020. As described previously, SNPs were hard-filtered with
VariantFiltration and SelectVariants tools integrated in the Genome Analysis Toolkit (GATK) (McKenna
et al., 2010). SNPs were removed if any of the following filter conditions applied: QUAL < 250; QD <
20.0; MQ < 30.0; –2 > BaseQRankSum > 2; –2 > MQRankSum > 2; –2 > ReadPosRankSum > 2; FS >
0.1. SNPs were excluded with vcftools version 0.1.17 and plink version 1.9 requiring a genotyping
rate > 90% and a minor allele frequency > 5% (https://www.cog-genomics.org/plink2, Chang et al.,
2015). Finally, we converted tri-allelic SNPs to bi-allelic SNPs by recoding the least frequent allele as
a missing genotype. Principal component analysis was performed using the gdsfmt and SNPRelate
packages in R (Zheng et al., 2012; Zheng et al., 2017). For a second principal component analysis
with a reduced set of random markers, we randomly selected SNPs with vcftools and the following
set of parameters: --maf 0.05 –thin 200,000 to obtain an approximately equivalent number of SNPs
as TE loci. Validation procedure for predicted TE insertions We then sorted and indexed the resulting bam file with samtools and converted
split junction reads with the function bam2hints in bamtools version 2.5.1 (Barnett et al., 2011). We
selected loci without overlapping spliced junction reads using the function intersect in bedtools with
the parameter -loj -v. We considered a TE as truly absent in an isolate if ngs_te_mapper did not
detect a TE and evidence for spliced junction reads were found, indicating that the isolate had no
inserted TE in this region. If the absence of a TE could not be confirmed by spliced junction reads, we
labeled the genotype as missing. Finally, we excluded TE loci with more than 20 % missing data from
further investigations (Figure 1D, Figure 1—figure supplement 1C). Population differentiation in TE frequencies p
q
We calculated Nei’s fixation index (FST) between pairs of populations using the R packages hierf-
stat and adegenet version 2.1.0 (Jombart, 2008; Jombart and Ahmed, 2011). To understand the
chromosomal context of TE insertion loci across isolates, we analyzed draft genome assemblies. We
generated de novo genome assemblies for all isolates using SPAdes version 3.5.0 with the parameter
--careful and a kmer range of ‘21, 29, 37, 45, 53, 61, 79, 87’ (Bankevich et al., 2012). We used
blastn to locate genes adjacent to TE insertion loci on genomic scaffolds of each isolate. We then
extracted scaffold sequences surrounding 10 kb up- and downstream of the localized gene with the
function faidx in samtools and reverse complemented the sequence if needed. Then, we performed
multiple sequence alignments for each locus across all isolates with MAFFT version 7.407 with param-
eter --maxiterate 1000 (Katoh and Standley, 2013). We performed visual inspections to ensure
correct alignments across isolates using Jalview version 2.10.5 (Waterhouse et al., 2009). To generate
phylogenetic trees of individual gene or TE loci, we extracted specific sections of the alignment using
the function extractalign in EMBOSS version 6.6.0 (Rice et al., 2000) and converted the multiple
sequence alignment into PHYLIP format with jmodeltest version 2.1.10 using the -getPhylip param-
eter. We then estimated maximum likelihood phylogenetic trees with the software PhyML version
3.0, the K80 substitution model, and 100 bootstraps on the ATGC South of France bioinformatics
platform (Guindon and Gascuel, 2003; Guindon et al., 2010; Darriba et al., 2012). Bifurcations with
a supporting value lower than 10 % were collapsed in TreeGraph version 2.15.0–887 beta, and trees
were visualized as circular phylograms in Dendroscope version 2.7.4 (Huson et al., 2007; Stöver
and Müller, 2010). For loci showing complex rearrangements, we generated synteny plots using 19
completely sequenced genomes from the same species using the R package genoplotR version 0.8.9
(Guy et al., 2010; Badet et al., 2020). We calculated the population-specific allele frequency for
TE loci and estimated the exponential decay curve with a self-starting Nls asymptomatic regression
model nls(p_loci ~ SSasymp(p_round, Asym, R0, lrc)) in R. We analyzed signatures of selective sweeps based on genome-wide SNPs using the extended
haplotype homozygosity (EHH) tests implemented in the R package REHH (Sabeti et al., 2007;
Gautier and Vitalis, 2012). We analyzed within-population signatures based on the iHS statistic and
chose a maximum gap distance of 20 kb. Population differentiation in TE frequencies We also analyzed cross-population signatures based on the
XP-EHH statistic for the following two population pairs: North America 1990 versus North America
2015, Europe 1999 versus Europe 2016. We defined significant selective sweeps as being among the
99.9th percentile outliers of the iHS and XP-EHH statistics. Significant SNPs at less than 5 kb were
clustered into a single selective sweep region adding ±2.5 kb. Finally, we analyzed whether TE loci in
the population pairs were within 10 kb of a region identified as a selective sweep by XP-EHH using
the function intersect from bedtools. Evaluation of singleton insertions g
To evaluate the reliability of singleton TE insertion loci, we analyzed singleton loci in isolates for
which we had both Illumina datasets and complete reference-quality genomes (Badet et al., 2020). From a set of 19 long-read PacBio reference genomes spanning the global distribution of Z. tritici,
one isolate each from Australia, Israel, North America (1990) and four isolates from Europe (1999)
were also included in the TE insertion screening. To assess the reliability of singleton TE insertions,
we first investigated structural variation analyses among the reference genomes (Badet et al., 2021:
Supplementary Data 1 and 2). The structural variation was called both based on split read mapping
of PacBio reads and pairwise whole-genome alignments. Using bedtools intersect, we recovered for
the 31 singleton TE loci in the seven analyzed genomes a total of 17 loci showing either an indel,
translocation, copy number polymorphism, duplication, inverted duplication, inversion, or inverted
translocation at the same location. We visually inspected the PacBio read alignment bam files against
the IPO323 reference genome using IGV version 2.4.16 (Robinson et al., 2011), and found a typical
coverage increase at the target site duplication, with most read mappings interrupted at the target
site duplication as expected for an inserted TE. For the 14 remaining TE loci, we extracted the region
of the predicted insertion and padded the sequence on both ends with an additional 500 bp using
samtools faidx. We used blast to identify a homologous region in the assembled reference-quality
genomes. Matching regions were inspected based on blastn for the presence of a TE sequence
matching the TE family originally detected at the locus. With this second approach, we confirmed
an additional five singletons to be true insertions. Both methods combined produced supportive
evidence for 22 of 31 singleton insertions (71%). We calculated the read coverage after mapping
to the reference genome IPO323 with bedtools genomecov for each PacBio long-read dataset and
calculated mean coverage for 500 bp regions around singleton TE insertions. Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 17 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics Genomic location of TE insertions To characterize the genomic environment of TE insertion loci, we split the reference genome into
non-overlapping windows of 10 kb using the function splitter from EMBOSS. TEs were located in
the reference genome using RepeatMasker providing consensus sequences from RepeatModeler
(http://www.repeatmasker.org/). To analyze coding sequence, we retrieved the gene annotation for
the reference genome (Grandaubert et al., 2015). We estimated the percentage covered by genes or
TEs per window using the function intersect in bedtools. Additionally, we calculated the GC content
using the tool get_gc_content (https://github.com/spundhir/RNA-Seq/blob/master/get_gc_content.
pl; Sachin, 2021). We extracted the number of TEs present in 1 kb windows around each annotated
core gene in the reference genome IPO323, using the function window in bedtools. We calculated
the relative distances between each gene and the closest TE with the function bedtools closest. For
the TEs inserted into genes, we used the function intersect in bedtools to distinguish intron and exon
insertions with the parameters -wo and -v, respectively. TEs that overlap more than one exon were
only counted once. For each 100 bp segment in the 1 kb windows as well as for introns and exons, we
calculated the mean number of observed TE insertions per base pair. We calculated the mean number
of TEs per window and calculated the log2 of the observed number of TE insertions divided by the
expected value. We extracted information about recombination hotspots from Croll et al., 2015. This
dataset is based on two experimental crosses initiated from isolates included in our analyses (1A5 ×
1E4, 3D1 × 3D7). The recombination rates were assessed based on the reference genome IPO323 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 18 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics and analyzed with the R/qtl package in R. We used bedtools intersect to compare both TE density in
IPO323 and TE insertion polymorphism with predicted recombination hotspots. and analyzed with the R/qtl package in R. We used bedtools intersect to compare both TE density in
IPO323 and TE insertion polymorphism with predicted recombination hotspots. Fungicide resistance assay To quantify susceptibility toward propiconazole, we used a previously published microtiter plate
assay dataset with three replicates performed for each isolate and concentration. Optical density was
used to estimate growth rates under different fungicide concentrations (0, 0.00006, 0.00017, 0.0051,
0.0086, 0.015, 0.025, 0.042, 0.072, 0.20, 0.55, 1.5 mg L–1) (Hartmann et al., 2020). We calculated
dose–response curves and estimated the half-maximal lethal concentration EC50 with a four-parameter
logistics curve in the R package drc (Ritz and Streibig, 2005). Core genome size estimation Accessory chromosomes show the presence/absence variation within the species and length poly-
morphism (Goodwin et al., 2011; Croll et al., 2013) and thus impact genome size. We controlled for
this effect by first mapping sequencing reads to the reference genome IPO323 using bowtie2 with
--very-sensitive-local settings and retained only reads mapping to any of the 13 core chro-
mosomes using seqtk subseq v1.3-r106 (https://github.com/lh3/seqtk/; Heng, 2021). Furthermore,
we found that different sequencing runs showed minor variation in the distribution of the per read GC
content. In particular, reads of a GC content lower than 30 % were under-represented in the Australian
(mean reads <30 % of the total readset: 0.05%), North American 1990 (0.07%) and Middle East (0.1%)
populations, and higher in the Europe 1999 (1.3%), North American 2015 (3.0%), and Europe 2016
(4.02%) populations (Figure 1—figure supplement 3). Library preparation protocols and Illumina
sequencer generations are known factors influencing the recovery of reads of varying GC content
(Benjamini and Speed, 2012). To control a potential bias stemming from this, we subsampled reads based on GC content to create
homogeneous datasets. For this, we first retrieved the mean GC content for each read pair using
geecee in EMBOSS and binned reads according to GC content. For the bins with a GC content <30%,
we calculated the mean proportion of reads from the genome over all samples. We then used seqtk
subseq to subsample reads of <30% to adjust the mean GC content among readsets. We generated
de novo genome assemblies using the SPAdes assembler version with the parameters --careful
and a kmer range of ‘21, 29, 37, 45, 53, 61, 79, 87’. The SPAdes assembler is optimized for the
assembly of relatively small eukaryotic genomes. We evaluated the completeness of the assemblies
using BUSCO v4.1.1 with the fungi_odb10 gene test set (Simão et al., 2015). We finally ran Quast
v5.0.2 to retrieve assembly metrics including scaffolds of at least 1 kb (Mikheenko et al., 2018). Acknowledgements We thank Andrea Sánchez Vallet, Anne C Roulin, Luzia Stalder, Adam Taranto, Emilie Chanclud, and
Alice Feurtey for helpful discussions and comments on previous versions of the manuscript. We also
thank the three reviewers for very helpful suggestions. We thank C Sarai Reyes-Avila for advice on
statistical analyses. DC is supported by the Swiss National Science (grants 31,003A_173265) and the
Fondation Pierre Mercier pour la Science. Additional information Funding
Funder
Grant reference number
Author
Schweizerischer
Nationalfonds zur
Förderung der
Wissenschaftlichen
Forschung
31003A_173265
Daniel Croll
Pierre Mercier pour la
science
Daniel Croll 19 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Decision letter and Author response Decision letter and Author response
Decision letter https://doi.org/10.7554/69249.sa1
A th
htt
//d i
/10 7554/69249
2 Decision letter https://doi.org/10.7554/69249.sa1 Author response https://doi.org/10.7554/69249.sa2 Supplementary files • Transparent reporting form • Transparent reporting form Grant reference number
Author Grant reference number
Author Funder Funder The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication. Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 19 of 25 Research article Research article Evolutionary Biology | Genetics and Genomics Author contributions Ursula Oggenfuss, Conceptualization, Data curation, Formal analysis, Investigation, Methodology,
Writing - original draft; Thomas Badet, Formal analysis; Thomas Wicker, Data curation, Supervision;
Fanny E Hartmann, Data curation, Resources, Supervision; Nikhil Kumar Singh, Leen Abraham, Petteri
Karisto, Christopher Mundt, Bruce A McDonald, Resources; Tiziana Vonlanthen, Investigation; Daniel
Croll, Conceptualization, Funding acquisition, Methodology, Project administration, Resources,
Supervision, Writing - original draft, Writing - review and editing Author ORCIDs Author ORCIDs
Ursula Oggenfuss http://orcid.org/0000-0001-9291-9185
Thomas Badet http://orcid.org/0000-0001-6130-441X
Fanny E Hartmann http://orcid.org/0000-0002-9365-4008
Nikhil Kumar Singh http://orcid.org/0000-0001-7236-9278
Petteri Karisto http://orcid.org/0000-0003-4807-0190
Christopher Mundt http://orcid.org/0000-0002-1216-7583
Bruce A McDonald http://orcid.org/0000-0002-5332-2172
Daniel Croll http://orcid.org/0000-0002-2072-380X Decision letter and Author response Decision letter and Author response Data availability Sequence data are deposited at the NCBI Sequence Read Archive under the accession numbers
PRJNA327615, PRJNA596434 and PRJNA178194. Transposable element consensus sequences are
available from https://github.com/crolllab/datasets (copy archived at https://archive.softwareheri-
tage.org/swh:1:rev:3647456a2f7ed986b690501d690f09d02d3f586e). The following dataset was generated: The following dataset was generated:
Author(s)
Year
Dataset title
Dataset URL
Database and Identifier
Hartmann FE,
McDonald BA, Croll
D
2019
Population sequencing
of Zymoseptoria tritici in
Switzerland and Oregon
(USA)
https://www.ncbi.nlm.
nih.gov/bioproject/
PRJNA596434
NCBI BioProject,
PRJNA596434 The following previously published datasets were used: The following previously published datasets were used:
Author(s)
Year
Dataset title
Dataset URL
Database and Identifier
Hartmann FE,
McDonald BA, Croll
D
2016
Population genomics of
Zymoseptoria tritici
https://www.ncbi.nlm.
nih.gov/bioproject/
PRJNA327615/
NCBI BioProject,
PRJNA327615
Croll D, McDonald
BA
2012
Zymoseptoria tritici
genome sequencing
https://www.ncbi.nlm.
nih.gov/bioproject/
PRJNA178194/
NCBI BioProject,
PRJNA178194 20 of 25 Oggenfuss et al. eLife 2021;0:e69249. DOI: https://doi.org/10.7554/eLife.69249 Research article Research article Research article Evolutionary Biology | Genetics and Genomics References DOI: https://doi.org/10.1186/s13059-018-1577-z, PMID: 30454069 gy
p
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The first comprehensive Health Sector Review and future Operational Plan of Liberia after the EBOLA crisis: Consolidated Operational Plan 2016-2017
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Foreword Liberia emerged out of 14 years (1989-2003) of brutal conflict that ravaged gains made in
every sector of the society. With one decade of relative peace and stability, Liberia restored
health services and started making enormous strives in childhood mortality reduction,
decline in AIDS related deaths and expansion of health services. Unfortunately, in 2014, the
country was hit with an incomparable Ebola outbreak that shattered gains in health status,
social values and the economy. The impact of the EVD outbreak was very grave, affecting households’ income, health status
and food security. The crisis depleted the health workforce, led to low utilization of health
services and desertion of health facilities by service providers. The economic forecast
shows that the outbreak is draining the finances of governments—increasing national
deficits due to additional expenditures incurred during the EVD crisis amidst drastic
shortfalls in domestic revenue. The Ministry of Health in 2007 developed a post conflict policy and strategic plan that
guided the health sector into restoration and recovery for five years (2007-2011). The five
years Plan were revised to ten years with new strategies to maintain gains and further
improve health outcomes. The 2011-2021 national health plan was launched in 2011 and
implemented until 2014 when Ebola exposed the health systems vulnerabilities. In order to
address the health systems fragilities, the health sector investment plan was formulated to
drive responsiveness and resiliency in the sector. The investment plan has nine vital areas
for improving the sector effectiveness, efficiency, and resilience. It is my conviction that despite the colossal challenges we have to surmount in the health
sector to attain the health related Sustainable Development Goals (SDGs) and resiliency, I
am confident that the FY 2016/17 operational will be instrumental in translating the
strategic priorities of the health sector recovery and development as stipulated in the
investment plan (2015-2021) into concrete actions. I trust that partnership will take us to
alignment and harmonization, and make the operational plan a reality. I am pleased to express my deep appreciation and sincere gratitude to all stakeholders,
partners, and MOH staff that contributed technical and financial support in the
development of the Operational Plan 2016/17. FY 2016/17 September 2016 September 2016 September 2016 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Dr. Bernice Dahn MD, MPH, FLCP
Minister of Health Minister of Health Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Acknowledgement The annual operational plan is the road map for the implementation of the post
EVD recovery plan and the health sector investment plan for building a resilient
health system. The overall objective of the plan is to ensure a functional and
resilient health system that guarantees its population an effective and equitable
health. The plan is a consolidation of both central and county levels work plans. The annual operational plan is the road map for the implementation of the post
EVD recovery plan and the health sector investment plan for building a resilient
health system. The overall objective of the plan is to ensure a functional and
resilient health system that guarantees its population an effective and equitable
health. The plan is a consolidation of both central and county levels work plans. The Ministry of Health is pleased to recognize the effort of all of those who
contributed to the development of the FY 2016/17 annual operational plan. Special
thanks are extended to members of the county health teams, MOH departments,
programs and divisions for their contributions towards the formulation of the
annual operational plan. Without their involvement, the consolidated operational
plan would not have been finalized. The MOH is grateful to the World Health Organization for providing financial and
technical support for the orientation and training workshops for county health
teams and partner and the elaboration of the annual consolidated operational plan
together with EPOS EU TA. Special gratitude is extended to USAID (FARA project) for providing funds for the
county level planning process. We want to express our appreciation to all NGOs, UN agencies for technical
support and county officials that participated in the planning exercise. Your
participation and contributions have made this document useful and inclusive. It is our hope and aspiration that the annual operational plan will be used as the
health sector development agenda for FY 2016/17 at all levels to build a system
that is responsive, effective and resilient. Let us solicit the financial and technical
resources to implement this operational plan together. 3 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Table of Contents Table of Contents
1.0
INTRODUCTION ......................................................................................................................6
1.1
BACKGROUND ................................................................................................................................... 6
1.2
HEALTH SECTOR GOVERNANCE AND MANAGEMENT ................................................................................ 6
2.0
PLANNING CYCLE AND PROCESSES ..........................................................................................8
2.1
PLANNING OBJECTIVES ....................................................................................................................... 8
2.2
PLANNING PROCESS ........................................................................................................................... 8
3.0
SITUATIONAL ANALYSIS AND PERFORMANCE FY 2015/16 ...................................................... 10
3.1
SITUATIONAL ANALYSIS ........................................................................................................... 10
3.2
PERFORMANCE FY 2015/16 ................................................................................................... 11
4.0
OPERATIONAL PLAN 2016/17 ................................................................................................ 12
4.1
HEALTH INFRASTRUCTURE ................................................................................................................. 12
4.2
HUMAN RESOURCES FOR HEALTH ...................................................................................................... 13
4.3
HEALTH CARE FINANCING ................................................................................................................. 14
4.4
EMERGENCY PREPAREDNESS AND RESPONSE (EPR) .............................................................................. 14
4.5
HEALTH SERVICE DELIVERY AND QUALITY OF CARE ................................................................................ 17
4.6
DRUGS AND SUPPLY CHAIN MANAGEMENT ......................................................................................... 31
4.7
HEALTH INFORMATION SYSTEMS, M&E AND RESEARCH ........................................................................ 32
4.8
COMMUNITY ENGAGEMENT .............................................................................................................. 34
4.9
LEADERSHIP AND GOVERNANCE ......................................................................................................... 34
5.0
COSTING AND BUDGETING .................................................................................................... 35
5.1
NATIONAL BUDGET................................................................................................................... 36
6.0
MONITORING AND REVIEW OF INVESTMENT PLAN ................................................................ 36
ANNEXES ............................................................................................................................................ ANNEX A: HEALTH INFRASTRUCTURE NEEDS FY 2016/17 ......................................................... 37
ANNEX B: SUPPLY CHAIN FY 2016/17 ACTIVITIES ............................................................................................ 38
ANNEX C: HEALTH SECTOR PERFORMANCE FRAMEWORK ................................................................................... 38
ANNEX D: COUNTY LEVEL PERFORMANCE FRAMEWORK ..................................................................................... 40 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Abbreviations
CHA
Community Health Assistant
CHW
Community Health Worker
CSA
Civil Service Agency
EVD
Ebola Virus Disease
EPR
Emergency Preparedness Response
EPI
Expanded Program on Immunization
FARA
Fixed Amount Reimbursement Arrangement
FHD
Family Health Division
FY
Fiscal Year
HR
Human Resources
HF
Health Facility
HIS
Health Information System
HMER
Health Information System, Monitoring and Evaluation and Research
HMIS
Health Management Information System
M&E
Monitoring and Evaluation
MFDP
Ministry of Finance and Development Planning
MOH
Ministry of Health
NACP
National AIDs & STDs Control Program
NDS
National Drug
OSC
One Stop Center
OP
Operational Plan
SOP
Standard Operating Procedure
TTMs
Trained Traditional Midwives Abbreviations Abbreviations CHA
Community Health Assistant
CHW
Community Health Worker
CSA
Civil Service Agency
EVD
Ebola Virus Disease
EPR
Emergency Preparedness Response
EPI
Expanded Program on Immunization
FARA
Fixed Amount Reimbursement Arrangement
FHD
Family Health Division
FY
Fiscal Year
HR
Human Resources
HF
Health Facility
HIS
Health Information System
HMER
Health Information System, Monitoring and Evaluation and Research
HMIS
Health Management Information System
M&E
Monitoring and Evaluation
MFDP
Ministry of Finance and Development Planning
MOH
Ministry of Health
NACP
National AIDs & STDs Control Program
NDS
National Drug
OSC
One Stop Center
OP
Operational Plan
SOP
Standard Operating Procedure
TTMs
Trained Traditional Midwives Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 1.1
Background The 10-year strategic plan (2011–2021) and the post Ebola crisis National Investment Plan
(2015–2021), provide an overall guide and orientation, while the instrument that is
required to ensure that the strategy is implemented is the operational plan (OP). The health sector recognizes its inherent health system challenges and weaknesses that
were further amplified by the impact of the Ebola crisis. The operational plan is the annual
roadmap to implement the National Investment Plan (2015-2021). The OP provides priorities activities and establishes targets that are linked to budget,
which is funded through a combination of known domestic and external resources. It was
formulated based on county and central levels consultations with various stakeholders. In view of the National Investment plan, the current operational plan, while focusing on
safe and quality health services, recognizes the need to invest on key support components
of the health system that enable service provision and fosters donor coordination and
alignment at decentralized and central levels of the health system. The process of planning
has also taken account of existing capacity and resources through integrating the
contributions of most development partners into a consistent framework, both in terms of
financial and of technical assistance. The plan intends to address issues related to recovery
through phased or incremental changes but in an equitable and sustainable manner. Furthermore, the recovery phase of the operational plan goes beyond the hardware
required to include the regulatory and implementation capacity building components. Moreover, products of the Annual Plan consist of aligning available funding with planned
activities, resources, and service delivery targets. 1.2
Health Sector Governance and Management The Liberian government comprises three separate branches, the Executive, Judiciary, and
House of Representatives and the Senate. Administratively, the country is divided into 15
counties and 73 political districts. The country is however demarcated into 89 health
districts for operational purposes. The superintendents are the heads of the counties. The
superintendents and other county functionaries (district commissioners, paramount chiefs,
clan chiefs, city majors and town chiefs) are appointed by the President. With the wave of
reforms in governance, the superintendents and other government functionaries in the
future are likely to be elected by the people if the Local Government Act - now before the 6 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 House - is passed into law. In the draft act, the county structure consists of 9 departments. The county health team becomes a department of health. 1.2.1 Structure of Ministry of Health The Minister of Health is appointed by the President and is not a member of the House of
Representatives or of the Senate. The minister is assisted by four deputies manning the
following departments: Health Services; Planning, Policy and Development; Administration,
and Public Health - recently included out of the aches of the Ebola crisis. There are several
assistant ministers heading bureaus and managers heading divisions and vertical programs
such as the National TB & Leprosy Control Program (NTLCP), the National AIDs & STDs
Control Program (NACP), etc. Minister There are four levels of supervision: (i) central level, which includes departments,
programs, divisions and units, (ii) county level, hospitals and lower level health units, (iii)
district level, and (iv) community based health services. The county health services and
community health services units of the ministry of health have been established to provide
direct support to the counties and community based services, respectively. While systems
for supervision and monitoring exist, there are enormous challenges. The fiscal year plan is
to redress issues of the essential services and other components of the Investment Plan. Strong involvement and engagement of communities and their representatives are expected
to play a critical role in the management and monitoring of the operational plan at all
levels of the health system. 1.2.1 Structure of Ministry of Health p
Health Services
Bureau of
Curative
Services
15 county health
and social
welfare teams
Bureau of
Preventive
Services
National
programs (e.g.,
TB, EPI,
Malaria, etc
Planning and
Statistics
Bureau of
Planning and
Policy
Bureau of Vital
Statistics
p
Administration
Bureau of Fiscal
Affairs
Bureau of
Administration
p
Public Health 7
Minister
Department of
Health Services
Bureau of
Curative
Services
15 county health
and social
welfare teams
Bureau of
Preventive
Services
National
programs (e.g.,
TB, EPI,
Malaria, etc
Department of
Planning, Policy
and
Development
Bureau of
Planning and
Policy
Bureau of Vital
Statistics
Department of
Administration
Bureau of Fiscal
Affairs
Bureau of
Administration
Department of
Public Health Minister
Department of
Health Services
Bureau of
Curative
Services
15 county health
and social
welfare teams
Bureau of
Preventive
Services
National
programs (e.g.,
TB, EPI,
Malaria, etc
Department of
Planning, Policy
and
Development
Bureau of
Planning and
Policy
Bureau of Vital
Statistics
Department of
Administration
Bureau of Fiscal
Affairs
Bureau of
Administration
Department of
Public Health Minister Department of
Planning, Policy
and
Development Department of
Health Services and
Development Bureau of
Preventive
Services Bureau of
Planning and
Policy Bureau of Vital
Statistics Bureau of
Administration Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 2.1
Planning Objectives 2.1
Planning Objectives The overall objectives of the operational plan are to ensure a functional and resilient health
system that guarantees its population an effective and equitable health. The Operational
Plan however, is to enhance implementation of the investment plan for recovery and
resilience through coherent planning and budgeting at different levels of the health care
delivery and management systems. The specific objectives are as follows:
1. Identify and measure needs,
2. Map available resources (HR, Infrastructure and financial);
3. Establish baselines and set targets for the priority activities of the recovery of the
investment plan;
4. Prioritize activities for implementation of programs and delivery of services during
the fiscal year; and
5. Develop a harmonized and integrated Annual Plan in line with the investment plan
(2015-2021). 6 The specific objectives are as follows:
1. Identify and measure needs,
2. Map available resources (HR, Infrastructure and financial);
3. Establish baselines and set targets for the priority activities of the recovery of the
investment plan;
4. Prioritize activities for implementation of programs and delivery of services during
the fiscal year; and
5. Develop a harmonized and integrated Annual Plan in line with the investment plan
(2015-2021). 6 The specific objectives are as follows:
1. Identify and measure needs,
2. Map available resources (HR, Infrastructure and financial);
3. Establish baselines and set targets for the priority activities of the recovery of the
investment plan;
4. Prioritize activities for implementation of programs and delivery of services during
the fiscal year; and
5. Develop a harmonized and integrated Annual Plan in line with the investment plan
(2015-2021). 6. The expected results of the annual operational plan, 2016/2017
1. Annual health sector Plans for 15 counties,
2. A consolidated national integrated Annual Plan FY 2016/217
2 2
Pl
i
P The expected results of the annual operational plan, 2016/2017 1. Annual health sector Plans for 15 counties, 2. A consolidated national integrated Annual Plan FY 2016/217 2.2
Planning Process In 2014, the Ministry of Health with support from its development partners reviewed the
implementation of its 10-year national health plan and strategy (2011-2021) and developed
a post EVD response National Health Investment Plan and Strategy (2015-2021) aimed at
restoring health care services and to incrementally enhance resilience and health security
in the health services delivery system. One fiscal year of implementing this resilience
strategy elapsed at the close of the FY 2015/16. To operationalize the National Health Investment Plan (2015-2021) the Department of
Planning, Research & Human Development is mandated to specify the investment plan into
core and comprehensive operational plans at community, health facility, county, and
national levels. The process involves the provision of technical support for the development
of each county’s operational plan from the levels of the community to health facility
including the districts that culminated into the counties’ operational plans for the ensuing
fiscal year. The central level support by extension is also aimed at building the capacities of
counties in planning, budgeting, data analyses and service delivery target setting. 8 8 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Additionally, the county planning process is comprehensive and entails the review of
counties performance over the preceding fiscal year with an assessment of the previous
plan to inform the development of the ensuing fiscal year. Coupled with this, the process
also includes the customization of the planning tools, templates and developing national
targets while at the same time aligning activities and resources at the county level with all
implementing partners providing services at all levels within the counties (community,
facility district, and county). The process of producing the Ministry of Health’s consolidated operational plan follows its
planning cycle aligned with that of the Government of Liberia. It begins with the situational
analysis, followed by the priority settings, options appraisal, programming and monitoring
and evaluation. Moreover, it focuses on possible health system diagnosis, including
bottleneck analysis, the enabling environment at operational levels that follows the select
of appropriate target setting and integration of resources and activities mappings to ensure
the harmonization and alignment at county and national levels. The process follows a consultative and participatory process to ensure that all key
stakeholders are involved. 2.2
Planning Process The activities and steps that follow are conducted both at county
and national levels to facilitate the development of the consolidated national health
operational plan. Step 1: Compile and share operational planning tools, guides, and reference
documents: 9
a. All key documents to be used in the process are collected, compiled and distributed
to all relevant stakeholders at county and national levels;
b. Existing MoH operational planning guides and revised tools are adapted to respond
to the needs of the planning exercises. Step 2: Prepare and orientate technical working groups consisting of MoH
health managers and partners for the operational planning exercise:
a. Set national targets that aligned with the national health investment plan;
b. Conduct activity and resource mapping (local, Government, donors and partners);
c. Adapt planning tools and guides to inform planning at all levels;
d. Identify national and county teams and technical assistance to support the
operational planning process and plan orientation sessions
e. Hold central and county levels operational planning orientation sessions
Step 3: Assess and mobilize resources, both on and off budget at all levels of
the health system
Step 4: Develop operational plans at various levels (facility, district, county
and central levels) a. All key documents to be used in the process are collected, compiled and distributed
to all relevant stakeholders at county and national levels;
b. Existing MoH operational planning guides and revised tools are adapted to respond
to the needs of the planning exercises. Step 2: Prepare and orientate technical working groups consisting of MoH
health managers and partners for the operational planning exercise: Step 4: Develop operational plans at various levels (facility, district, county
and central levels) Step 4: Develop operational plans at various levels (facility, district, county
and central levels) Ministry of Health Consolidated Work Plan 2016/17
a. Update county situational analyses with improved data/information;
b. Establish targets for main service delivery components aligned with the national
indicative targets;
c. Identify priority strategies/actions to achieve targets;
d. Analyze health service delivery systemic components (human resources for health,
financing, infrastructure, supply chain) and set objectives;
e. Estimate resource requirements to implement planned priority interventions
f. Establish objectives, activities and funding
Step 5: Finalize, consolidate, and implement national health operational plan
(The consolidated plan guides the sector for the ensuing fiscal year)
a. Policy and Planning Unit collaborates with Central MoH Departments, Divisions,
Units and Programs to develop central level plans;
a. Operational plans get reviewed and feed backs provided until plans meet the
standards and deliverables;
b. The Policy and Planning Unit coordinates with technical team to consolidate national
health operational plan for dissemination and consequent implementation Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 a. Update county situational analyses with improved data/information;
b. Establish targets for main service delivery components aligned with the national
indicative targets;
c. Identify priority strategies/actions to achieve targets;
d. Analyze health service delivery systemic components (human resources for health,
financing, infrastructure, supply chain) and set objectives;
e. Estimate resource requirements to implement planned priority interventions
f. Establish objectives, activities and funding e. Estimate resource requirements to implement planned priority interventions
f. Establish objectives, activities and funding Step 5: Finalize, consolidate, and implement national health operational plan
(The consolidated plan guides the sector for the ensuing fiscal year) a. Operational plans get reviewed and feed backs provided until plans meet the
standards and deliverables; b. The Policy and Planning Unit coordinates with technical team to consolidate national
health operational plan for dissemination and consequent implementation 3.0 Situational Analysis and Performance FY 2015/16 3.1
Situational Analysis 3.1
Situational Analysis In 2015, the health sector led by the MOH developed a post EVD recovery plan and an
investment plan to restore basic health service and build a health care delivery system that
is resilient. The investment plan identified nine (9) critical health system areas for
investment that will drive the system to be more responsive, efficient and resilient. There are currently, 727 health facilities in Liberia of which 64% are public. These
facilities provide 71% of Liberians geographical access within 1 hour of walking distance or
within 5KM of walking distance. Though nationally, 29% of the population lacks physical
access to health care, 20% (3 out 15) of the counties have over 50% of their population
living beyond 5KM or 1hour walking distance within reach of a health facility. The 2013
Liberia Demographic and Health Survey results revealed that 65% of households do not
drive by cars but walk to health facilities in case of need. The 2016 Health Workforce
Census results show that 35% of health facilities lack electricity while 32% do not have
water supplies. The health workforce census documented 16,064 health workers of which 10,672 are
within the employed of the MOH. One-third of the public health workers are clinical and
their distribution is skewed towards urban areas. There acute shortage of critical health
workers such as physicians, midwives, lab technicians and specialist doctors (ie: surgeons,
pediatricians, psychiatrics, etc). 10 10 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Drugs and medical supplies is an essential component of the health system. However, this
area is characterized by irregular supply of drugs and medical supplies, limited number of
personnel, (pharmacists), to prescribe, quantify and dispense medicines to patients. Due to
inadequate budgetary allocation to procure and distribute drugs and medical supplies,
patients are often given prescription to buy unavailable essential medicines. Monitoring, supervision and mentoring has been weak and irregular. Furthermore, the
capacity of the HMIS is still inadequate for example data collection and timely reporting
from the service delivery sites have remained incomplete and less integrated. Information
use culture at the collection and intermediate aggregation levels has remained weak. The utilization of health services in Liberia is poor due to limited access and/or poor
quality of health services and patients and providers’ relationship. 3.1
Situational Analysis In 2015/16, utilization
rate was 1.06, which is only 21% of the optimal utilization rate (5.0). Antenatal care (4
visits) was 57.5% and only one-third of postnatal mothers received care. Half (51%) of the
pregnant women delivered in health facilities and 60% of children under age one were
fully immunized with all basic vaccines. 4.1
Health Infrastructure 4.1
Health Infrastructure Health infrastructure is the second priority pillar of the health sector investment plan that
allows access to health care. Expanding access to quality health care through the
construction, renovation and improvement of health facilities is critical for reaching the 1.2
million Liberians that are derived of basic health services. Investment in this pillar is
enormous and requires both domestic and external resources. Objective:
To increase physical access to the EPHS services. Objective:
To increase physical access to the EPHS services. To accomplish this objective, ten major activities have been earmarked for the fiscal year
2016/17 excluding the National Drug Service Warehouse and the health workers housing
unit projects. Below is the list of the 2016/17 planned activities: 3.2
Performance FY 2015/16 The health sector performance was appalling in 2014 due to the devastation caused by EVD
in the country. However, during the restoration and recovery stages of the investment plan
(2015), basic health services were restored and utilization increased. The sector has made
progress since the cessation of the EVD crisis, although few of the health service delivery
targets were not achieved. Table 3.1 presents service delivery summary accomplishments. On the other hand, HMIS report submission increased from 70% in 2014 to 78% in 2015/16
and timeliness improved from 41% to 57% over the same period. The proportion of health
facilities with basic utilities (ie: water and electricity) increased from 55% in 2014 to 66%
(HR census 2016). Table 3.1: Health Service Delivery indicators, targets and achievement in FY
2015/16
#
Health Service Delivery Indicators
Baseline
Targets
FY 2015/16
Achievement
1
%
of
children
under
1yr
fully
immunized
46%
64%
60%
2
Penta -3
63%
75%
65%
3
Measles
58%
64%
64%
4
ANC 1st Visits
61%
75%
75% 11
Table 3.1: Health Service Delivery indicators, targets and achievement in FY
2015/16
#
Health Service Delivery Indicators
Baseline
Targets
FY 2015/16
Achievement
1
%
of
children
under
1yr
fully
immunized
46%
64%
60%
2
Penta -3
63%
75%
65%
3
Measles
58%
64%
64%
4
ANC 1st Visits
61%
75%
75% Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 5
ANC 4th visits and more
46%
51%
58%
6
Institutional Delivery
40%
45%
52%
7
%
of
deliveries
by
Skilled
birth
attendants
39%
42%
51%
8
% of Pregnant women receiving second
dose IPTp
39%
44%
41%
9
% of postnatal mothers that received
PNC
29%
34%
33% The number of health facilities providing health services increased by 71 from 2014 – 2015
(656 in 2014 to 727 in 2015). Additionally, the public health workforce increased from
10,052 in 2014 to 10,673 in 2016. The number of health workers placed on Government of
Liberia payroll increased from 5,920 in 2014 to 7,214 in 2016. 4.0 Operational Plan 2016/17 Central Level Activities 1. Hold Consultative meeting with Civil Service Agency and Ministry of Finance and
Development Planning to establish a singular payroll system. 1. Hold Consultative meeting with Civil Service Agency and Ministry of Finance and
Development Planning to establish a singular payroll system. 2. MOH and MOFDP establish and validate mobile money accounts for salary
disbursement of salaries and manage queries and complaints. 3. MOH, CSA and technical assistance costs to develop remuneration packages options
analysis and proposal. 2. MOH and MOFDP establish and validate mobile money accounts for salary
disbursement of salaries and manage queries and complaints. 2. MOH and MOFDP establish and validate mobile money accounts for salary
disbursement of salaries and manage queries and complaints. 3. MOH, CSA and technical assistance costs to develop remuneration packages options
analysis and proposal. 3. MOH, CSA and technical assistance costs to develop remuneration packages options
analysis and proposal. 4. Explore remuneration package CHWs 4. Explore remuneration package CHWs Central Level Activities 12
Central Level Activities
1. Construct national drug depot (NDS Warehouse)
2. Assess and complete 16 abandoned clinics
3. Build 168 staff housing units
County Level Activities
1. Construct 29 primary clinics
2. Construct 47 maternal waiting homes
3. Construct 41 incinerators
4. Construct 34 triages 12
Central Level Activities
1. Construct national drug depot (NDS Warehouse)
2. Assess and complete 16 abandoned clinics
3. Build 168 staff housing units
County Level Activities
1. Construct 29 primary clinics
2. Construct 47 maternal waiting homes
3. Construct 41 incinerators
4. Construct 34 triages 12
Central Level Activities
1. Construct national drug depot (NDS Warehouse)
2. Assess and complete 16 abandoned clinics
3. Build 168 staff housing units
County Level Activities
1. Construct 29 primary clinics
2. Construct 47 maternal waiting homes
3. Construct 41 incinerators
4. Construct 34 triages Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 5. Fence 52 health facilities including Phebe Hospital 5. Fence 52 health facilities including Phebe Hospital
6. Construct 26 clinics laboratory
7. Build 17 district health teams’ offices
8. Renovate 20 health facilities County Level Activities Target: Ensure the availability of adequate health workforce with inclusive
capacity building, supervision and performance appraisal systems at county level. Recruit clinical and non-clinical staffers at facility, district and county levels 1. Recruit clinical and non-clinical staffers at facility, district and county levels
2. Conduct in-service infection prevention and control at county levels Conduct in-service infection prevention and control at county levels 2. Conduct in-service infection prevention and control at county levels 3. Ensure county, district and facility levels staffers supervision, appraisal monitoring
and performance 3. Ensure county, district and facility levels staffers supervision, appraisal monitoring
and performance 13 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17
4. Conduct evidence-based capacity building sessions for staffers at county, district
and facility levels
4.3
Health Care Financing
Objective:
Establish sustainable health financing systems that will ensure sufficient
and predictable resource generation, risk pooling mechanisms and strategic
purchasing of services. 4. 4.3
Health Care Financing 4.3
Health Care Financing Objective:
Establish sustainable health financing systems that will ensure sufficient
and predictable resource generation, risk pooling mechanisms and strategic
purchasing of services. Objective:
Establish sustainable health financing systems that will ensure sufficient
and predictable resource generation, risk pooling mechanisms and strategic
purchasing of services. Implementation of the below noted activities will require collaboration between all health
financing functions within the Ministry of Health and partners, specifically the Health
Financing Unit (Planning department), Office of Financial Management (Administration
department), the Performance-based Financing Unit (Health Services department), FARA
Management office and Pool Fund Management Office. Central Level Activities 14
Central Level Activities
1. Evaluate effectiveness of PBF in Liberia to date-full impact evaluation study
depending on available resources and feasibility-SWOT analysis
2. Finalize fiscal space analysis and disseminate by end of FY 2016/ 2017
3. Finalize resource allocation formula in consultation with stakeholders and apply to
FY 2017/18 budget
4. Train selected staffers of 15 CHTS in planning, costing and budgeting (activity based
costing)
5. Finalize legal proceedings and legislate Liberia Health Equity Fund (LHEF) Act
6. Conduct publicity and advocacy in 4 counties on Revolving Drug Fund (RDF) and
LHEF
County Level Activities
1. Support county level capacity building in financial management and auditing
2. Develop strategy to mobilize domestic resources and ensure financial sustainability
to support county level operational plan
3. Establish fixed assets management systems at county level
4. Provide short term financial management training for financial officers at county
level
4.4
Disease Prevention and Control
Objective:
Strengthen national core capacities in compliance with International Health
Regulation (2005) requirements capable to timely detect, investigate and
response to epidemic prone diseases and other health related events. Central Level Activities 14
County Level Activities
1. Support county level capacity building in financial management and auditing
2. Develop strategy to mobilize domestic resources and ensure financial sustainability
to support county level operational plan
3. Establish fixed assets management systems at county level
4. Provide short term financial management training for financial officers at county
level
4.4
Disease Prevention and Control
Objective:
Strengthen national core capacities in compliance with International Health
Regulation (2005) requirements capable to timely detect, investigate and
response to epidemic prone diseases and other health related events. Central Level Activities Central Level Activities Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 1. Train and deploy 2000 Community Health Assistants to implement CEBS
2. Finalize IDSR technical guidelines and other relevant operational tool (Reporting
forms, CEBS manual, health facility SOP on IDSR, Maternal death SOP, etc)
3. Reproduce and distribute training modules and simulation tools for PoE
4. Develop and disseminate monthly IDSR dash board
5. Produce and disseminate weekly and quarterly epidemiological bulletin
6. Establish functional IDSR Situational Awareness rooms in 16 EOCs (National and 15
Counties)
7. Conduct annual IDSR/IHR program implementation review meetings
8. Develop national risk communication plan for public health events
9. Central Level Activities Conduct external assessment of IHR core capacities
10. Conduct health risk and vulnerability assessment /mapping
11. Develop risk communication operational plan
12. Produce and air prevention messages using local radio (IDSR jingles, drama, etc)
13. Print and disseminate surveillance reporting forms to all levels
14. Print and disseminate Patient Care report form for 100 Ambulances
15. Print and disseminate IDSR Technical Guidelines to 1,000 health facilities
16. Print and disseminate CEBS manual to all 1,000 facilities and PoEs
17. Print and disseminate IDSR strategic Plan to all counties and district offices
18. Print and disseminate 100 Ambulance Guidelines
19. Print and disseminate National and County EPR Plans
20. Print and disseminate relevant SOPs, EMS Protocol and Guidelines
21. Print and disseminate Job Aides: CEBS, Clinical
22. Develop, reproduce, and disseminate IDSR priority disease media kits to media
houses. 1. Train and deploy 2000 Community Health Assistants to implement CEBS
2. Finalize IDSR technical guidelines and other relevant operational tool (Reporting
forms, CEBS manual, health facility SOP on IDSR, Maternal death SOP, etc)
3. Reproduce and distribute training modules and simulation tools for PoE
4. Develop and disseminate monthly IDSR dash board
5. Produce and disseminate weekly and quarterly epidemiological bulletin
6. Establish functional IDSR Situational Awareness rooms in 16 EOCs (National and 15
Counties)
7. Conduct annual IDSR/IHR program implementation review meetings
8. Develop national risk communication plan for public health events
9. Conduct external assessment of IHR core capacities
10. Conduct health risk and vulnerability assessment /mapping
11. Develop risk communication operational plan
12. Produce and air prevention messages using local radio (IDSR jingles, drama, etc)
13. Print and disseminate surveillance reporting forms to all levels
14. Print and disseminate Patient Care report form for 100 Ambulances
15. Print and disseminate IDSR Technical Guidelines to 1,000 health facilities
16. Print and disseminate CEBS manual to all 1,000 facilities and PoEs
17. Print and disseminate IDSR strategic Plan to all counties and district offices
18. Print and disseminate 100 Ambulance Guidelines
19. Print and disseminate National and County EPR Plans
20. Print and disseminate relevant SOPs, EMS Protocol and Guidelines
21. Print and disseminate Job Aides: CEBS, Clinical
22. Develop, reproduce, and disseminate IDSR priority disease media kits to media
houses. Specific Objective 1: Improve IDSR data and specimen management, biosafety regulations,
ensure interoperability with DSIS Activities 1. Provide logistical support to 3 laboratories
2. Central Level Activities Support RRT simulation activities across target levels
3. Support internal functionality of PH emergency operation centers to coordinate the
epidemic preparedness and response at national and county levels (Stationeries,
fuel, internet connectivity)
4. Develop national and county specific EPR and disease specific contingency plans
5. Work with partners to provide counties emergency contingency fund
6. Procure and deploy preparedness stocks in all counties for all priority public health
events: Cholera, EVD, Rabies, Bloody Diarrhea, Lassa Fever, Yellow Fever,
Meningitis 3. Support internal functionality of PH emergency operation centers to coordinate the
epidemic preparedness and response at national and county levels (Stationeries,
fuel, internet connectivity) 6. Procure and deploy preparedness stocks in all counties for all priority public health
events: Cholera, EVD, Rabies, Bloody Diarrhea, Lassa Fever, Yellow Fever,
Meningitis 7. Conduct midterm review and update of national and county specific epidemic
preparedness and response plans Specific Objective 3: Institutional support, capacity building, advocacy, and communication. Project coordination, fiduciary management, monitoring and evaluation, data generation,
and knowledge management Central Level Activities Validate, reproduce and disseminate national public health lab strategic plan (2017-
2022)
3. Procure IDSR lab. Sample collection kits for peripheral health facilities
4. Train and mentor IDSR focal persons at National, County, District and Health
facilities
5. Scale up IDSR Surveillance (eDEWS) implementation in 11 counties
6. Improve rapid sharing of public health and scientific information and data
7. Purchase and distribute reagents, lab supplies, and equipment Cholera, lassa Fever,
Yellow Fever, EVD, Measles, Shigella, Rabies, AFP (support transportation and
sample referral) 1. Provide logistical support to 3 laboratories
2. Validate, reproduce and disseminate national public health lab strategic plan (2017-
2022)
3. Procure IDSR lab. Sample collection kits for peripheral health facilities
4. Train and mentor IDSR focal persons at National, County, District and Health
facilities
5. Scale up IDSR Surveillance (eDEWS) implementation in 11 counties
6. Improve rapid sharing of public health and scientific information and data
7. Purchase and distribute reagents, lab supplies, and equipment Cholera, lassa Fever,
Yellow Fever, EVD, Measles, Shigella, Rabies, AFP (support transportation and
sample referral) 7. Purchase and distribute reagents, lab supplies, and equipment Cholera, lassa Fever,
Yellow Fever, EVD, Measles, Shigella, Rabies, AFP (support transportation and
sample referral) 15 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Specific Objective 2: Strengthen emergency preparedness and response and enhance cross-
sector coordination and collaboration 1. Establish, train and deploy Rapid Response Teams (RRT) at National, County
District Levels. 2. Support RRT simulation activities across target levels
3. Support internal functionality of PH emergency operation centers to coordinate
epidemic preparedness and response at national and county levels (Stationer
fuel, internet connectivity)
4. Develop national and county specific EPR and disease specific contingency plans
5. Work with partners to provide counties emergency contingency fund
6. Procure and deploy preparedness stocks in all counties for all priority public he
events: Cholera, EVD, Rabies, Bloody Diarrhea, Lassa Fever, Yellow Fe
Meningitis
7. Conduct midterm review and update of national and county specific epide
preparedness and response plans
Specific Objective 3: Institutional support, capacity building, advocacy, and communicat
Project coordination, fiduciary management, monitoring and evaluation, data generat
and knowledge management
C
t
l L
l A ti iti 1. Establish, train and deploy Rapid Response Teams (RRT) at National, County and
District Levels. 2. Support RRT simulation activities across target levels 2. Central Level Activities Central Level Activities
1. Provide credentialing and capacity building in surveillance staff, including Field
Epidemiology
Laboratory
Training
Programs
(FELTP-Frontline:
3
months;
intermediate- 9 moths; and Advance-masters)), and PhD programs targeting
Hospital, CHOs, National staff, CSO, MOA,
2. Work with the pre-service institutions (Medical and para-medical) to build HR
instructional capacity and strengthen curriculum
3. Train DSOs in Dead Body Swabbing and safe Specimen collection, packaging and
transport
4. Support Short Course (International) in Emergency Management/IMS for All
Incident Command Managers at national and county levels
5. Train 3 national, 15 Counties and 92 District RRTs
6. Train 15 county health promotion focal persons in Risk/outbreak communication
7. Cross border coordination meeting at all levels
8. Provide incentives for DSO
County Level Activities
1. Train traditional healers in community case definition of priority diseases
2. Provide refresher training on IDSR for health facility staff
3. Conduct weekly surveillance coordination meeting
4. Conduct RRT refresher training for surveillance Team
5. Refresher training to all HCW on IPC standard precautions 1. Provide credentialing and capacity building in surveillance staff, including Field
Epidemiology
Laboratory
Training
Programs
(FELTP-Frontline:
3
months;
intermediate- 9 moths; and Advance-masters)), and PhD programs targeting
Hospital, CHOs, National staff, CSO, MOA, County Level Activities 16 Ministry of Health Consolidated Work Plan 2016/17
6. Conduct quarterly mentorship on IPC standard precautions
7. Supply facilities with IEC/BCC Materials
8. To conduct health talk using available IEC/BCC materials
9. Conduct epidemiological investigation of rumors/suspected disease of epidemic
potential
10. Conduct EDEWs Supervision
11. Establish data storage for surveillance
12. Continue Community Events Based Surveillance
13. Ensure the supply of PPE for EPR purpose
14. Establish facilities EPR team and conduct in-service training
15. Liaise with County Health Team to provide the updated EPR plan to the district and
health facilities. 16. Work with county pharmacist to supply and maintain the minimum stock of EPR
supply at all levels
17. Work with partner to provide logistic supports (fuel & stationaries) for district EPR
activities. 18. Provide communication equipment (phones, VHF Radio etc) for surveillance
activities
19. Review IDSR plan every 6 months to evaluate and improve the performance of
surveillance and response systems and provide feedback within and across levels of
the health system
4.5
Health Service Delivery and Quality of Care
4.5.1 Improve Health of Mothers, and Newborns services
Objective:
To improve availability and readiness of quality of and demand for
maternal, newborn, adolescent and reproductive health services to
improve access and coverage. Target 1: Improve coverage of family planning with couple year of protection
in all the 15 counties. Central Level Activities
1. Conduct mapping of community based family planning distributors
2. Train/refresh CBD to scale up distribution points
3. Provide RH commodity storage boxes (wooden) for Community Based Distributors
4. Conduct Post Partum Family Planning Training
5. Conduct training/refresher to strengthen and scale up EPI/FP Integration in all
fifteen counties
6. Support provision of IUCD insertion kits to health care facilities
7. Provide financial support for NDS for Quarterly supply of RH commodities Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 6. Conduct quarterly mentorship on IPC standard precautions
7. Supply facilities with IEC/BCC Materials
8. To conduct health talk using available IEC/BCC materials
9. Conduct epidemiological investigation of rumors/suspected disease of epidemic
potential
10. Conduct EDEWs Supervision
11. Establish data storage for surveillance
12. Continue Community Events Based Surveillance
13. Ensure the supply of PPE for EPR purpose
14. Establish facilities EPR team and conduct in-service training
15. Liaise with County Health Team to provide the updated EPR plan to the district and
health facilities. 16. County Level Activities Work with county pharmacist to supply and maintain the minimum stock of EPR
supply at all levels
17. Work with partner to provide logistic supports (fuel & stationaries) for district EPR
activities. 18. Provide communication equipment (phones, VHF Radio etc) for surveillance
activities
19. Review IDSR plan every 6 months to evaluate and improve the performance of
surveillance and response systems and provide feedback within and across levels of
the health system 4.5.1 Improve Health of Mothers, and Newborns services
Objective:
To improve availability and readiness of quality of and demand for
maternal, newborn, adolescent and reproductive health services to
improve access and coverage. Target 1: Improve coverage of family planning with couple year of protection
in all the 15 counties. Central Level Activities
1. Conduct mapping of community based family planning distributors
2. Train/refresh CBD to scale up distribution points
3. Provide RH commodity storage boxes (wooden) for Community Based Distributors
4. Conduct Post Partum Family Planning Training
5. Conduct training/refresher to strengthen and scale up EPI/FP Integration in all
fifteen counties
6. Support provision of IUCD insertion kits to health care facilities
7. Provide financial support for NDS for Quarterly supply of RH commodities 4.5.1 Improve Health of Mothers, and Newborns services
Objective:
To improve availability and readiness of quality of and demand for
maternal, newborn, adolescent and reproductive health services to
improve access and coverage. j
,
,
p
improve access and coverage. Target 1: Improve coverage of family planning with couple year of protection
in all the 15 counties. Central Level Activities Central Level Activities 1. Conduct refresher training and TOT in BLSS/EmONC quarterly
2. Review and consolidate 15 County RH supervisors and partners work plans and
consolidate FY 2016/17
3. Ensure a sufficient and reliable supply of safe blood for CEMoNC
4. Build new clinics and upgrade select clinics to health centers to improve access to
care. 5. Deploy community base certified midwives to underserved areas to care for women
without access to facilities. Target 2: Coverage to basic and comprehensive Emergency Obstetric and
Neonatal Care (EmONC) and essential Maternal and Newborn care increased in
nine counties (health centers and hospitals) Target 2: Coverage to basic and comprehensive Emergency Obstetric and
Neonatal Care (EmONC) and essential Maternal and Newborn care increased in
nine counties (health centers and hospitals) Central Level Activities Provide financial support for NDS for Quarterly supply of RH commodities 17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 8. Provide support for printing of revised family planning strategy
9. Review, print and disseminate to operationalize the family planning road map
10. Support development of messages, radio talk show, jingles/dramas to educate on the
side-effects of family planning commodity to reduce myths
11. Provide vehicle to support RMNCAH supportive
12. Provide maintenance for vehicles
13. Support 1 TA for RH commodities quantification
14. Procure 1 Laptop for data management (PPMR) at FHD level
15. Print and disseminate Mother and child health cards to the 15 counties
16. Conduct quarterly mentoring in all counties
County level Activities
1. Provide motivational package for gCHVs providing CBD services
2. Provide logistical support for mentors to implement FP activities at health facility
and community levels
3. Establish/reactivate condom distribution points in 300 communities
4. Implement providers initiated counseling on family planning in all routine health
services at all levels
5. Identify and train CBD to pilot community Depo/injectable administration in three
counties
6. Integrated family planning, EPI, and MCH outreach
Target 2: Coverage to basic and comprehensive Emergency Obstetric and
Neonatal Care (EmONC) and essential Maternal and Newborn care increased in
nine counties (health centers and hospitals)
Central Level Activities 8. Provide support for printing of revised family planning strategy
9. Review, print and disseminate to operationalize the family planning road map
10. Support development of messages, radio talk show, jingles/dramas to educate on the
side-effects of family planning commodity to reduce myths
11. Provide vehicle to support RMNCAH supportive
12. Provide maintenance for vehicles
13. Support 1 TA for RH commodities quantification
14. Procure 1 Laptop for data management (PPMR) at FHD level
15. Print and disseminate Mother and child health cards to the 15 counties
16. Conduct quarterly mentoring in all counties County level Activities 1. The FHD will review, revise and print the midwifery constitution and disseminate
copies to all facilities and stakeholders. 2. Validation, printing and dissemination of standards for midwifery practice will be
done including monitoring of its use. 2. Validation, printing and dissemination of standards for midwifery practice will be
done including monitoring of its use. 18 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 3. FHD will provide support for strengthening implementation of safe delivery services
under the National Health Policy & Plan related to maternal, newborn and child
health through promotion of delivery kits to facilities. 3. FHD will provide support for strengthening implementation of safe delivery services
under the National Health Policy & Plan related to maternal, newborn and child
health through promotion of delivery kits to facilities. 4. Conduct Emergency Obstetrics and Neonatal Care (EmONC) training - basic and
comprehensive to nurses and midwives in 6 counties. Central Level Activities Central Level Activities
1. Provide program management, strengthened coordination and collaboration
2. Provide on-site training in adherence counseling skills and ensure acceptance
attitude for knowing your HIV status for pregnant women. 3. Train service providers on Option B+, TTMs/TBA, mother peers, and exposed infants
for PCR Target 6: Strengthen national capacity to address gender-based violence using
a multi-sectoral approach and the provision of high quality services to
survivors. Target 6: Strengthen national capacity to address gender-based violence using
a multi-sectoral approach and the provision of high quality services to
survivors. Central Level Activities
1. Support to the 12 existing One Stop Centers (OSC)
2. Procure and distribute Rape treatment (PEP/KIT3)
3. Provide transportation incentive for OSC Personnel
4. Refurbish two additional OSCs in two counties (Lofa and Nimba)
5. Train clinical staff in the management of GBV/SGBV
6. Produce medical reporting form-10000 copies
7. Supervise and mentor staff at all OSCs
County level Activity
1. Implement, supervise and monitor performance
Target 7: Prevention, management and control of PMTCT strengthened at
national and county level
Central Level Activities
1. Provide program management, strengthened coordination and collaboration
2. Provide on-site training in adherence counseling skills and ensure acceptance
attitude for knowing your HIV status for pregnant women. 3. Train service providers on Option B+, TTMs/TBA, mother peers, and exposed infants
for PCR Central Level Activities
1. Support to the 12 existing One Stop Centers (OSC)
2. Procure and distribute Rape treatment (PEP/KIT3)
3. Provide transportation incentive for OSC Personnel
4. Refurbish two additional OSCs in two counties (Lofa and Nimba)
5. Train clinical staff in the management of GBV/SGBV
6. Produce medical reporting form-10000 copies
7. Supervise and mentor staff at all OSCs
County level Activity
1. Implement, supervise and monitor performance
Target 7: Prevention, management and control of PMTCT strengthened at
national and county level County Level Activities 19
County Level Activities
1. Provide HIV Care, Treatment and Support services (Antiretroviral Therapy - ART)
for HIV positive pregnant women and children
2. Strengthen and provide Mother to mother peer support services (preventing lost to
follow-up) - father and adolescent
3. Provide supportive supervision, on-site mentoring and ensure data accuracy
4. Strengthen community based organizations and structures to provide community
awareness, sensitization and mobilization on eMTCT/pediatric and adolescent HIV
Target 8: Improving health and education with emphasis on reduced maternal
and child mortality and education achievement services and as well enhanced y
1. Provide HIV Care, Treatment and Support services (Antiretroviral Therapy - ART)
for HIV positive pregnant women and children
2. Strengthen and provide Mother to mother peer support services (preventing lost to
follow-up) - father and adolescent
3. Provide supportive supervision, on-site mentoring and ensure data accuracy
4. Strengthen community based organizations and structures to provide community
awareness, sensitization and mobilization on eMTCT/pediatric and adolescent HIV Target 8: Improving health and education with emphasis on reduced maternal
and child mortality and education achievement services and as well enhanced Target 8: Improving health and education with emphasis on reduced maternal
and child mortality and education achievement services and as well enhanced 19 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17
national capacity for treatment and social reintegration of obstetric fistula. Central Level Activities
1. Produce 25 copies magazines of fistula survivors success stories
2. Provide support for surgical outreach in hard-to-reach counties
3. Maintain fistula facilities and services (including patients feeding, laundry services,
cleaning)
4. Provide salary payment for project staff
5. Provide support to operational activities
County Level Activity
1. Implement robust mobilization campaigns in 3 hard-to-reach counties
4.5.2 Improve Child Health
Objective:
To improve availability and readiness of child health services to
improve access and coverage
Target 1: Reviewed and revised the national Child Survival Strategy (2008-
2011)
Central Level Activities
1. Undertake comprehensive assessment of process actors and context
2. Define goals and priorities
3. Validate a national policy
Target 2: Minimum 75% of the monthly target of children under 1 year in all
counties vaccinated by August 2015 (for all antigens to achieve 85%),
Central Level Activities
1. Provide quarterly financial support to 534 HF for outreach Vaccination Teams for 12
months @ US50.00
2. Conduct refresher training on immunization in practice
3. County Level activities y
1. Periodic Intensification of Routine Immunization (PIRI), Round 3
2. Continue regular immunization with outreach services
3. Training for HF personnel on immunization in practice (IIP)
4. Conduct quarterly monitoring and supportive supervision to district and HFs
(provide US$150/month for 12 months), y
1. Periodic Intensification of Routine Immunization (PIRI), Round 3
2. Continue regular immunization with outreach services
3. Training for HF personnel on immunization in practice (IIP)
4. Conduct quarterly monitoring and supportive supervision to district and HFs
(provide US$150/month for 12 months), Target 3: At least 85% of all 15 counties will attain all EPI surveillance
indicators by December 2016 County Level Activities TOT for 45 counties participants, 15 national, 6 facilitators
4. Conduct quarterly periodic intensification of routine immunization (PIRI) in all
counties
5. Conduct national micro-planning exercise
6. Implement Urban Immunization Strategy
7. Support social mobilization and communication for urban immunization with
Montserrado county
8. Support the development/production of messages
9. Procure 100 motorbikes for integrated outreach services national capacity for treatment and social reintegration of obstetric fistula. Central Level Activities
1. Produce 25 copies magazines of fistula survivors success stories
2. Provide support for surgical outreach in hard-to-reach counties
3. Maintain fistula facilities and services (including patients feeding, laundry services,
cleaning)
4. Provide salary payment for project staff
5. Provide support to operational activities
County Level Activity
1. Implement robust mobilization campaigns in 3 hard-to-reach counties
4.5.2 Improve Child Health
Objective:
To improve availability and readiness of child health services to
improve access and coverage
Target 1: Reviewed and revised the national Child Survival Strategy (2008-
2011)
Central Level Activities
1. Undertake comprehensive assessment of process actors and context
2. Define goals and priorities
3. Validate a national policy
Target 2: Minimum 75% of the monthly target of children under 1 year in all
counties vaccinated by August 2015 (for all antigens to achieve 85%),
Central Level Activities
1. Provide quarterly financial support to 534 HF for outreach Vaccination Teams for 12
months @ US50.00
2. Conduct refresher training on immunization in practice
3. TOT for 45 counties participants, 15 national, 6 facilitators
4. Conduct quarterly periodic intensification of routine immunization (PIRI) in all
counties
5. Conduct national micro-planning exercise
6. Implement Urban Immunization Strategy
7. Support social mobilization and communication for urban immunization with
Montserrado county
8. Support the development/production of messages
9. Procure 100 motorbikes for integrated outreach services 20 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 10. Conduct quarterly cold chain and vaccine management monitoring & supervision
visits
11. Produce, printing and distribution of EPI Monitoring tools (i.e. child health cards,
ledgers, tally booklets, monitoring charts, summary forms and job-aids)
12. Train CCO and CSFP on equipment maintenance and vaccine management
13. Procure bundle vaccines and other supplies. 14. Distribute bundle vaccines to county depots. Central Level Activities Improve documentation and timely reporting to the central level
4. Enhance stakeholder coordination at county and below, on monthly and quarterly
bases
Target 5: At least 95% of all 554 HFs have bundle vaccines and supplies
available with functional cold chain equipment at all times
Central Level Activities
1. Forecast and Procure bundle vaccines
2. Expand cold chain thru the procurement and installation of additional solar direct
drive (SDD), WICR, refrigerators, cold boxes, etc. Target 6: Central and 15 county program management improved
Central Level Activities
1. Build Capacity of county and health facility EPI Management Team
2. Conduct mid-term and end of period programme evaluation and planning Ministry of Health Consolidated Work Plan 2016/17 County level Activities
1. Intensify and strengthen AFP surveillance nationwide
2. Conduct EPI biannual surveillance supervisory visits to priority sites
3. Conduct regular quarterly cross border meeting on immunization activities
Objective:
To ensure that at least 90% of all EPI data (i.e. Absolute numbers &
Coverage rates) from health facilities are verified by the end of the year. Target 4: Immunization data quality improved from 85% to 95% completion,
by the end 2015. Central Level Activities
1. Train health workers (CHDD, CSFP, Data Manager & CCO) on District Vaccination
Data Monitoring Tool (DVD-MT) from all counties,
2. Conduct quarterly data harmonization and validation. 3. Reinforce and recognize good practices publically
County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Improve documentation and timely reporting to the central level
4. Enhance stakeholder coordination at county and below, on monthly and quarterly
bases
Target 5: At least 95% of all 554 HFs have bundle vaccines and supplies
available with functional cold chain equipment at all times
Central Level Activities
1. Forecast and Procure bundle vaccines
2. Expand cold chain thru the procurement and installation of additional solar direct
drive (SDD), WICR, refrigerators, cold boxes, etc. Target 6: Central and 15 county program management improved
Central Level Activities
1. Build Capacity of county and health facility EPI Management Team
2. Conduct mid-term and end of period programme evaluation and planning County level Activities County level Activities Coverage rates) from health facilities are verified by the end of the year. Target 4: Immunization data quality improved from 85% to 95% completion,
by the end 2015. Central Level Activities
1. Central Level Activities 1. Provide regular logistics support and equipment for the conduct of active
surveillance activities at counties and districts,
2. Support outbreak investigation and response
3. Procure data management and ICT equipment (e.g. Lap top, back-up, antivirus, etc)
for County and National levels
4. Provide financial support for NCC, NEC, and NPEC activities
5. Conduct quarterly surveillance visits to rotavirus sentinel site at Redemption
Hospital
6. Develop immunization supply chain (iSCM) SOPs
7. Procure and install continuous temperature monitoring device at EPI Regional Cold
Stores
8. Conduct temperature mapping study of cold/freezer rooms at national and regional
stores; and temperature monitoring study in vaccine supply chain in accordance
with WHO protocol. 9. Conduct cold chain inventory assessment and develop equipment replacement plan
10. Procure fuel for County Generators
11. Procure fuel for County Vehicles for vaccine distribution
12. Provide financial support to procure immunization supplies and spare parts for
motorcycles maintenance for district & HF 150 @ $20/month
13. Support running and maintenance of central and 2 regional cold room
14. Procure one 4 X 4 utility truck for delivery of assorted immunization supplies and
one refrigerated truck for vaccine transportation; three Toyota 4x4 pick-up and one
4x4 Nissan Jeep 2. Provide financial support to procure immunization supplies and spare parts for
motorcycles maintenance for district & HF 150 @ $20/month 14. Procure one 4 X 4 utility truck for delivery of assorted immunization supplies and
one refrigerated truck for vaccine transportation; three Toyota 4x4 pick-up and one
4x4 Nissan Jeep 21 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17
County level Activities
1. Intensify and strengthen AFP surveillance nationwide
2. Conduct EPI biannual surveillance supervisory visits to priority sites
3. Conduct regular quarterly cross border meeting on immunization activities
Objective:
To ensure that at least 90% of all EPI data (i.e. Absolute numbers &
Coverage rates) from health facilities are verified by the end of the year. Target 4: Immunization data quality improved from 85% to 95% completion,
by the end 2015. Central Level Activities
1. Train health workers (CHDD, CSFP, Data Manager & CCO) on District Vaccination
Data Monitoring Tool (DVD-MT) from all counties,
2. Conduct quarterly data harmonization and validation. 3. Reinforce and recognize good practices publically
County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Central Level Activities Train health workers (CHDD, CSFP, Data Manager & CCO) on District Vaccination
Data Monitoring Tool (DVD-MT) from all counties,
2. Conduct quarterly data harmonization and validation. 3. Reinforce and recognize good practices publically
County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Improve documentation and timely reporting to the central level
4. Enhance stakeholder coordination at county and below, on monthly and quarterly
bases
Target 5: At least 95% of all 554 HFs have bundle vaccines and supplies
available with functional cold chain equipment at all times
Central Level Activities
1. Forecast and Procure bundle vaccines
2. Expand cold chain thru the procurement and installation of additional solar direct
drive (SDD), WICR, refrigerators, cold boxes, etc. Target 6: Central and 15 county program management improved
Central Level Activities
1. Build Capacity of county and health facility EPI Management Team
2. Conduct mid-term and end of period programme evaluation and planning
County level Activities
1. Train community and health facility managers on basics of health services planning
and monitoring County level Activities Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 2. Undertake regular supportive supervision Objective:
Strengthen community based health services to improve access and coverage
of essential services for communities and families that reside beyond 5 KM. Target 1: Establish support systems to strengthen implementation of quality
services (HR, M&E, supply chain and operations, supervision, performance and
quality improvement) Central Level Activities
1. Develop, define, standardize and validate minimum set of indicators (including
community births and deaths) in collaboration with programs
2. Develop, field test and finalize data collection and reporting tools for CHAs and
CHSSs
3. Develop CBIS database and modules in affiliated systems (iHRIS, LMIS, DHIS2, etc)
4. Develop SOPs for CBIS data management (data reporting, analysis, use and
feedback) and integration with other systems Including CEBS, LMIS, iHRIS, etc)
5. Hold validation workshop for CBIS tools and SOPs
6. Print and supply monitoring & evaluation materials (including CBIS SOPs, indicator
guidelines/definitions, data collection and reporting forms to counties and health
facilities)
7. Conduct training and roll-out of CBIS at in all 15 counties
8. Carry out bi-annual joint coaching & mentoring visits to CHA implementation sites
Target 2: Recruitment & Training of 2000 CHAs, 300 CHSSs and 100 Master
Trainers
Central Level Activities
1. Develop and validate training modules and guideline
2. Facilitate and hire master trainers
3. Train HSS from the 15 counties
4. Train CHAs
5. Deploy and manage work of CHAs
6. Develop supervision checklist and train CHSS
7. Print curriculum, training SOPs, job aids, tools, and training materials Central Level Activities 1. Develop, define, standardize and validate minimum set of indicators (including
community births and deaths) in collaboration with programs 2. Develop, field test and finalize data collection and reporting tools for CHAs and
CHSSs 3. Develop CBIS database and modules in affiliated systems (iHRIS, LMIS, DHIS2, etc)
4. Develop SOPs for CBIS data management (data reporting, analysis, use and
feedback) and integration with other systems Including CEBS, LMIS, iHRIS, etc)
5. Hold validation workshop for CBIS tools and SOPs 6. Print and supply monitoring & evaluation materials (including CBIS SOPs, indicator
guidelines/definitions, data collection and reporting forms to counties and health
facilities) 8. Carry out bi-annual joint coaching & mentoring visits to CHA implementation sites 23 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Central Level Activities Central Level Activities
1. Launch Community Health Assistant program at National Level
2. Develop a dissemination guide and fact sheet for dissemination at county and local
level
3. Print policy, strategic plan, implementation guide for dissemination,
4. Conduct Dissemination Workshops & Tool kit orientation (implementation guide,
TORs, recruitment guidelines, etc.) for Revised Community Health Services Policy &
Strategic Plan AND Launch CHA Program in all counties
5. Establish and hold monthly coordination meeting for Community Health Partners,
Chaired by Director of CHSD
6. Organize & host annual review of the community health program
County level Activities
1. Establish county and health facility coordination mechanisms among implementing
entities,
2. Strengthen health facility boards
3. Support CHAs and health facility health workers integrate advocacy and social
mobilization,
4. Supervise implementation at community and health facility
5. Undertake bimonthly monitoring and review at health facility and community level
6. Organize biannual review at county level
4.5.4 Improve Coverage of Health Related Nutrition Services
Objective:
Strengthen integration and analysis of nutrition program information and
surveillance system. Central Level Activities 1. Launch Community Health Assistant program at National Level 2. Develop a dissemination guide and fact sheet for dissemination at county and local
level 2. Develop a dissemination guide and fact sheet for dissemination at county and local
level 3. Print policy, strategic plan, implementation guide for dissemination,
4. Conduct Dissemination Workshops & Tool kit orientation (implementation guide,
TORs, recruitment guidelines, etc.) for Revised Community Health Services Policy &
Strategic Plan AND Launch CHA Program in all counties
5. Establish and hold monthly coordination meeting for Community Health Partners,
Chaired by Director of CHSD Target 3: Strengthen national advocacy, coordination, partnerships, and
leadership at all levels Target 3: Strengthen national advocacy, coordination, partnerships, and
leadership at all levels Central Level Activities Central Level Activities
1. Collect and collate monthly nutrition information not included in the current HMIS
using MoH endorsed nutrition reporting tools
2. Organize monthly forums to discuss and address bottlenecks identified in the
nutrition program
3. Support development and dissemination of quarterly nutrition dashboard County level Activities 4.5.4 Improve Coverage of Health Related Nutrition Services
Objective:
Strengthen integration and analysis of nutrition program information and
surveillance system. Central Level Activities
1. Collect and collate monthly nutrition information not included in the current HMIS
using MoH endorsed nutrition reporting tools 4.5.4 Improve Coverage of Health Related Nutrition Services
Objective:
Strengthen integration and analysis of nutrition program information and
surveillance system. 4.5.5 Improve Mental Health Services 4.5.5 Improve Mental Health Services 4.5.5 Improve Mental Health Services
Objective 1:
Enhance the capacity of the National Health Coordination unit, thereby
improving mechanisms for coordination, collaboration and monitoring all
mental health related activities by June 2017. Objective 1:
Enhance the capacity of the National Health Coordination unit, thereby
improving mechanisms for coordination, collaboration and monitoring all
mental health related activities by June 2017. Target 1: Capacity of the National Health Coordination Unit built
Thereby improving mechanisms for coordination, collaboration and monitoring a Target 1: Capacity of the National Health Coordination Unit built
Thereby improving mechanisms for coordination, collaboration and monitoring all mental
health related activities. Thereby improving mechanisms for coordination, collaboration and monitoring all mental
health related activities. County Level Activities 1. Print the updated National Mental Health policy & Strategic Plan to include
implementation framework and county specific plan. 2. Ensure the provision of additional one 4X4 vehicle to capacitate mental health unit
to conduct supervision and other mental health activities
3. Conduct quarterly National Mental Health & Psychosocial Coordination Meeting
4. Support to EVD survivors to be able to communicate the challenges and tell their
stories through individual advocacy, and participatory research through photo voice
programs
5. To
conduct
rapid
situation
analysis
&
strengthen
Mental
health
data
reporting/recording systems integrated with existing HMIS and other information
management systems as well as, designing software/ database for Mental Health
Information system
Objective 2:
Increase the clinical capacity of Mental Health Professional 1. Print the updated National Mental Health policy & Strategic Plan to include
implementation framework and county specific plan. 2. Ensure the provision of additional one 4X4 vehicle to capacitate mental health unit
to conduct supervision and other mental health activities
3. Conduct quarterly National Mental Health & Psychosocial Coordination Meeting
4. Support to EVD survivors to be able to communicate the challenges and tell their
stories through individual advocacy, and participatory research through photo voice
programs
5. To
conduct
rapid
situation
analysis
&
strengthen
Mental
health
data
reporting/recording systems integrated with existing HMIS and other information
management systems as well as, designing software/ database for Mental Health
Information system Objective 2:
Increase the clinical capacity of Mental Health Professional
Target 2: Additional 1500 PHC workers trained
Central Level Activities
1. Revise the National Mental Health Policy and update the strategic plan with co
specification of county level structure and an overall timeframe
2. Ensure that all county referral hospitals are well prepared with trained PH
workers with mhGAP-IG materials to host mentally ill patients
3. Print and distribute 500 copies of the validated mhGAP Materials and launch
4. Institute supervision mechanisms to follow up on mhGAP trainees
5. Increase in the # of Mental Health Clinicians
County level Activities
1. Conduct in service training at the primary care level for additional 1500 PH
workers using mhGAP materials by Dec. 15, 2016
2. Conduct training for new cadre of the mental Health Clinicians (Child & Adolescen Target 2: Additional 1500 PHC workers trained Central Level Activities
1. Revise the National Mental Health Policy and update the strategic plan with cost,
specification of county level structure and an overall timeframe
2. Ensure that all county referral hospitals are well prepared with trained PHC
workers with mhGAP-IG materials to host mentally ill patients
3. Print and distribute 500 copies of the validated mhGAP Materials and launch
4. Institute supervision mechanisms to follow up on mhGAP trainees
5. Increase in the # of Mental Health Clinicians Central Level Activities
1. Revise the National Mental Health Policy and update the strategic plan with cost,
specification of county level structure and an overall timeframe
2. Ensure that all county referral hospitals are well prepared with trained PHC
workers with mhGAP-IG materials to host mentally ill patients
3. Print and distribute 500 copies of the validated mhGAP Materials and launch
4. Institute supervision mechanisms to follow up on mhGAP trainees
5. Increase in the # of Mental Health Clinicians County Level Activity y
y
1. Organize monthly forums to discuss and address bottlenecks identified in the
nutrition program 1. Organize monthly forums to discuss and address bottlenecks identified in the
nutrition program 24 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 County level Activities 1. Conduct in service training at the primary care level for additional 1500 PHC
workers using mhGAP materials by Dec. 15, 2016 1. Conduct in service training at the primary care level for additional 1500 PHC
workers using mhGAP materials by Dec. 15, 2016 1. Conduct in service training at the primary care level for additional 1500 PHC
workers using mhGAP materials by Dec. 15, 2016 onduct training for new cadre of the mental Health Clinicians (Child & Adolesc 25 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Objective 3:
Advocacy and awareness of mental Health issues through celebration of Mental
Health illness reduced misconceptions, common fears to reduce stigma and
discrimination for people living with mental illnesses, substance use disorders
and epilepsy. County Level Activities 1. Celebrate key mental health days such as World Mental Health Day, World Epilepsy
Day, World Drug Day, World Children Mental Health Day and work with resources in
the community (e.g. CHWs, religious leaders & traditional healers) to raise
awareness on mental, neurological and substance abuse disorders and to identify
and refer clients 1. Celebrate key mental health days such as World Mental Health Day, World Epilepsy
Day, World Drug Day, World Children Mental Health Day and work with resources in
the community (e.g. CHWs, religious leaders & traditional healers) to raise
awareness on mental, neurological and substance abuse disorders and to identify
and refer clients 1. Celebrate key mental health days such as World Mental Health Day, World Epilepsy
Day, World Drug Day, World Children Mental Health Day and work with resources in
the community (e.g. CHWs, religious leaders & traditional healers) to raise
awareness on mental, neurological and substance abuse disorders and to identify
and refer clients
2. Mental Health Unit (MHU) to undertake mental health promotion activities
3. MHU to celebrate International Children MH Day
4. MHU and DEA to celebrate World Drug Day
5. Celebrate International Mental Health Day and work with structures in community
(eg CHWs, religious leaders & traditional healers) to raise awareness on mental,
neurological and substance abuse disorders and to identify and refer clients
6. Conduct additional community healing dialogue for 1,500 EVD affected survivors,
family and community Members
7. Raise awareness on mental illnesses, substance use disorders and epilepsy (Radio
Advent, UNMIL & ELBS)
Objective 4:
To improve the accessibility and availability of quality mental health treatme
and services including epilepsy and substance abuse disorders management
all persons at all levels of the health care provision. Ensure the Improvemen
accessibility, availability, distribution and utilization of cost effecti
psychotropic medications. 2. Mental Health Unit (MHU) to undertake mental health promotion activities
3. MHU to celebrate International Children MH Day
4. MHU and DEA to celebrate World Drug Day
5. Celebrate International Mental Health Day and work with structures in community
(eg CHWs, religious leaders & traditional healers) to raise awareness on mental,
neurological and substance abuse disorders and to identify and refer clients
6. Conduct additional community healing dialogue for 1,500 EVD affected survivors,
family and community Members
7. Target 4: Accessibility and availability of quality mental health treatment and
services improved This should include improvement of epilepsy and substance abuse disorders management of
all persons at all levels of the health care provision. County Level Activities Raise awareness on mental illnesses, substance use disorders and epilepsy (Radio
Advent, UNMIL & ELBS) Objective 4:
To improve the accessibility and availability of quality mental health treatment
and services including epilepsy and substance abuse disorders management of
all persons at all levels of the health care provision. Ensure the Improvement,
accessibility, availability, distribution and utilization of cost effective
psychotropic medications. County Level Activities 1. Advocate for uninterrupted drug supply chain of essential medicines for mental and
neurological disorders to be captured on the National Essential Medicines List
2. Conduct regular supportive supervision and monitor available stocks of psychotropic
medication and mental health services at each level of service provision 1. Advocate for uninterrupted drug supply chain of essential medicines for mental and
neurological disorders to be captured on the National Essential Medicines List 1. Advocate for uninterrupted drug supply chain of essential medicines for mental and
neurological disorders to be captured on the National Essential Medicines List 2. Conduct regular supportive supervision and monitor available stocks of psychotropic
medication and mental health services at each level of service provision 2. Conduct regular supportive supervision and monitor available stocks of psychotropic
medication and mental health services at each level of service provision 26 Ministry of Health Consolidated Work Plan 2016/17
3. MHU to work with Supply Chain Management Unit (SCMU) with forecasting and
quantification tools for mental and neurological health care products at national and
county levels. 4. MHU to work with SCMU in strengthening procurement and the distribution based
on needs and request
5. Provide MHPSS to children affected by EVD in 15 counties
6. Mental Health Unit to work with LMHRA to regulate, evaluate and register of
essential medicines for mental and neurological disorders
7. Establish mechanisms to support to EVD survivors to be able to communicate the
challenges and tell their stories through individual advocacy, and participatory
research through photo voice programs remain available
8. Conduct Psychotropic drugs monthly inventory from 15 counties
County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Improve documentation and timely reporting to the central level
4. Enhance stakeholder coordination at county and below, on monthly and quarterly
bases
4.5.6 Improve Hygiene & Environmental Services
Objective:
To
improve
accessibility
to,
quality
of
hygiene
and
environmenta
determinants of health and related services. Target 1: Increase hygiene awareness and ensure access to acceptable
Sanitation with 200 households trigger CLTS, 50% access to sanitation and
35% practice improved hygiene
Central Level Activities
1. Develop/produce and disseminate National Hygiene promotion guidelines
2. Revise CLTS guidelines Strengthen National capacity to manage CLTS
3. County Level Activities Increase access to CLTS triggering and ODF monitoring, capacity building of NTCU staff,
Re-activation of CSC 12 routines monitoring of CLTS Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17
3. MHU to work with Supply Chain Management Unit (SCMU) with forecasting and
quantification tools for mental and neurological health care products at national and
county levels. 4. MHU to work with SCMU in strengthening procurement and the distribution based
on needs and request
5. Provide MHPSS to children affected by EVD in 15 counties
6. Mental Health Unit to work with LMHRA to regulate, evaluate and register of
essential medicines for mental and neurological disorders
7. Establish mechanisms to support to EVD survivors to be able to communicate the
challenges and tell their stories through individual advocacy, and participatory
research through photo voice programs remain available
8. Conduct Psychotropic drugs monthly inventory from 15 counties
County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Improve documentation and timely reporting to the central level
4. Enhance stakeholder coordination at county and below, on monthly and quarterly
bases
4.5.6 Improve Hygiene & Environmental Services
Objective:
To
improve
accessibility
to,
quality
of
hygiene
and
environmental
determinants of health and related services. Target 1: Increase hygiene awareness and ensure access to acceptable
Sanitation with 200 households trigger CLTS, 50% access to sanitation and
35% practice improved hygiene
Central Level Activities
1. Develop/produce and disseminate National Hygiene promotion guidelines
2. Revise CLTS guidelines Strengthen National capacity to manage CLTS
3. Increase access to CLTS triggering and ODF monitoring, capacity building of NTCU staff,
Re-activation of CSC, 12 routines monitoring of CLTS
4.5.7 County Health Services
Objective:
To strengthen structures for partnership & coordination at CHT and Central
levels. 3. MHU to work with Supply Chain Management Unit (SCMU) with forecasting and
quantification tools for mental and neurological health care products at national and
county levels. 3. MHU to work with Supply Chain Management Unit (SCMU) with forecasting and
quantification tools for mental and neurological health care products at national and
county levels. 3. County Level Activities
1. Conduct independent integrated supportive supervision to districts, and health
facilities
2. Monthly meetings with CHDCC
3. Improve documentation and timely reporting to the central level
4. County Level Activities Enhance stakeholder coordination at county and below, on monthly and quarterly
bases 27
4.5.6 Improve Hygiene & Environmental Services
Objective:
To
improve
accessibility
to,
quality
of
hygiene
and
environmental
determinants of health and related services. Target 1: Increase hygiene awareness and ensure access to acceptable
Sanitation with 200 households trigger CLTS, 50% access to sanitation and
35% practice improved hygiene
Central Level Activities
1. Develop/produce and disseminate National Hygiene promotion guidelines
2. Revise CLTS guidelines Strengthen National capacity to manage CLTS
3. Increase access to CLTS triggering and ODF monitoring, capacity building of NTCU staff,
Re-activation of CSC, 12 routines monitoring of CLTS
4.5.7 County Health Services
Objective:
To strengthen structures for partnership & coordination at CHT and Central
levels. 4.5.6 Improve Hygiene & Environmental Services
Objective:
To
improve
accessibility
to,
quality
of
hygiene
and
environmental
determinants of health and related services. Target 1: Increase hygiene awareness and ensure access to acceptable
Sanitation with 200 households trigger CLTS, 50% access to sanitation and
35% practice improved hygiene
Central Level Activities
1. Develop/produce and disseminate National Hygiene promotion guidelines
2. Revise CLTS guidelines Strengthen National capacity to manage CLTS
3. Increase access to CLTS triggering and ODF monitoring, capacity building of NTCU staff,
Re-activation of CSC, 12 routines monitoring of CLTS
4.5.7 County Health Services
Objective:
To strengthen structures for partnership & coordination at CHT and Central
levels. 4.5.6 Improve Hygiene & Environmental Services 4.5.7 County Health Services 4.5.7 County Health Services Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Target: Structures for coordination and partnership revitalized and monitored Central Level Activities Central Level Activities Central Level Activities
1. Attend monthly prison health coordination meetings to strengthen collaboration
2. Develop County Heath Team Partners data base and update to ensure proper
coordination and partnership
County Level Activities
1. Review and harmonize program work plans for implementation at county levels
2. Coordinate with QMU, CHTs and Secondary PBF hospitals to establish QI Committees
3. Attend monthly prison health coordination meetings to strengthen collaboration
4. Develop County Heath Team Partners data base and update to ensure proper
coordination and partnership
Specific Objective 2: To ensure implementation of the EPHS
Target 1: Routine use of the EPHS for Health Service implemented
Central Level Activities
1. Support the implementation of EPHS package
2. Receive all requests including liquidation from CHTs, pass requests to appropriate
unit and ensure prompt action is taken
County Level Activities
1. Monitor and evaluate primary health care activities in the county
Specific Objective 3: To strengthen systems through capacity building activities at Central
MOHSW and county levels
Target 1: Systems for capacity building activities are strengthened at Central
MOSHW and county levels
Central Level Activities
1. Work closely with Contracting-in Coordinator to finalize Capacity Building
2. Provide supportive guidance on the design and implementation of CB activities
within the counties
County Level Activities
1. Provide supportive guidance on the design and implementation of CB activities
within the counties
2. Monitor the implementation of CB plan Central Level Activities
1. Attend monthly prison health coordination meetings to strengthen collaboration 1. Attend monthly prison health coordination meetings to strengthen collaboration 1. Attend monthly prison health coordination meetings to strengthen colla 2. Develop County Heath Team Partners data base and update to ensure proper
coordination and partnership 2. Develop County Heath Team Partners data base and update to ensure proper
coordination and partnership y
1. Review and harmonize program work plans for implementation at county levels
2. Coordinate with QMU, CHTs and Secondary PBF hospitals to establish QI Committees
3. Attend monthly prison health coordination meetings to strengthen collaboration
4. Develop County Heath Team Partners data base and update to ensure proper
coordination and partnership Specific Objective 2: To ensure implementation of the EPHS Target 1: Routine use of the EPHS for Health Service implemented
Central Level Activities
1. Support the implementation of EPHS package
2. Receive all requests including liquidation from CHTs, pass requests to appropriate
unit and ensure prompt action is taken Central Level Activities 1. Work closely with Contracting-in Coordinator to finalize Capacity Building
2. Provide supportive guidance on the design and implementation of CB activities
within the counties y
1. Provide supportive guidance on the design and implementation of CB activities
within the counties 1. Provide supportive guidance on the design and implementation of CB activities
within the counties 2. Monitor the implementation of CB plan 28 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Specific Objective 4: To improve prison health activities and ensure that prisons are
provided with quality health care within the 15 counties provided with quality health care within the 15 counties
Target 1: Prison Health activities are improved
Central Level Activities
1. Conduct quarterly monitoring visits for Protection Officers & Health workers in 15
counties
2. Provide essential hygienic materials for inmates
County level Activity
1. Provide essential hygienic materials for inmates
Specific Objective 5: To strengthen contracting mechanisms for the delivery of the EPHS
Target 1: Harmonization of contracting mechanisms
Central Level Activities
1. Organize meeting with all donors’/fund holders for the implementation of various
contract schemes
2. Standardize all contracting mechanisms approach
3. Develop zero draft of the contracting guidelines and tool
4. Pretesting of the zero draft of the contracting guidelines and tool
5. Validation of revised contracting-in guidelines and tool
6. Conduct end of contracts Performance appraisal for all contracts
County level Activities
1. Conduct readiness assessment for county to be contracted in
2. Conduct training for CHTs on the guidelines and tool
3. Capacity building mentoring and coaching for contracting-in
4. Quarterly monitoring of county readiness for contracting
5. Conduct end of contracts Performance appraisal for all contracts
4.5.8 National Health Promotion
Specific Objective 1: Improved coordination among major stakeholders in promoting
healthy practices by the end of 2016
Target 1: Health Promotion Policy, Strategic plan and Communication Strategy
Finalized, Validated, printed and disseminated
Central Level Activities County Level Activities 1. Initiate Partner Mapping in all Counties
2. Awareness campaign on Non communication disease in all 15 counties
3. Development of messages and materials to create awareness and sensitize the
general public to prevent stigmatization of Ebola survivors
4. Introduction of HPV Vaccine and Rota Vaccine 1. Initiate Partner Mapping in all Counties
2. Awareness campaign on Non communication disease in all 15 counties
3. Development of messages and materials to create awareness and sensitize the
general public to prevent stigmatization of Ebola survivors
4. Introduction of HPV Vaccine and Rota Vaccine Specific Objective 2: Strengthen and sustain Community engagement, to identify health
needs and take actions Central Level Activities Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 30
1. Complete and Validate Health Promotion Policy, Strategic plan and Communication
Strategy,
2. Develop and disseminate Health Promotion Policy Briefs and conduct policy
dissemination events at the county level. 3. Develop National Emergency risk Communication Strategy
4. Initiate Partner Mapping in all Counties
5. Establish Health promotion technical working groups at county and District levels
6. Conduct joint assessment on risk communication capacity
7. Training of ECAP2 NGO network on health messaging using LLA training
methodology
8. Initiate Bio-safety and Traffic Health hazard awareness and sensitization
9. Assessment on knowledge on Cervical Cancer and ROTA Vaccine (Diarrhea Vaccine)
10. Development of messages and materials to create awareness and sensitize the
general public for all Vaccine Preventable Diseases
11. Under the leadership of the HPTWG/Messages and Materials Development (MMD)
working group, draft and disseminate RMNCH message guide. 12. Development of messages and materials to create awareness and sensitize the
general public to prevent stigmatization of Ebola survivors
13. Review and revise pretest questionnaire for message and materials development
14. Identify, recruit and train volunteers for pretesting of messages and materials
15. Introduction of HPV Vaccine and Rota Vaccine Launching Nation wide campaign on
Healthy life brand and airing of radio spots to increase demand of and utilization of
health services
County Level Activities
1. Initiate Partner Mapping in all Counties
2. Awareness campaign on Non communication disease in all 15 counties
3. Development of messages and materials to create awareness and sensitize the
general public to prevent stigmatization of Ebola survivors
4. Introduction of HPV Vaccine and Rota Vaccine
Specific Objective 2: Strengthen and sustain Community engagement, to identify health
needs and take actions
Target 1: Community Stakeholders will be aware and sensitized to disseminate
information to community members
Central Level Activities
1. Dialogue with Community stakeholders (Chiefs, Religious, Traditional, Youths
leaders and women groups
2. Radio Distance Learning Program to enable CHVs to implement more effective
health promotion and social mobilization activities leading to improved health
practices and return to RMNCH services 1. Complete and Validate Health Promotion Policy, Strategic plan and Communication
Strategy,
2. Develop and disseminate Health Promotion Policy Briefs and conduct policy
dissemination events at the county level. 3. Develop National Emergency risk Communication Strategy
4. Initiate Partner Mapping in all Counties
5. Central Level Activities Establish Health promotion technical working groups at county and District levels
6. Conduct joint assessment on risk communication capacity
7. Training of ECAP2 NGO network on health messaging using LLA training
methodology
8. Initiate Bio-safety and Traffic Health hazard awareness and sensitization
9. Assessment on knowledge on Cervical Cancer and ROTA Vaccine (Diarrhea Vaccine)
10. Development of messages and materials to create awareness and sensitize the
general public for all Vaccine Preventable Diseases
11. Under the leadership of the HPTWG/Messages and Materials Development (MMD)
working group, draft and disseminate RMNCH message guide. 12. Development of messages and materials to create awareness and sensitize the
general public to prevent stigmatization of Ebola survivors
13. Review and revise pretest questionnaire for message and materials development
14. Identify, recruit and train volunteers for pretesting of messages and materials
15. Introduction of HPV Vaccine and Rota Vaccine Launching Nation wide campaign on
Healthy life brand and airing of radio spots to increase demand of and utilization of
health services 1. County level Activity 1. Dialogue with Community stakeholders (Chiefs, Religious, Traditional, Youths
leaders and women groups Specific Objective 3: Empower Media to Inform and educate the public to promote healthy
life style by the end of 2016 life style by the end of 2016 Central Level Activities 1. Dialogue with Community stakeholders (Chiefs, Religious, Traditional, Youths
leaders and women groups 1. Dialogue with Community stakeholders (Chiefs, Religious, Traditional, Youths
leaders and women groups 2. Radio Distance Learning Program to enable CHVs to implement more effective
health promotion and social mobilization activities leading to improved health
practices and return to RMNCH services 2. Radio Distance Learning Program to enable CHVs to implement more effective
health promotion and social mobilization activities leading to improved health
practices and return to RMNCH services 30 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 3. Continue the orientation of select CHVs and their supervisors on the community
engagement tool, Bridges of Hope
4. Training of 1,500 CHCs on health messaging and community health risk reduction
plan 3. Continue the orientation of select CHVs and their supervisors on the community
engagement tool, Bridges of Hope 3. Continue the orientation of select CHVs and their supervisors on the community
engagement tool, Bridges of Hope
4. Training of 1,500 CHCs on health messaging and community health risk reduction
l 4. Training of 1,500 CHCs on health messaging and community health risk reduction
plan Central level Activities 1. Strengthen partnership with Media in health promotion Activities
2. Conduct Radio/TV shows, print news letter and provide information to the public
3. Continue and maintain the Dey Say rumor tracker system, expand network of users,
conduct roundtables with local media, orient CHWs and CHVs to Dey Say use,
mentor journalists on the use of Dey Say
4. Establish Resource Center/ Documentation
4.6
Drugs and Supply Chain Management 1. Strengthen partnership with Media in health promotion Activities
2. Conduct Radio/TV shows, print news letter and provide information to the public
3. Continue and maintain the Dey Say rumor tracker system, expand network of users,
conduct roundtables with local media, orient CHWs and CHVs to Dey Say use,
mentor journalists on the use of Dey Say 1. Strengthen partnership with Media in health promotion Activities
2. Conduct Radio/TV shows, print news letter and provide information to the public
3. Continue and maintain the Dey Say rumor tracker system, expand network of users,
conduct roundtables with local media, orient CHWs and CHVs to Dey Say use,
mentor journalists on the use of Dey Say 4. Establish Resource Center/ Documentation 4.6
Drugs and Supply Chain Management Drugs and medical supplies is an essential component of the investment plan for building a
resilient health care in Liberia. This pillar is under-funded, with insufficient capacity to
effectively deliver and maintain commodities and supplies at the service delivery levels. These factors result in frequent stock out, distribution of prescriptions to patients and low
utilization of health services. Objective:
To put in place a cost-effective and efficient supply chain management
systems for essential medicines and supplies, including PPEs. Objective:
To put in place a cost-effective and efficient supply chain management
systems for essential medicines and supplies, including PPEs. To achieve this objective, 18 major activities have been earmarked for the fiscal year
2016/17. Below is the list of the 2016/17 planned activities: Central Level Activities
1. Develop and decentralize LMIS
2. Evaluate Interim Approach
3. Distribute drug and medical supplies from NDS
4. Assess drug national and counties drug depots
5. Conduct six counties drug depots (Lofa, Grand Kru, Sinoe, Bomi, Grand Bassa and
Grand Cape Mt)
6. Build drug shelves in 350 health facilities and at Supply Chain Offices Central Level Activities Train supply chain officers and program managers on reporting, supervision,
monitoring, quantification and supply chain management
11. Dispose of expired pharmaceuticals and medical equipment without harming the
environment and the community
12. Procure drugs and medical supplies
13. Procure motorcycles a for supply chain officers
14. Procure vehicles for county pharmacists
15. Train dispenser on rational use of drugs and supply chain management
16. Conduct quarterly monitoring and audit County Level Activities
1. Conduct last mile drug and medical supplies distribution
2. Procure Lab reagent
3. Conduct quarterly monitoring and supervision
4. Procure essential drugs and medical supplies Health Information System, Research and M&E are the fulcrum for the evidence-based
management that the Ministry of Health subscribes to. The HIS, M&E and Research Units
have set objectives and earmarked key activities that are geared towards strengthening
data collection, information generation and inquiry to support management decision
making, implementation tracking and performance monitoring. The objectives and key
activities include: Central Level Activities 31 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 7. De-junk and incinerate health facilities and depots expire drugs
8. Automate the LMIS into the general HMIS of the MoH
9. Procure ICT equipment (Laps, desktops, scanners, printers, etc)
10. Train supply chain officers and program managers on reporting, s
monitoring, quantification and supply chain management
11. Dispose of expired pharmaceuticals and medical equipment without ha
environment and the community
12. Procure drugs and medical supplies
13. Procure motorcycles a for supply chain officers
14. Procure vehicles for county pharmacists
15. Train dispenser on rational use of drugs and supply chain management
16. Conduct quarterly monitoring and audit
County Level Activities
1. Conduct last mile drug and medical supplies distribution
2. Procure Lab reagent
3. Conduct quarterly monitoring and supervision
4. Procure essential drugs and medical supplies
4.7
Health Information Systems, M&E and Research 7. De-junk and incinerate health facilities and depots expire drugs
8. Automate the LMIS into the general HMIS of the MoH
9. Procure ICT equipment (Laps, desktops, scanners, printers, etc)
10. Train supply chain officers and program managers on reporting, supervision,
monitoring, quantification and supply chain management
11. Dispose of expired pharmaceuticals and medical equipment without harming the
environment and the community
12. Procure drugs and medical supplies
13. Procure motorcycles a for supply chain officers
14. Procure vehicles for county pharmacists
15. Train dispenser on rational use of drugs and supply chain management
16. Conduct quarterly monitoring and audit 7. De-junk and incinerate health facilities and depots expire drugs
8. Automate the LMIS into the general HMIS of the MoH
9. Procure ICT equipment (Laps, desktops, scanners, printers, etc)
10. Train supply chain officers and program managers on reporting, supervision,
monitoring, quantification and supply chain management
11. Dispose of expired pharmaceuticals and medical equipment without harming the
environment and the community
12. Procure drugs and medical supplies
13. Procure motorcycles a for supply chain officers
14. Procure vehicles for county pharmacists
15. Train dispenser on rational use of drugs and supply chain management
16. Conduct quarterly monitoring and audit 7. De-junk and incinerate health facilities and depots expire drugs
8. Automate the LMIS into the general HMIS of the MoH
9. Procure ICT equipment (Laps, desktops, scanners, printers, etc)
10. Central Level Activities 1. Hold Monthly HMER Technical working groups with all national programs and
technical partners 1. Hold Monthly HMER Technical working groups with all national programs and
technical partners 2. Hold Quarterly HMER Coordination Committee Meeting with senior MOH manager
and representative of donor institutions. 2. Hold Quarterly HMER Coordination Committee Meeting with senior MOH manager
and representative of donor institutions. 3. Map key partners for research, local and international to identify opportunities for
collaboration and support for research 3. Map key partners for research, local and international to identify opportunities for
collaboration and support for research 4. Train county M&E staff in monitoring and evaluation concept and practices for
effective M&E at the lower levels 4. Train county M&E staff in monitoring and evaluation concept and practices for
effective M&E at the lower levels 32 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 5. Train county and District Health Teams on Data Use in ongoing management
decision making
6. Mentor County Health M&E team to master core M&E skills and execute their
functions with efficiency and effectiveness, and transfer skills down to the district
levels
7. Train District Health Team on data validation, analysis and interpretation
8. Decentralize DHIS-2 to district levels on an incremental basis as District Health
Teams developed. 9. Produce and disseminate revised national M&E Policy and strategy
10. Validate, produce and disseminate MOH indicator reference book to all stakeholders
including the CHTs and Districts offices. 11. Mobilize resources for logistics to facilitate core M&E activities to the decentralized
levels
12. Develop unique code for ID for every health facility in collaboration with key
stakeholders including LISGIS and Liberia Medical and Dental Council. 13. Produce master facility registry capturing all health facilities in the country
indicating their facility types and GPS coordinates. 14. Work towards the development of the seven sub-information systems on an
incremental basic with standards and capabilities to interoperate and exchange data
15. Train 20 health managers on research methodologies, analysis and report writing. 16. Train 15 health managers on the use of statistical packages and technical writing
skills
17. Establish health research repository
18. Produce quarterly dashboard and scorecards using selected core indicators to
measure MOH’s overall and key programs performance
19. Central Level Activities Produce quarterly dashboard and scorecards using selected core indicators to
measure MOH’s overall and key programs performance
19. Produce quality of performance report to inform management on some of the factors
influencing and or impeding progress on service delivery, quality of care, and health
system strengthening; and to document those enablers, challenges, lessons, and
good
practices to inform remedial management actions. 20. Conduct quarterly verification of implementation and monitoring visits to counties
monitor counties’ implementation of the NHPP as expressed in their annual
operational plan, looking at CHTs, Facilities and communities as well a NGO
partners’ activities. 21. County M&E Teams to conduct routine data verification, monitoring and M&E
supervision to the districts and facilities levels
22. County M&E to produce quarterly reports to inform CHTs of their performances as
well as central MOH on where each county stand on progress towards their annual
targets and activities plan. 23. Conduct annual nation review of the health system to take stock of performance for
the year in review and fine-tune operation plan for the following year. 24. Conduct quarterly review at central to look at output and assess progress towards
national annual target and key investment activities 5. n District Health Team on data validation, analysis and interpretation 8. Decentralize DHIS-2 to district levels on an incremental basis as District Health
Teams developed. Develop unique code for ID for every health facility in collaboration with key
stakeholders including LISGIS and Liberia Medical and Dental Council. Produce master facility registry capturing all health facilities in the country
indicating their facility types and GPS coordinates. 16. Train 15 health managers on the use of statistical packages and technical writing
skills . Produce quarterly dashboard and scorecards using selected core indicators to
measure MOH’s overall and key programs performance 9. Produce quality of performance report to inform management on some of the factors
influencing and or impeding progress on service delivery, quality of care, and health
system strengthening; and to document those enablers, challenges, lessons, and
good
practices to inform remedial management actions. 20. Conduct quarterly verification of implementation and monitoring visits to counties
monitor counties’ implementation of the NHPP as expressed in their annual
operational plan, looking at CHTs, Facilities and communities as well a NGO
partners’ activities. 21. County M&E Teams to conduct routine data verification, monitoring and M&E
supervision to the districts and facilities levels 22. Central Level Activities Produce quality of performance report to inform management on some of the factors
influencing and or impeding progress on service delivery, quality of care, and health
system strengthening; and to document those enablers, challenges, lessons, and
good
practices to inform remedial management actions. 20. Conduct quarterly verification of implementation and monitoring visits to counties
monitor counties’ implementation of the NHPP as expressed in their annual
operational plan, looking at CHTs, Facilities and communities as well a NGO
partners’ activities. 21. County M&E Teams to conduct routine data verification, monitoring and M&E
supervision to the districts and facilities levels
22. County M&E to produce quarterly reports to inform CHTs of their performances as
well as central MOH on where each county stand on progress towards their annual
targets and activities plan. 23. Conduct annual nation review of the health system to take stock of performance for
the year in review and fine-tune operation plan for the following year. 24. Conduct quarterly review at central to look at output and assess progress towards
national annual target and key investment activities 5. Train county and District Health Teams on Data Use in ongoing management
decision making
6. Mentor County Health M&E team to master core M&E skills and execute their
functions with efficiency and effectiveness, and transfer skills down to the district
levels
7. Train District Health Team on data validation, analysis and interpretation
8. Decentralize DHIS-2 to district levels on an incremental basis as District Health
Teams developed. 9. Produce and disseminate revised national M&E Policy and strategy
10. Validate, produce and disseminate MOH indicator reference book to all stakeholders
including the CHTs and Districts offices. 11. Mobilize resources for logistics to facilitate core M&E activities to the decentralized
levels
12. Develop unique code for ID for every health facility in collaboration with key
stakeholders including LISGIS and Liberia Medical and Dental Council. 13. Produce master facility registry capturing all health facilities in the country
indicating their facility types and GPS coordinates. 14. Work towards the development of the seven sub-information systems on an
incremental basic with standards and capabilities to interoperate and exchange data
15. Train 20 health managers on research methodologies, analysis and report writing. 16. Train 15 health managers on the use of statistical packages and technical writing
skills
17. Establish health research repository
18. Central Level Activities Print and disseminate the revised National Health Sector Decentralization Policy & Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 25. County conduct quarterly data and performance review meetings involving facilities,
districts and local authorities to discuss success and failures and look at assess
strategies against challenges 25. County conduct quarterly data and performance review meetings involving facilities,
districts and local authorities to discuss success and failures and look at assess
strategies against challenges 4.8
Community Engagement
Objective:
Strengthen community awareness on health risks and their engagement and
linkages with the health system. 34
Objective:
Strengthen community awareness on health risks and their engagement and
linkages with the health system. Central Level Activities
1. Develop community engagement Policy and Strategy
2. Conduct stakeholders’ orientation on community engagement policy and strategy
County level Activities
1. Engage TTMs to refer pregnant women from communities to health facilities
2. Conduct awareness and public education on ENA by TTMs and gCHVs
3. Support gCHVs/CHA to deliver integrated community health services
4. Conduct monthly meeting with CHDC to take ownership of health facilities
5. Conduct IDSR refresher training for gCHVs, TTMs and community leaders in
community event based surveillance
4.9
Leadership and Governance
Objective: Strengthen governance, leadership and management capacities at all levels to
implement the national and county plans. Central Level Activities
1. Finalize and validate the Ministry of Health organizational structure
2. Finalize the organizational structure of the CHTs
3. Develop, review and validate County Health Boards Mandate, Membership and TORs
(operational manual)
4. Establish, finalize and validate organizational structures of DHTs
5. Reactivate /establish district health boards
6. Review and align the Ministry of Health Decentralization Policy and Strategy to the
National Health Investment Plan (2016-2021)
7. Print and disseminate the revised National Health Sector Decentralization Policy &
Plan (2016-2021)
Target 1: 15 County Annual plans and the consolidated national Plan 2016/17
with an effective feedback Mechanism from the central to counties, districts,
and communities are developed. Objective: Strengthen community awareness on health risks and their e
linkages with the health system. Central Level Activities County M&E to produce quarterly reports to inform CHTs of their performances as
well as central MOH on where each county stand on progress towards their annual
targets and activities plan. 22. County M&E to produce quarterly reports to inform CHTs of their performances as
well as central MOH on where each county stand on progress towards their annual
targets and activities plan. 23. Conduct annual nation review of the health system to take stock of performance for
the year in review and fine-tune operation plan for the following year. 23. Conduct annual nation review of the health system to take stock of performance for
the year in review and fine-tune operation plan for the following year. 24. Conduct quarterly review at central to look at output and assess progress towards
national annual target and key investment activities 24. Conduct quarterly review at central to look at output and assess progress towards
national annual target and key investment activities 33 Ministry of Health Consolidated Work Plan 2016/17
25. County conduct quarterly data and performance review meetings involving facilities,
districts and local authorities to discuss success and failures and look at assess
strategies against challenges
4.8
Community Engagement
Objective:
Strengthen community awareness on health risks and their engagement and
linkages with the health system. Central Level Activities
1. Develop community engagement Policy and Strategy
2. Conduct stakeholders’ orientation on community engagement policy and strategy
County level Activities
1. Engage TTMs to refer pregnant women from communities to health facilities
2. Conduct awareness and public education on ENA by TTMs and gCHVs
3. Support gCHVs/CHA to deliver integrated community health services
4. Conduct monthly meeting with CHDC to take ownership of health facilities
5. Conduct IDSR refresher training for gCHVs, TTMs and community leaders in
community event based surveillance
4.9
Leadership and Governance
Objective: Strengthen governance, leadership and management capacities at all levels to
implement the national and county plans. Central Level Activities
1. Finalize and validate the Ministry of Health organizational structure
2. Finalize the organizational structure of the CHTs
3. Develop, review and validate County Health Boards Mandate, Membership and TORs
(operational manual)
4. Establish, finalize and validate organizational structures of DHTs
5. Reactivate /establish district health boards
6. Review and align the Ministry of Health Decentralization Policy and Strategy to the
National Health Investment Plan (2016-2021)
7. County Level Activities County Level Activities
1. Conduct quarterly county health board meetings
2. Orientate County Health Board members on their roles and responsibilities (CSH)
3. Conduct quarterly district health coordination meetings
4. Conduct Bi-annual County Review Meeting with (CSH) in collaboration with
partners
5. Conduct quarterly Health Board Meeting
6. Organize CHC meetings and disseminate revised community health policy
7. Training of Communities Leaders and OICs on the EPHS Components
8. Conduct monthly meeting with gCHVs and TTMs in the communities
9. Train DHT on supervision and reporting
10. Refresher training for OIC, CM, Registrar, and Dispenser on data management
11. Work through the District Superintendent/Commissioner to establish district health
committees
12. Conduct bi-annual operational plan review meeting to review the county work plan,
identify progress, gaps and address the gaps
13. Work with DHTs to engage community leadership on taking ownership of health
facilities Central Level Activities Target 1: 15 County Annual plans and the consolidated national Plan 2016/17
with an effective feedback Mechanism from the central to counties, districts,
and communities are developed. Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 1. Develop standardized guides and tools for formulating the annual operational plan. 1. Develop standardized guides and tools for formulating the annual operational plan. 2. Apply a bottom up approach to develop annual operation plans for the district and
counties with the participation of all stakeholders 2. Apply a bottom up approach to develop annual operation plans for the district and
counties with the participation of all stakeholders
3. Facilitate and conduct horizontal plan for the central MOH and a consolidated pp y
p
pp
p
p
p
counties with the participation of all stakeholders
3. Facilitate and conduct horizontal plan for the central MOH and a consolidated
annual plan for the whole health sector for the fiscal year 3. Facilitate and conduct horizontal plan for the central MOH and a consolidated
annual plan for the whole health sector for the fiscal year 5.0 Costing and Budgeting Below is a
description of the draft health sector FY 2016/17 budget. Table 5.2 : Health Sector Government draft Budget FY 2016/17
Health Sector Expending Entities
FY 2016/17 Budget
Ministry of Health
57,126,248
John F. Kennedy Medical Center
6,295,156
Phebe Hospital and School of Nursing
2,130,956
Liberia Institute of Bio-Medical Research
487,778
Liberia Board for Nursing and Midwifery
188,628
Liberia Pharmacy Board
189,938
Liberia Medical and Dental Council
387,358
Liberia College of Physician and Surgeons
1,117,500
Liberia Medical and Health Products Regulation
458,079
National Aids Commission
844,367
Jackson F. Doe Hospital
2,835,468
Total Budget
72,061,476 5.0 Costing and Budgeting The amount of US$ 149.89 million is required to fully implement the FY 2016/17
operational plan of the national investment plan for building a resilient health system. The
MOH financing unit conducted a resource mapping within the sector and has identified US$
251,513,495 as commitment from the Government of Liberia (US$ 72, million) and partners
(US$ 179.45 million) for the fiscal year. 35
Table 5.1: Estimated Budget and Committed Resources by Investment Areas
#
Investment Pillars
Estimated Cost
Committed Partners
&
Donors
Resources
1
Fit for Purpose motivated workforce
21.3 million
2
Re-engineer health infrastructure
10.9 million
Emergency
Preparedness
and 35
Table 5.1: Estimated Budget and Committed Resources by Investment Areas
#
Investment Pillars
Estimated Cost
Committed Partners
&
Donors
Resources
1
Fit for Purpose motivated workforce
21.3 million
2
Re-engineer health infrastructure
10.9 million
Emergency
Preparedness
and Ministry of Health Consolidated Work Plan 2016/17
3
Response
29.7 million
4
Health care financing
3.8 million
5
Quality of Health Services
57.2 million
6
Drugs and Medical Supplies
13.9 million
7
Comprehensive Information System
and Research
0.387 million
8
Leadership and Governance
11.3 million
9
Community Engagement
1.4 million
Total
US$
US$ 149.89 million
5.1
National Budget
The approved FY 2016/17 National Health sector budget is US$ 72 million of which the
Ministry of Health has US$ 57 million. The budget will be used to fund activities in the
operational plan at the national, county, district and health facility levels. Below is a
description of the draft health sector FY 2016/17 budget. Table 5.2 : Health Sector Government draft Budget FY 2016/17
Health Sector Expending Entities
FY 2016/17 Budget Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17
3
Response
29.7 million
4
Health care financing
3.8 million
5
Quality of Health Services
57.2 million
6
Drugs and Medical Supplies
13.9 million
7
Comprehensive Information System
and Research
0.387 million
8
Leadership and Governance
11.3 million
9
Community Engagement
1.4 million
Total
US$
US$ 149.89 million
5.1
National Budget 5.1
National Budget 5.1
National Budget The approved FY 2016/17 National Health sector budget is US$ 72 million of which the
Ministry of Health has US$ 57 million. The budget will be used to fund activities in the
operational plan at the national, county, district and health facility levels. 6.0 Monitoring and Review of Investment Plan This operational health plan will be monitored using the performance framework available
in Annex C. The list of core output and short term outcome indicators contained in the
framework will be used to track performance at every level of the health system. The
performance framework will guide all stakeholders including partners to monitor and
review the health system for the fiscal year. District and facility teams will focus on service
delivery and community engagement indicators, while county and central levels will focus
on indicators in their monitoring and reviews. 36 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Reviews will take place quarterly and annually. At the decentralized level, review will be
done quarterly involving service providers, health managers and local authorities. This
quarterly review will look back at performance over the previous three months at the end
of the quarters. It will focus on successes and failures, weakness and strengths, good
practices and learn lessons to improve results in the subsequent quarters. At the central
level, quarterly review will be done looking at performance on the core list of indicators
and the implementation of central level planned activities and achievement of key
deliverables in the Investment Plan. At the end on the fiscal year, a comprehensive review
will be done using a mixed of methodologies and gauge the sector’s performance for the
fiscal year ended. Outcomes of the review will form the agenda for the annual health
review conference of all stakeholders in the sector. This annual meeting will take place
preferably in October will document progress towards 2021 and re-align the MOH priorities
towards achievements of the milestones set forth in the National Health Plan and the
Investment Plan for Building a Resilient Health Plan. Annex C presents national and county
levels performance framework. Reviews will take place quarterly and annually. At the decentralized level, review will be
done quarterly involving service providers, health managers and local authorities. This
quarterly review will look back at performance over the previous three months at the end
of the quarters. It will focus on successes and failures, weakness and strengths, good
practices and learn lessons to improve results in the subsequent quarters. 6.0 Monitoring and Review of Investment Plan At the central
level, quarterly review will be done looking at performance on the core list of indicators
and the implementation of central level planned activities and achievement of key
deliverables in the Investment Plan. At the end on the fiscal year, a comprehensive review
will be done using a mixed of methodologies and gauge the sector’s performance for the
fiscal year ended. Outcomes of the review will form the agenda for the annual health
review conference of all stakeholders in the sector. This annual meeting will take place
preferably in October will document progress towards 2021 and re-align the MOH priorities
towards achievements of the milestones set forth in the National Health Plan and the
Investment Plan for Building a Resilient Health Plan. Annex C presents national and county
levels performance framework. Annex A: Health Infrastructure Needs FY 2016/17
#
County
Clinic
Maternal
Home
Staff
Quarter
DHT /CHT
Office
Lab
Incinerators
Fence HF
Triag
e
Cost
1
Bomi
2
22
22
2
Bong
5
127,200
3
Gbarpolu
2
3
7
3
4
6
5
8
4
Grand Bassa
4
21
20
8
22
10
775,150
5
Grand Cape Mt
2
1
2
192,950
6
Grand Gedeh
4
4
3
149,400
7
Grand Kru
10
1
2
8
Lofa
10
9
Margibi
10
Maryland
11
Montserrado
5
7
1
8
2
12
Nimba
2
10
7
8
18
13
Rivercess
3
6
14
River Gee
6
1
12
15
Sinoe
7
2
5
2
775,600
Total
39
47
69
17
26
41
52
34
2,020,300 Annex A: Health Infrastructure Needs FY 2016/17 37 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Annex B: Supply Chain FY 2016/17 Activities
# Activity
Cost
1 Develop and decentralize LMIS
100,000
2 Evaluate Interim Approach
27,700
3 Distribute drug and medical supplies from NDS
200,000
4 Assess drug national and counties drug depots
20,000
5
Conduct six counties drug depots (Lofa, Grand Kru, Sinoe, Bomi, Grand Bassa
and Grand Cape Mt)
900,000
6 Build drug shelves in 350 health facilities and at Supply Chain Offices
245,000
7 De-junk and incinerate health facilities and depots expire drugs
200,000
8 Automate the LMIS into the general HMIS of the MoH
350,000
9 Procure ICT equipment (Laps, desktops, scanners, printers, etc)
100,000
10
Train supply chain officers and program managers on reporting, supervision,
monitoring, quantification and supply chain management
100,000
11
Dispose expired pharmaceuticals and medical equipment without harming the
environment and the community
150,000
12 Conduct last mile drug distribution
250,000
13 Procure drugs and medical supplies
3,000,000
14 Procure Lab reagent
100,000
15 Conduct quarterly monitoring and audit
200,000
16 Procure motorcycles a for supply chain officers
70,000
17 Procure vehicles for county pharmacists
525,000
18 Train dispenser on rational use of drugs and supply chain management
100,000
Total
6,637,700 Annex B: Supply Chain FY 2016/17 Activities
# Activity
Cost
1 Develop and decentralize LMIS
100,000
2 Evaluate Interim Approach
27,700
3 Distribute drug and medical supplies from NDS
200,000
4 Assess drug national and counties drug depots
20,000
5
Conduct six counties drug depots (Lofa, Grand Kru, Sinoe, Bomi, Grand Bassa
and Grand Cape Mt)
900,000
6 Build drug shelves in 350 health facilities and at Supply Chain Offices
245,000
7 De-junk and incinerate health facilities and depots expire drugs
200,000
8 Automate the LMIS into the general HMIS of the MoH
350,000
9 Procure ICT equipment (Laps, desktops, scanners, printers, etc)
100,000
10
Train supply chain officers and program managers on reporting, supervision,
monitoring, quantification and supply chain management
100,000
11
Dispose expired pharmaceuticals and medical equipment without harming the
environment and the community
150,000
12 Conduct last mile drug distribution
250,000
13 Procure drugs and medical supplies
3,000,000
14 Procure Lab reagent
100,000
15 Conduct quarterly monitoring and audit
200,000
16 Procure motorcycles a for supply chain officers
70,000
17 Procure vehicles for county pharmacists
525,000
18 Train dispenser on rational use of drugs and supply chain management
100,000
Total
6,637,700 Annex B: Supply Chain FY 2016/17 Activities 38
Annex C: Health Sector Performance Framework
No. 37 Indicators
Baseline
2015/2016
Baseline
Year
Data
Sources
Target
FY 2016/17
1
Percentage of pregnant mothers attending 4 ANC
visits
50%
2015/16
HMIS
76%
2
Percentage of pregnant mothers receiving IPT-2
37%
2015/16
HMIS
60%
3
Percentage of HIV positive pregnant women initiated
on ARV prophylaxis or ART to reduce the risk of MTCT
TBD
HMIS
60% Annex C: Health Sector Performance Framework
No. 37 Indicators
Baseline
2015/2016
Baseline
Year
Data
Sources
Target
FY 2016/17
1
Percentage of pregnant mothers attending 4 ANC
visits
50%
2015/16
HMIS
76%
2
Percentage of pregnant mothers receiving IPT-2
37%
2015/16
HMIS
60%
3
Percentage of HIV positive pregnant women initiated
on ARV prophylaxis or ART to reduce the risk of MTCT
TBD
HMIS
60% Annex C: Health Sector Performance Framework 38 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 4
Percentage of deliveries attended by skilled personnel
50%
2015/16
HMIS
72%
5
Percentage of infants fully immunized
52%
2015/16
HMIS
75%
6
Percentage of children zero to five months of age
exclusively breast fed
TBD
TBD
7
TB case detection rate (all forms)
56%
2015/16
HMIS
75%
8
Proportion of children one year old immunized
against measles
63%
2015/16
HMIS
70
9
Treatment Success rate among smear positive TB
cases (Under Directly Observed Treatment Short
Course)
72%
2015/16
HMIS
85
10
% of health facilities meeting minimum IPC standards
TBD
QU
100%
11
Percentage of population living within 5 km from the
nearest health facility
71%
2013
DHS
80%
12
Functional Health facilities per 10,000 population
1.8
2016
HR
Census
2
13
Percentage of health facilities with all utilities, ready
to provide services (water, electricity)
64%
2016
SARA
80%
14
Number of counties with funded outbreak
preparedness and response plans
Non Funded
90%
15
Number of counties reporting event based
surveillance data
100%
2016
DCP
weekly
EPI
Report
100%
16
Percentage of health facilities with no stock-outs of
tracer drugs at any given time (amoxicillin,
cotrimoxazole, paracetamol, ORS, iron folate, ACT, FP
commodity)
TBD
SATA
85%
17
OPD consultations per inhabitant per year
1.08
2016
HMIS
2.0
18
Skilled health workforce (physicians, nurses,
midwives, physician assistants) per 1,000 persons
4,756
2016
HR
Census
11.0
19
Proportion of health facilities with at least Two
skilled health workers
NA
N/A
20
Proportion of health workers on government payroll
6,272
2015/16
GOL
payroll
N/A
21
Timeliness of HMIS reports
Quarterly
2016
HMIS
75%
22
Proportion of facilities that submitted HMIS reports
2016
HMIS
75%
23
Per capita public health expenditure in USD
US$ 11.23
2015
MOH AR
US$70
24
Public expenditure in health as % of total public
expenditure
US$ 12.4
2015
MOH AR
15% 39 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 Annex D: County Level Performance Framework
#
County
ANC 4 Visits
IPTp2
Institutional Delivery
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
77%
85%
47%
55%
57%
62%
2
Bong
77%
79%
73%
79%
78%
80%
3
Gbarpolu
33%
48%
23%
53%
29%
41%
4
Grand Bassa
67%
70%
41%
55%
52%
60%
5
Grand Cape
Mt
47%
52%
35%
46%
46%
50%
6
Grand Gedeh
67%
70%
41%
55%
52%
60%
7
Grand Kru
8
Lofa
9
Margibi
49%
0%
37%
0%
42%
0%
10
Maryland
11
Montserrado
44%
45%
22%
25%
24%
30%
12
Nimba
44%
85%
40%
70%
46%
80%
13
Rivercess
14
River Gee
15
Sinoe
73%
78%
53%
62%
61%
66%
National
50%
76%
37%
60%
50%
72%
#
County
Delivery by SBA
Fully Immunized
Penta-3
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
57
62
71
91
76
82
2
Bong
57
62
71
91
76
82
3
Gbarpolu
29
41
0
0
82
94
4
Grand Bassa
44
50
43
50
57
60
5
Grand Cape
Mt
46
50
55
60
74
78
6
Grand Gedeh
44%
50%
43%
50%
57%
60%
7
Grand Kru
8
Lofa
9
Margibi
42%
0%
71%
0%
82%
0%
10
Maryland
11
Montserrado
24%
30%
53%
60%
62%
68%
12
Nimba
46%
80%
40%
13
Rivercess
14
River Gee
15
Sinoe
61%
66%
68%
76%
92%
94%
National
50%
72%
52%
75% Annex D: County Level Performance Framework
#
County
ANC 4 Visits
IPTp2
Institutional Delivery
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
77%
85%
47%
55%
57%
62%
2
Bong
77%
79%
73%
79%
78%
80%
3
Gbarpolu
33%
48%
23%
53%
29%
41%
4
Grand Bassa
67%
70%
41%
55%
52%
60%
5
Grand Cape
Mt
47%
52%
35%
46%
46%
50%
6
Grand Gedeh
67%
70%
41%
55%
52%
60%
7
Grand Kru
8
Lofa
9
Margibi
49%
0%
37%
0%
42%
0%
10
Maryland
11
Montserrado
44%
45%
22%
25%
24%
30%
12
Nimba
44%
85%
40%
70%
46%
80%
13
Rivercess
14
River Gee
15
Sinoe
73%
78%
53%
62%
61%
66%
National
50%
76%
37%
60%
50%
72% Annex D: County Level Performance Framework #
County
Delivery by SBA
Fully Immunized
Penta-3
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
57
62
71
91
76
82
2
Bong
57
62
71
91
76
82
3
Gbarpolu
29
41
0
0
82
94
4
Grand Bassa
44
50
43
50
57
60
5
Grand Cape
Mt
46
50
55
60
74
78
6
Grand Gedeh
44%
50%
43%
50%
57%
60%
7
Grand Kru
8
Lofa
9
Margibi
42%
0%
71%
0%
82%
0%
10
Maryland
11
Montserrado
24%
30%
53%
60%
62%
68%
12
Nimba
46%
80%
40%
13
Rivercess
14
River Gee
15
Sinoe
61%
66%
68%
76%
92%
94%
National
50%
72%
52%
75% 40 40 Ministry of Health Consolidated Work Plan 2016/17 Ministry of Health Consolidated Work Plan 2016/17 #
County
Measles
HMIS Reporting Rate
HMIS Reporting
Timeliness
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
66%
70%
100%
100%
100%
100%
2
Bong
95%
96%
100%
100%
100%
100%
3
Gbarpolu
63%
79%
100%
100%
100%
100%
4
Grand Bassa
69%
75%
93%
100%
97%
100%
5
Grand Cape
Mt
60%
60%
94%
100%
97%
100%
6
Grand Gedeh
48%
54%
93%
100%
93%
100%
7
Grand Kru
8
Lofa
9
Margibi
73%
-
-
-
-
-
10
Maryland
11
Montserrado
60%
68%
-
-
-
-
12
Nimba
68%
70%
80%
90%
80%
90%
13
Rivercess
63%
65%
100%
100%
100%
100%
14
River Gee
15
Sinoe
85%
90%
100%
100%
100%
100%
National
63%
70%
75%
75%
#
County
PNC within 2 wks
Utilization Rate
ANC 1st Visits
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
56%
60%
-
-
86%
90%
2
Bong
46%
50%
-
-
86%
90%
3
Gbarpolu
20%
40%
-
-
41%
59%
4
Grand Bassa
33%
40%
-
-
80%
89%
5
Grand Cape
Mt
56%
60%
-
-
71%
75%
6
Grand Gedeh
37%
40%
-
-
70%
75%
7
Grand Kru
8
Lofa
9
Margibi
10
Maryland
11
Montserrado
28%
30%
90%
90%
12
Nimba
78%
80%
94%
96%
13
Rivercess
34%
40%
73%
75%
14
River Gee
15
Sinoe
45%
49%
79%
85%
National
-
-
-
-
-
- #
County
Measles
HMIS Reporting Rate
HMIS Reporting
Timeliness
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
66%
70%
100%
100%
100%
100%
2
Bong
95%
96%
100%
100%
100%
100%
3
Gbarpolu
63%
79%
100%
100%
100%
100%
4
Grand Bassa
69%
75%
93%
100%
97%
100%
5
Grand Cape
Mt
60%
60%
94%
100%
97%
100%
6
Grand Gedeh
48%
54%
93%
100%
93%
100%
7
Grand Kru
8
Lofa
9
Margibi
73%
-
-
-
-
-
10
Maryland
11
Montserrado
60%
68%
-
-
-
-
12
Nimba
68%
70%
80%
90%
80%
90%
13
Rivercess
63%
65%
100%
100%
100%
100%
14
River Gee
15
Sinoe
85%
90%
100%
100%
100%
100%
National
63%
70%
75%
75% #
County
PNC within 2 wks
Utilization Rate
ANC 1st Visits
Baselines
Targets
Baselines
Targets
Baselines
Targets
1
Bomi
56%
60%
-
-
86%
90%
2
Bong
46%
50%
-
-
86%
90%
3
Gbarpolu
20%
40%
-
-
41%
59%
4
Grand Bassa
33%
40%
-
-
80%
89%
5
Grand Cape
Mt
56%
60%
-
-
71%
75%
6
Grand Gedeh
37%
40%
-
-
70%
75%
7
Grand Kru
8
Lofa
9
Margibi
10
Maryland
11
Montserrado
28%
30%
90%
90%
12
Nimba
78%
80%
94%
96%
13
Rivercess
34%
40%
73%
75%
14
River Gee
15
Sinoe
45%
49%
79%
85%
National
-
-
-
-
-
- 41
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https://econpapers.repec.org/scripts/redir.pf?u=https%3A%2F%2Fredfame.com%2Fjournal%2Findex.php%2Faef%2Farticle%2Fdownload%2F5343%2F5569;h=repec:rfa:aefjnl:v:8:y:2021:i:5:p:18-28
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Challenges in Trade openness, Governance and Industrial Productivity in West African Economic and Monetary Union (WAEMU) Countries
|
Applied economics and finance
| 2,021
|
cc-by
| 6,696
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Applied Economics and Finance
Vol. 8, No. 5; September 2021
ISSN 2332-7294
E-ISSN 2332-7308
Published by Redfame Publishing
URL: http://aef.redfame.com
Challenges in Trade openness, Governance and Industrial Productivity in
West African Economic and Monetary Union (WAEMU) Countries
Kossi AYENAGBO1 & Mamadou BOUKARI1
1
Department of Economics, University of Kara, LaRSEG/FaSEG, Togo
Correspondence: Kossi AYENAGBO, Department of Economics, University of Kara, LaRSEG/FaSEG, Togo.
Received: May 10, 2021
doi:10.11114/aef.v8i5.5343
Accepted: July 7, 2021
Available online: September 3, 2021
URL: https://doi.org/10.11114/aef.v8i5.5343
Abstract
Inward-looking development strategies can lead to marginalization and slow growth especially for the small African
domestic markets. However, when weak economies try to participate in the global economy studies in Southeast Asia
show they end with significant challenges. Therefore, this paper analyzed the effects of trade openness on industrial
development in West African Economic and Monetary Union (WAEMU) countries. However, due to data availability,
the study covered seven countries over the 1996 – 2018 period. The pooled-mean group method was used in the
analysis. The results of the analysis showed that, in the long run, trade openness did not benefit the development of the
industrial sector in all the countries studied. However, in the short run, the results revealed the specificities of each
country. These short-run results showed that trade openness has a positive and significant effect on the industry added
values observed in countries such as Burkina Faso, Niger and Togo. The results also showed that government
inefficiency has a negative impact on the development of the industrial sector in the long -run for all the countries
studied. Furthermore, the indicator capturing the degree of freedom of corruption had a positive impact on the
development of the industrial sector in the short or long run. Therefore, active engagement with the forces of
globalization need strategic approaches in their integration in developing countries.
Keywords: Africa, industrial development, trade openness, panel data, pooled-mean group method
JEL Classification Codes : H52, 055, L16, F18
1. Introduction
The globalization of the world economy is perhaps the most important trend which underlies all the current conditions
for the economic development of a country (Ernest Aryeetey et al., 1998). It offers excellent opportunities to all
countries, especially those in Sub-Saharan Africa, enabling them to increase their productivity and to accelerate their
economic development. One of the most ways for Sub-Saharan Africa, and the West African Economic and Monetary
Union (WAEMU), to participate in global economic integration is in the enhancing of their integration of these
countries into international trade. In this system, international trade, opportunities are offered to countries as well as to
individual companies.
Indeed, by creating large markets, international trade allows small businesses to reach their optimal size, which would
lower average costs as well as to lower prices for consumers. However, the constraints to the development of the
industrial sector in the WAEMU countries have remained enormous. We can cite, among others, the low availability
and quality of physical and technological infrastructure, the absence of investment supervision structures and business
assistance, the insufficiency of public and private investments, difficulties of access to commodity markets, the high
costs of production factors, difficulties in accessing the so-called conventional bank financing, problems of political
stability and corruption, weak capacity of institutions to carry out reforms and guarantee them, the inconsistencies in
industrial policies, and the dysfunction of judicial systems. Indeed, the level of industrialization of a country is
generally measured by the relative contribution of industrial production to GDP or by the proportion of people
employed in a sector of activity. Thus, the rise of the high-added-value industrial sector lead to lower unemployment,
increased labor productivity and, consequently, higher income (Alderson, 1999). Industrialization therefore plays a
decisive role in the development of a country.
However, the level of industrialization of the WAEMU countries remains low. Despite their efforts, most of the
WAEMU countries have encountered obstacles in their industrial development. The trade openness of these countries
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thus appeared as an opportunity to overcome the industrial constraints they face by pooling efforts. By trade openness
we hereby mean the enlargement of the market within a given area by the elimination of tariff and non-tariff barriers,
and the adoption of appropriate reforms to promote the development of trade through the free movement of people
goods and services and capital.
According to Abdellatif et al. (2019), theoretical analyses argue that trade openness could boost the performance of the
industrial sector, especially for countries that do not have a solid industrial base. This is how the WAEMU countries, each
relying on its comparative advantages, should thus take advantage of the vast market to increase their industrial
production and meet greater demands, develop their specializations, and get used to competition. In addition, this vast
market offers opportunities for small businesses which will be able to increase their production and consequently lead to a
consequent increase in national production. According to UNCTAD (2017), in 2016, intra-regional trade between the
countries of the Economic Community of West African States (ECOWAS) amounted to 11.4 billion against 34.7 billion
dollars for the countries of the Southern African Development Community (SADC). Moreover, the WAEMU countries
have opened up considerably to the outside world through trade which has continued to increase. Total exports of goods
by value of WAEMU countries stood at 14.986.8 billion in 2018, up 1.4% from the level recorded in 2017 (Central Bank
of West Africa States/ CBWAS, 2019). It is expected to stimulate growth and development through the intensification and
diversification of industries in the community. Indeed, ECOWAS countries, like many regions of the world, see trade
openness as a powerful tool to promote inclusive growth through the development of the industrial sector. However,
despite numerous regional initiatives and trade agreements, the level of development of the industrial sector in this area
remains very low overall. In view of the industrialization situation in the WAEMU countries and given their degree of
trade openness to the rest of the world, a reflection can be carried out concerning the effects of these international
exchanges on industrialization. As such, we ask the question, what is the effect of trade openness on the development of
the industrial sector in the countries of the WAEMU zone? More precisely, has regional opening really had a positive or
negative impact on the industrial sector between these countries?
Therefore, the objective of this research was set to analyze the effects of trade openness on industrial production in
WAEMU countries. We hypothesized firstly that trade openness could positively affect the added-value of industrial
production. Secondly, that improving the quality of governance through reduction of corruption and government
efficiency are the main channels through which trade openness affects industrial production.
2. Knowledge Gaps in Literature Review
Theoretical analyses within the framework of international trade theory and growth theory provide evidence that trade
openness positively affects the development of the industrial sector. Thus, the theoretical models of Krugman (1979a),
Helpman and Krugman (1985) predicted that trade openness is likely to increase the productivity of firms by inducing
economies of scale. According to the authors, trade liberalization leads companies to increase their exports, increase the
scale of production and, in turn, lower production costs. Therefore, this mechanism leads to increased industrial
production. However, according to Adam Smith (1776), international trade allows the expansion of the domestic market
and the improvement of the division of labor and this leads to an increase in productivity. For David Ricardo (1817) and
Heckscher-Ohlin (1933), the country's trade openness is determined by its level of productivity or its level of
technology. Therefore, countries should specialize in producing goods which are most competitive.
According to the work of Topalova and Khandelwal (2011), trade openness leads to increased productivity either
through trade competitiveness which is due to a reallocation of resources to the most productive sector or through entry
of qualitative products and technological efficiency which ensures higher productivity. The work of of Rowthorn et al.
(2004) and Graciela Chichilnisky (1994) showed that North-South trade would be beneficial for manufacturing
industries in countries of the South. Kim's (2000) work on the link between trade openness and total factor productivity
growth in Korean manufacturing showed that trade liberalization has a positive impact on productivity. By analyzing
the effects of trade openness on the productivity of Ecuadorian manufacturing sectors in 2006, Wong's results show a
positive and significant effect of trade openness on the productivity of manufacturing industries at the level of
export-oriented industries in the 2000s following the implementation of trade reforms, but a decline in productivity after
2000 due to economic events. Dijkstra (2000) in examining the effects of trade liberalization on industrial development
in Latin America, suggested that there may be a trade-off between static efficiency (X-efficiency, efficient allocation of
resources) and dynamic efficiency in particular , for countries which do not have a developed industrial base. A review
of the empirical evidence on several efficiency indicators shows that the effects of X-efficiency and efficient allocation
of resources do occur but are not very strong. Other factors may be more important in generating productivity growth
and structural change. Even for those Latin American countries (Argentina, Brazil, Mexico) with a more developed
industrial base, the dynamic effectiveness of trade liberalization does not happen automatically. The work of Tahir et al.
(2016) on the link between trade openness and the development of the industrial sector in six South Asian countries,
showed that trade openness has a positive and significant influence on the industrial sector. According to these authors,
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this positive impact is due to the factors of production (human capital and investment) constituting an incentive effect
which have contributed to the development of industrial added-value in these countries. These results are confirmed by
the work of Kapri (2016), which showed similar the positive effects of trade openness on industrial productivity.
Studies on a Korean manufacturer, according to the above author, the liberalization of trade in China has led to
productivity gains at the level of Korean manufacturing companies, such as a 1% cut in tariffs leads to a 0.92 increase in
total factor productivity.
Abdellatif et al. (2019) studied in a context of trade liberalization, the impact of trade openness on the development of
the industrial sector in developing countries: the case of Sub-saharan Africa by adopting the macro-econometric
approach in panel data for the period 1980-2016. Thus, these results showed that trade openness positively and
significantly influences the industrial sector of the countries of the region considered. Other determinants such as
education and macroeconomic stability also contribute to the development of the sector. The decomposition of the
sample into groups of countries according to their level of income made it possible for this study to arrive at
heterogeneous results between the Sub-saharan countries on the impact of trade openness on the performance of the
industrial sector.
3. Some Stylized Facts about Trade Openness and Industrialization
Between 1996 and 2018, it is observed external trade liberalization within WAEMU countries. The latter is
characterized by open trade in countries that tend to impose only low barriers to entering its market. This also reflects
the emphasis that has been placed on trade facilitation, trade promotion and support for improving competitiveness.
Regarding the evolution of the degree of trade openness, it is observed over the period from 1996 to 2018 three (03)
phases of evolution of the activity of industrial production. From 1996 to 1998, a sharp drop in industrial added-value
was observed reaching a lower level of 18.19489 as a percentage of GDP in 1998. Between 1998 and 2000, there was a
strong growth in industrial added-value with a value from 20.80 as a peak in 2000 and this production remained stable
until 2007 but a fall was observed from the years 2008. This downward trend in industrial production could be
explained by the advent of the financial crisis of 2008 (the subprime crisis) which affected several countries around the
world. There is more or less a linear upward trend over the period from 2009 to 2012. On the other hand, the trend
remains relatively normal until 2018. This shows that there is still a lot of effort to be made in the WAEMU zone on
structural transformation issues.
80
21.5
21
20.5
20
19.5
19
18.5
18
17.5
17
16.5
70
60
50
40
30
20
10
0
ouv
vai_PIB
Figure 3.1. Figure 1 shows the evolution of the added-value of industrial production as a percentage of GDP and the
degree of trade openness as a percentage of GDP within the WAEMU countries from 1996 to 2018.
Source: Author based on data from the World Bank's WDI
4. Methodology
4.1 Theoretical Framework of the Model
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The theoretical framework of the model is based on developments in endogenous growth theories.
Therefore, using Cobb Douglas production function to analyze this subject is justified as follows. Often, in order to
present and analyze the economic growth of a country or group of countries such as the WAEMU countries, which are
open to commerce, it is sufficient to apply formulas that have already proven to be reliable in market economies. This is
the case, especially with the Cobb Douglas theoretical model (production function). The analytical possibilities offered
by this function consist in juxtaposing potential and actual growth. When technical progress is such that there is both an
elasticity of substitution σ between capital and labour equal to unity at any point in the function and constant returns to
scale. So, to analyze the effect of trade openness on industrial development in the WAEMU zone, we are inspired by the
Cobb Douglas production function, whose technological progress is neutral in the sense of Hicks:
yt At K t Lt
Where 0
[1]
1, 0 1, 1 ,
y t represents the total output (national income or GDP) of the industrial sector at time t, At, Kt and Lt denote
respectively the total factor productivity, the capital stock and the labor stock
represent the parameters less than unity.
4.2 Specification of the Empirical Model and Description of Variables
We follow the methodological framework of Crespi and Geuna (2008, 2005) and Tahir (2016), to specify the empirical
model. In order to test the effect of trade openness on industrial production, we used econometric estimates with data
from 07 WAEMU countries covering the period 1996-2018.
The following equation is considered:
y it 0 1 y it 1 2 ouv _ comit 3 inv it hit X it u it
[2]
In order to identify the transmission channels of trade openness, we sought to analyze the interactive effects between the
degree of freedom from corruption and trade openness on industrial development. For this we establish the following
equation:
yit 0 1 yit1 2 ouv * corrupit uit
[3]
Where: yit, an industry's contribution to growth is generally measured by industrial added-value which reflects the
development and performance of the industrial sector. We measure the development of industrialization by the
industrialization rate, which is the ratio of the added-value of industries to the GDP (Ngoa Tabi and Atangana Ondoa,
2013; Hossein and Weiss, 1999). ouv_com: designates the commercial opening captured as the ratio between total
export and import to GDP (RODRIGUEZ & RODRIK, 2000); inv represents Investment and is captured by the Gross
fixed capital formation (GFCF) in percentage of GDP at constant price. These are all explanatory variables that will
allow us to assess the influence of modernity on industrialization. tx_GDP: is the GDP growth rate (annual%) in level
and squared, in order to test the U-shaped relationship between GDP and industrialization or deindustrialization
postulated by Clark (1957). Institutional quality: Industrial development is based on institutional factors that promote a
stable politico-legal climate in order to encourage the business climate and promote investment. We captured it by the
Corruption Perception Index (corrup) and the government effectiveness (effic). The Corruption Perception Index is
constructed from several optional surveys of experts commenting on their perception of the level of corruption in the
public sectors. Countries are ranked on a scale from 0 to 10; 0 indicates a high degree of corruption and 10 indicates a
low degree of corruption. We expect a negative sign (-) for the coefficient of corrup. The government effectiveness
(effic), is a variable that assesses the quality of services, the quality of bureaucracy, the competence of civil servants, the
policy and the government's credibility in respecting its commitments to economic and political actors. This variable is
between -4.5 and 4.5 where a high value indicates high government efficiency and therefore attracts foreign investors.
The expected sign is positive (+).
4.3 Presentation of the Study Sample and Data Source
The sample is composed of the countries of the West African Economic and Monetary Union (WAEMU). These
countries are: Benin, Burkina Faso, Côte d'Ivoire, Guinea-Bissau, Mali, Niger, Senegal and Togo. The study period
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covered annual analysis from 1996 to 2018. For reasons of data availability, the study will cover seven (07) countries as
Guinea-Bissau will be excluded during econometric estimates. In addition, the choice of these countries is justified by
the fact that they have the same economic characteristics, the same historical past (colonization and sub-regional
integration) and also have the same monetary policy regime. We use data from several sources (World Development
Indicators, perspective.usherbrooke.ca/bilan database, Worldwide Governance Indicators).
4.4 Estimation Techniques
4.4.1 Stationarity Tests
Before moving on to the estimations, we studied the stochastic properties of the data, in particular the unit root tests.
Stationarity tests are necessary to avoid the spurious regression problem. For Panel data, these tests constitute
hypotheses made on the dependence of interindividual error terms (N‘guessan, 2019; Hurlin and Mignon, 2007).
Authors such as Hurlin and Mignon (2007) report the fundamental differences between Panel and time series
stationarity tests. With regard to the tests of stationarity on time series, the statistical tests have non-standard and
conditional asymptotic distributions specific to each model, while the statistical tests of stationarity of the Panel models
admit the asymptotic distribution of laws normal.
The first empirical results were tested by Levin and Lin (1992). To solve interindividual correlation problems, we very
often use two tests. These are the first-generation tests developed by Maddala and Wu (1999) and a second-generation
test by Pesaran (2007).
4.4.2 Cointegration Test
Usually, time-series cointegration tests are used. However Pedroni (1995,1999, 2001, 2004), Kao (1999) and
(Westerlund et al., 2007) have proposed cointegration tests that apply to Panel data. The use of cointegration
techniques in panel data makes it possible to test for the presence of long-term relationships between integrated
variables. Pedroni offers seven (7) statistics to test cointegration in panel data: four are based on the within dimension
(intra) and three are based on the between (inter) dimension (Kos àMougnol and Kamajou, 2016). The advantage of the
Westerlund test is that it can be applied to unbalanced panels and, is applicable even in case of inter-individual
dependencies, with the bootstrapping procedure.
4.4.3 Pooled Mean-Group Method (Aggregate Group Average)
The Pooled Mean Group estimator, developed by Pesaran, Shin and Smith (1998, 1999) is part of the class of dynamic
panel models in which it is assumed that the number of observations T is as large as that of individuals N. This
estimator admits that the model constant, as well as the short-term coefficients and the variances of the errors, can differ
between individuals. The method is based on the dynamic panel specification of the Auto Regressive Distributive Lags
(ARDL) model of the following form:
yit j 1 ij yi ,t-j j 0 ij X i ,t-j i it
p
q
[4]
Where the number of individuals i = 1,2, ... ... N; the number of period t = 1, 2,… ... T; is a vector with explanatory
variables; are the vectors of the coefficients; are scalars and is the country specific effect. A main characteristic of
cointegrated variables is their reaction to any deviation from the long-term equilibrium. This characteristic implies an
error correction model in which the short-term dynamics of the system variables are influenced by the deviation from
the equilibrium. In this case, equation [1] can be re-parameterized as follows:
yit i yi ,t-1 'i X it j 1 ij yi ,t-j j 0 ij X i ,t-j i
p
q
[5]
The parameter is the rate of error correction of the adjustment term (adjustment coefficient). If, then there is no
evidence of the presence of a long-term relationship. If, then there is an error correction, which implies that the
variables and are cointegrated. This parameter is supposed to be significantly negative under the previous assumption
that the variables show a return to long-run equilibrium.
5. Empirical Results
5.1 Analysis of Descriptive Statistics
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In analyzing the results of econometric estimations, it is important to take a look at the descriptive analysis of the
natural behavior of the data available to us. At first glance, we see a large gap between certain variables such as
industrial added-value in US $ (constant value), trade openness, gross fixed capital formation, and institutional variables
(government efficiency), growth rate of GDP. We used the industrial added-value as our dependent variable. On
average, this dependent variable is about 20% of the GDP of the WAEMU countries. The minimum value was recorded
in Niger (11.26) in 2006 and the maximum in Benin (29.72) in 2001. The Min and Max values of the gross fixed capital
formation variable which represents the investments supposed to boost industrialization are, respectively, 8.25 and
38.89 in percentage of GDP. The minimum was recorded in Ivory Cost and the maximum in Niger.
Togo has the highest ratio of openness (118.10%) compared to the lowest level recorded by Burkina Faso (30.73%)
over the period of study.
Table 1. Descriptive statistics of the model variables
Variable
Industrial Added Value
Trade openness ratio
GDP growth
Gross capital Formation
Corruption index
Government Effectiveness
Obs
161
161
161
161
161
133
Mean
20.22
62.16
4.67
20.38
2.91
-0.75
Std. Dev.
3.74
16.45
3.06
6.60
0.43
0.35
Min
11.26
30.73
-4.39
8.25
2.09
-1.55
Max
29.72
118.10
15.38
38.89
3.91
0.08
Source : Author
The government effectiveness is in units of a standard normal distribution, with mean zero, standard deviation of one,
and running from approximately -2.5 to 2.5, with higher value corresponding to better governance. The average value of
this indicator is equal to -0.75, showing that the efficiency level of the WAEMU countries is bad.
5.2 Unit root test results
To determine the order of integration of the series studied, we retained one of the most widely used unit root tests
namely the test of Levin-Lin and Chu (2002). The test suggests that only the variable EFF_GOV (government
efficiency) is stationary in level while the other variables have a unit root. Note that all these variables are stationary in
first difference. This result allows us to conclude that the conditions for implementing the ARDL type cointegration
model for series I (0) or I (1) are met.
Table 2. Unit root test of study variables (stationarity)
Variables utilized
Levin-Lin and Chu stationarity test (VAI)
GBP Growth rate (TX_GDP)
Trade Openness (OUVCOM)
Investment (GFCF)
Government efficiency EFF_GOV)
Corruption freedom index (corrup)
Levin-Lin and Chu Stationarity test
In Level I(0)
p-value
Decision
In first difference I(1)
p-value
Decision
0.089
Non stationnary
0.000
stationnary
0.604
0.370
0.448
Non stationnary
Non stationnary
Non stationnary
0.000
0.000
0.000
stationnary
Stationnary
Stationnary
0.002
Stationnary
0.000
Stationnary
0.001
Stationnary
0.000
Stationnary
Source : Author
5.3 The Results of the Cointegration Test
The series relating to the variables OUVCOM. TCR_GDP; GFCF and VAI being integrated of order 1. The second step
of our empirical approach consists in testing the existence of a possible cointegration relation. The cointegration test
used is that proposed by Pedroni (1999). Indeed the cointegration of the variables depends on the value of the
probability associated with each test statistic. All statistical tests reject the null hypothesis of non-cointegration. In other
words, the series are cointegrated. The second step is to estimate the long-term relationship. Following our approach,
we will make the estimates using the ―Pooled Mean Group‖ method.
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Vol. 8, No. 5; 2021
Table 3. The results of the cointegration test of exogenous variables with dependent variable
Modified Phillips-Perron t
Phillips-Perron t
ADF t
Pedroni cointegration test
t- Statistics
1.52
-4.20
-4.05
P-value
0.060
0.000
0.000
Source : Author
5.4 The Results of the Estimates by the Pooled-Mean Group (PMG) Method
ARDL-type regressions show that all the variables are cointegrated. Indeed, the adjustment coefficient is negative and
significant. Industrial added-value and trade openness (ouv_com) evolve together in the long term. There is therefore a
stable equilibrium relationship between trade openness and industrial added value. According to the results obtained in
the long run, trade openness is not beneficial for the growth of production industry in all seven WAEMU countries
considered. Thus, when trade openness increases by one point, this leads to a decrease of 0.16 point in industrial value
added. This result reflects the weak capacity of industries in the zone to face competition from products from foreign
industries and also the small number of manufacturing industries established in the zone. On the other hand, in the short
run, trade openness has a positive impact on the growth of industrial added-value in all the countries studied. These
results corroborate those of Tahir et al (2016). Topalova and Khandelwal (2011) and Njikam (2009) which show that
trade openness has a positive and significant influence on the industrial sector. Specifically, in the short run, trade
openness has a positive and significant impact on industrial development in countries like Burkina Faso. Niger and
Togo. In the case of Togo, the one point increase in trade openness leads to 0.23 point increase in industrial added value.
This effect is greater for the case of Burkina Faso and Niger and respectively of the order of 0.44% and 1.15%. These
results reflect the partial removal of a number of tariff barriers in these countries.
With regard to the investment captured here by gross fixed capital formation (gfcf), it has a positive and significant
long-term effect on the growth of industrial added-value for all of the seven WAEMU countries. This result does not
collaborate with the work of Rodrik (1992) and Miyagiwa and Ohno (1995) who show that trade openness reduces the
market share of domestic firms which encourages them to invest less. This has a negative impact on their productivity
and therefore on their competitiveness. However. the contribution of public and private investment to industrial
development in this study could be explained by the increase in the public investment effort due to the fall in public
debt, the fall in borrowing costs and the increase in revenue in all WAEMU countries. However, the opposite effect
occurs in the short -run in the countries studied except Benin. The results show that investment has a negative effect on
industrial productivity in the short run. This result shows that most industries do not have access to credit. Thus, the
narrowness of the volume of loans granted to local businesses is explained on the one hand by the presence of
information asymmetry which is a result of the unavailability of strong resources to guarantee these loans and of
another side of the weak development of the banking system in this region. This result is aligned with the study by Fjos.
Grunfeld and Green (2010).
In the short run, government efficiency evolves in the opposite direction with the industrial added-value of all WAEMU
countries. This shows that the government in these countries is making efforts in terms of supervision and orientation of
administrative files. It is about the creation of industries or efforts to improve the business climate. On the other hand, in
the short run, the coefficient assigned to government efficiency is positive and significant for Ivory Cost and Senegal
and has the opposite effect for Benin. In the long –run, the GDP growth rate is positive but not significant. This shows
that the growth rate of GDP does not contribute substantially to industrial added-value for all of the seven countries of
the WAEMU zone studied. On the other hand, in the short -run, the GDP growth rate has a positive and significant
impact on industrial added-value in Niger and leads to an increase in it. This result can be considered reasonable insofar
as the gains from trade liberalization (whether in terms of industrial development or welfare) are assumed to be
concentrated in countries with advanced income and the most competitive such as high growth countries.
Tables 4. Results with the Pooled-Mean Group estimator: PMG
Long-Run impact
Variables
Trade Openness
Coefficients (Z-Value)
-0.16**
(lOUVCOM)
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Vol. 8, No. 5; 2021
(2.04)
0.263***
(4.77)
0.002
(0.56)
-0.288***
(4.79)
105
169.13
Investment (GFCF)
Growth rate of GDP (tcr-GDP)
Government efficiency (Eff-Gov)
N-Observation
Log Likelihood
VARIABLES
WAEMU
BENIN
Adjustment
Coefficient
-0.48***
(2.97)
-0.09*
(1.87)
Specifics short- term impact
BURKINA
COTE
D’IVOIRE
-0.44**
-1.26***
(2.25)
(7.52))
Trade
Openness
(lOUVCOM)
Investment (GFCF)
0.238
(1.39)
-0 .2
(1.34)
0.002
(1.19)
0.07
(1.00)
-0.08
(0.87)
0.22**
(2.55)
0.002
(0.39)
-0.204**
(2.38)
0.44**
(2.08)
-0.007
(0.002)
0.004
(0.40)
-0.059
(0.21)
Rate
Growth GDP (GDP
Efficiency
Government
(Eff-Gov)
Constante
0.04
(0.48)
-0.197**
(2.90)
0.001
(0.65)
0.4***
(5.18)
MALI
NIGER
SENEGAL
TOGO
-0.06
(0.56)
-0.17
(0.95)
-0.62**
(2.22)
-0.73***
(3.03)
-0.175
(1.02)
-0.081
(0.4)
0.007
(0.14)
-0.05
(0.29)
1.15*
(1.90)
-1.02**
(2.29)
0.01*
(1.66)
0.10
(0.26)
0.05
(0.25)
-0.33*
(1.92)
-0.004
(0.76)
0.20**
(2.01)
0.23**
(2.82)
-0.008
(0.07)
-0.009
(0.10)
0.12
(0.65)
1.30***
0.231
1.12**
3.69***
0.16
0.44
1.77**
1.71***
(2.77)
(1.62)
(2.30)
(6.71)
(0.51)
(1.05)
(2.31)
(2.88)
*** ; ** ; * ; * indicate the significance at the respective thresholds of 1%; 5% and 10. value in parenthesis means t-student
***; **; * indicate the significance at the respective thresholds of 1%; 5% and 10. value in parenthesis means t-Student
Source : Author
The results of the interactive analysis between trade openness and the degree of corruption are presented in the table
below. This result shows that the channel through which trade openness is favorable to long-term industrial productivity
growth is the degree of freedom from corruption. Estimates show that in the long- run, trade openness has a positive and
significant effect on industrial productivity through corruption. When the degree of corruption decreases by one point, it
results in an increase in added-value of 0.04%. This reflects the effect of corruption on the business climate and plays
an important role on industrial production. Thus, the work done on public investment in Sub-saharan Africa shows
that when countries control corruption, it is ensure political stability and public investment would be revived and this
could increase industrial productivity. Likewise, in the short–run, trade openness has a positive and significant impact
on the development of the industrial sector in Côte d'Ivoire by reducing the degree of corruption.
Table 5. Results of the interactive effect between corruption and trade openness on industrial added -value
Long -run Impact
Variables
OUV*CORRUP
Coefficients (Z-Value)
0.04***
(3.10)
154
194 .83
N-Observation
Log Likelihood
VARIABLES
WAEMU
BENIN
Adjustment
Coefficient
-0.38***
(8.46)
-0.31**
(2.57)
Sepecific Short -run Impact
BURKINA
COTE
D’IVOIRE
-0.44**
-0.38***
(2.25)
(3.04))
OUV*CORRUP
0.02*
(1.79)
0.94***
(9.35)
0.05
(0.59)
0.82**
(2.57)
0.03
(0.66)
1.2**
(2.55)
Constant
0.06**
(2.52)
1.4***
(2.88)
MALI
NIGER
TOGO
-0.33**
(2.25)
SENEG
AL
-0.33
(1.62)
-0.23*
(1.68)
0.03
(0.96)
0.59*
(1.69)
-0.01
(0.22)
0.70**
(2.11)
-0.03
(0.89)
0.89*
(1.65)
0.03
(1.61)
1.33***
(3.90)
-0.59***
(3.94)
Source : Author
6. Discussions and Conclusions
This research enriches the economic literature on the effects of trade opening. Most studies have analyzed the
microeconomic and macroeconomic consequences of trade openness and the enrollment rate in secondary education but
25
Applied Economics and Finance
Vol. 8, No. 5; 2021
do not often address the issue in terms of industrial production in WAEMU countries. On the other hand, this research
reveals the channels through which trade openness manifests.
The econometric analyzes showed that in the long -run trade openness is not beneficial for development of the industrial
sector in all the countries studied. However, in the short–run, the results reveal specificities to each country. In the short
–run, trade openness has a positive and significant effect on the industrial added-value observed in countries such as
Burkina Faso. Niger and Togo. Another channel through which trade openness can positively affect the development of
the industrial sector is through the quality of institutions. Indeed. the results show that government inefficiency
negatively impacts the development of the industrial sector in the long -run for all the countries studied. Thus, in the
short -run, this effect was more pronounced in Benin. The indicator capturing the degree of freedom of corruption was
found to have a positive impact on the development of the industrial sector. The research was limited by the availability
of data as indicated in the results section.
In order to promote good development of the industrial sector in the WAEMU zone. the governments and
decision-makers of these countries must implement a kind of protectionism in order to protect their infant or fragile
industries from foreign competition. This is one way of enhancing local development of industries as they mature for
competitive trade opening. However, these protectionist trade policies can prove difficult to implement by because they
shall contradict the rules of the World Trade Organization. Therefore, developing nations should work out stimulus
models to spur progressive industrial development. This can be by interactively customising and applying the above
findings to improve the quality of governance and institution establishment. Studies of these models in Southeast Asian
situations show that the sub-Saharan Africa policy-makers could learn and adapt and customise them for accelerated
profitable globalization participation.
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Accuracy of stroke volume measurement with phase-contrast cardiovascular magnetic resonance in patients with aortic stenosis
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Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124
https://doi.org/10.1186/s12968-021-00814-4 Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124
https://doi.org/10.1186/s12968-021-00814-4 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Abstract Background: Phase contrast (PC) cardiovascular magnetic resonance (CMR) in the ascending aorta (AAo) is widely
used to calculate left ventricular (LV) stroke volume (SV). The accuracy of PC CMR may be altered by turbulent flow. Measurement of SV at another site is suggested in the presence of aortic stenosis, but very few data validates the
accuracy or inaccuracy of PC in that setting. Our objective is to compare flow measurements obtained in the AAo and
LV outflow tract (LVOT) in patients with aortic stenosis. Methods: Retrospective analysis of patients with aortic stenosis who had CMR and echocardiography. Patients with
mitral regurgitation were excluded. PC in the AAo and LVOT were acquired to derive SV. LV SV from end-systolic and
end-diastolic tracings was used as the reference measure. A difference ≥ 10% between the volumetric method and
PC derived SVs was considered discordant. Metrics of turbulence and jet eccentricity were assessed to explore the
predictors of discordant measurements. Results: We included 88 patients, 41% with bicuspid aortic valve. LVOT SV was concordant with the volumetric
method in 79 (90%) patients vs 52 (59%) patients for AAo SV (p = 0.015). In multivariate analysis, aortic stenosis flow
jet angle was a strong predictor of discordant measurement in the AAo (p = 0.003). Mathematical correction for the
jet angle improved the concordance from 59 to 91%. Concordance was comparable in patients with bicuspid and
trileaflet valves (57% and 62% concordance respectively; p = 0.11). Accuracy of SV measured in the LVOT was not
influenced by jet eccentricity. For aortic regurgitation quantification, PC in the AAo had better correlation to volumet‑
ric assessments than LVOT PC. Conclusion: LVOT PC SV in patients with aortic stenosis and eccentric jet might be more accurate compared to the
AAo SV. Mathematical correction for the jet angle in the AAo might be another alternative to improve accuracy. Keywords: Phase contrast, Aortic stenosis, Valvular heart disease Accuracy of stroke volume measurement
with phase‑contrast cardiovascular magnetic
resonance in patients with aortic stenosis Ezequiel Guzzetti†, Hugo‑Pierre Racine†, Lionel Tastet, Mylène Shen, Eric Larose, Marie‑Annick Clavel,
Philippe Pibarot and Jonathan Beaudoin* Background Phase-contrast (PC) is the main approach to quantify
blood flow parameters with cardiovascular magnetic res-
onance (CMR). It is widely accepted and used to assess
valvular and congenital heart diseases [1, 2]. Flow volume
can be measured by the acquisition of a cross-sectional
image of the vessel or area of interest, in which fluid
velocity is calculated for every pixel [1, 3]. However, the *Correspondence: jonathan.beaudoin@criucpq.ulaval.ca
†Ezequiel Guzzetti and Hugo-Pierre Racine contributed equally as first
authors *Correspondence: jonathan.beaudoin@criucpq.ulaval.ca
†Ezequiel Guzzetti and Hugo-Pierre Racine contributed equally as first
authors Institut Universitaire de Cardiologie et de Pneumologie de Québec,
Université Laval, 2725 Chemin Sainte‑Foy, Québec, QC G1V‑4G5, Canada RESEARCH Open Access Cardiovascular magnetic resonance measurements Cardiovascular magnetic resonance measurements
CMR was performed using 1.5 and 3T CMR scanners
(Achieva or Ingenia, Philips Healthcare). Cardiac mor-
phology and function were assessed by balanced steady-
state free precession sequences at 30 phases per cardiac
cycle in held end-expiration. Standard planes included
8–14 contiguous parallel short-axis (8 mm thickness,
0 mm gap) covering the entire cardiac volume, 2-cham-
ber, 4-chamber and two orthogonal LVOT planes. Typical parameters at 1.5T were TR/TE 3.2/1.6 ms, flip
angle 60º, and NEX of 1, in-plane spatial resolution of
1.6 × 2 mm. Equivalent acquisition parameters at 3T
were TR/TE 2.8/1.3 ms, flip angle 45°, and NEX of 1, in-
plane spatial resolution of 1.7 mm × 2 mm, 7 mm slice
thickness, 0 mm gap. LV volumes and LVEF were meas-
ured by contour analysis of end-diastolic and end-systolic
phases of the short-axis stack. LV SVVM was calculated
as the difference between LV end-diastolic and end-sys-
tolic volumes. To reflect different practices of CMR post-
processing, LV volumes and SV were computed with and
without including the papillary muscles and major tra-
beculations in the blood pool. Our objectives are to compare flow measurements
obtained by PC in the ascending aorta (SVAAo) and the
LVOT (SVLVOT) in patients with various degrees of aortic
stenosis. We have selected a population without signifi-
cant mitral regurgitation so that SV obtained by volu-
metric method (SVVM) from LV tracings can be used as
a reference. . Open Access T © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 2 of 10 patients signed a written informed consent at the time of
inclusion. accuracy of PC may be altered by the presence of tur-
bulent flow. Such turbulence typically occurs in patients
with heart valve diseases and stenotic or regurgitant jets,
which are associated with flow acceleration/deceleration
and intravoxel dephasing [4–6]. Regurgitant or stenotic
valves are also associated with flow eccentricity, increas-
ing the difficulty to optimize the correct imaging plane. Doppler echocardiographic measurements All Doppler echocardiographic examinations were
acquired
using
commercially
available
ultrasound
machines (iE33 and EPIQ, Philips Healthcare, Best, Neth-
erlands) and according to the current recommendations
of the American Society of Echocardiography [13, 14]. Images were analyzed offline in a core laboratory. Aortic
regurgitation and mitral regurgitation were graded using
a multiparametric approach as suggested by guidelines
[14, 15]. All patients with more than mild mitral regur-
gitation severity were excluded for the purpose of this
study. fi
PC in the ascending aorta (AAo) is widely used to cal-
culate forward stroke volume (SV) and regurgitant aortic
flow; those variables are essential to assess mitral and/
or aortic regurgitation [5, 7]. Aortic regurgitation can be
measured directly from the PC sequence, while mitral
regurgitation is computed as the difference between aor-
tic SV and left ventricular (LV) SV obtained volumetri-
cally from a short axis stack. Blood flow quantification
in the AAo is however potentially altered in the presence
of aortic valve disease, and SV measurement at another
site [pulmonary valve or left ventricular outflow tract
(LVOT)] is sometime suggested [5, 8, 9]. However, it is
not clear if and at which severity the presence of aortic
valve disease can invalidate AAo PC measurements [10,
11], and there are few data comparing SV obtained from
different sites in the presence of valve disease. These
issues are relevant as patients with multiple valve diseases
are frequently encountered and increasingly assessed by
CMR [5, 7].l Patient population A total of 88 patients prospectively recruited in the ongo-
ing PROGRESSA study (NCT 01679431) between 2011
and 2015 were retrospectively analyzed. Included patients
had either aortic stenosis (Vmax > 2 m/s), bicuspid aor-
tic valve (with or without stenosis), and controls without
valve disease. Patients were excluded if they had symp-
tomatic aortic stenosis, any mitral valve disease (mitral
stenosis or > trace mitral regurgitation), LV ejection frac-
tion (LVEF) < 50%, rheumatic valve disease or endocar-
ditis, previous aortic/mitral valve repair or replacement,
previous ascending aorta repair or replacement, if they
were pregnant/lactating or if they had contraindications
to gadolinium. More details about inclusion/exclusion
criteria were previously described [12]. Patients under-
went transthoracic echocardiogram (TTE) and CMR
within 3 months. The study was approved by the Ethics
Committee of the Quebec Heart and Lung Institute and Using double-oblique long-axis views of the LVOT
and aortic valve, through-plane PC imaging was per-
formed during breath-hold at two sites: (1) LVOT,
5–10 mm below the aortic annulus in mid-systole and
(2) AAo, 10-mm above the aortic annulus (Fig. 1). The
imaging PC sequence was planned parallel to the aor-
tic valve annulus plane as previously described [16]. Flow imaging parameters consisted of TR/TE = 4.29–
4.92/2.52–3.05 ms, flip angle = 15°, 24 phases, pixel
spacing = 1.32–2.07 mm, slice thickness = 10 mm,
acquisition matrix = 256 × 208. For each patient, peak
aortic jet velocity measured by TTE was used as a start-
ing point to define CMR encoding velocity in the AAo
[CMR encoding velocity = (1.25–1.5) × peak jet veloc-
ity] with further adjustment in case of aliasing. Forward Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 3 of 10 Fig. 1 Locations of Phase-contrast flow measurements. A, B show double oblique orthogonal planes of the left ventricular (LV) outflow tract (LVOT)
with the corresponding slice planes for the LVOT (blue lines) and ascending aorta (green lines). Phase-contrast images for measurement of flow at
the LVOT and ascending aorta are shown in C, D Fig. 1 Locations of Phase-contrast flow measurements. A, B show double oblique orthogonal planes of the left ventricular (LV) outflow tract (LVOT)
with the corresponding slice planes for the LVOT (blue lines) and ascending aorta (green lines). Patient population Phase-contrast images for measurement of flow at
the LVOT and ascending aorta are shown in C, D All analyses were done with cvi42 software (version
5.6.4, Circle Cardiovascular Imaging, Calgary, Alberta,
Canada). systolic flows (SVAAo and SVLVOT) were computed
using semi-automated tracings. Regurgitant volume
was measured by PC at both sites and also estimated
using the difference between right ventricular (RV) and
LV SVs as these patients did not have significant mitral
or tricuspid regurgitation [15]. We have evaluated the
eccentricity of the aortic stenosis jet by assessing the
angle between the aortic valve plane and the aortic jet
in double-oblique long axis images. An angle of 90°
reflects a jet flow parallel to the vessel orientation, and
lower angles reflects jet eccentricity (Fig. 2). The angle
was measured in 2 cross sectional planes, and the low-
est measured angle was registered. Mathematical cor-
rection of the measured SVAAo for the eccentricity
angle was performed [corrected flow = measured flow/
sin(angle)] as illustrated in Additional file 1: Figure S1). systolic flows (SVAAo and SVLVOT) were computed
using semi-automated tracings. Regurgitant volume
was measured by PC at both sites and also estimated
using the difference between right ventricular (RV) and
LV SVs as these patients did not have significant mitral
or tricuspid regurgitation [15]. We have evaluated the
eccentricity of the aortic stenosis jet by assessing the
angle between the aortic valve plane and the aortic jet
in double-oblique long axis images. An angle of 90°
reflects a jet flow parallel to the vessel orientation, and
lower angles reflects jet eccentricity (Fig. 2). The angle
was measured in 2 cross sectional planes, and the low-
est measured angle was registered. Mathematical cor-
rection of the measured SVAAo for the eccentricity
angle was performed [corrected flow = measured flow/
sin(angle)] as illustrated in Additional file 1: Figure S1). Statistical analyses Normal distribution of continuous variables was assessed
using the Shapiro–Wilk test. Continuous data were
expressed as mean ± standard deviation or median [inter-
quartile range], and categorical variables as percentages. Correlation and agreement (95% confidence intervals)
between SVAAo and SVLVOT as compared with SVVM were
assessed by Spearman correlations and Bland–Altman
comparisons [17]. Paired Student t tests were used to
test for significance of any overestimation or underesti-
mation. A margin of error of ± 10% between PC derived
SV and SVVM was considered concordant measurements, Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 4 of 10 Fig. 2 Assessment of jet eccentricity. A, B double-oblique long-axis of a bicuspid aortic valve with an eccentric jet (jet angle 65 degrees). C, D
trileaflet aortic valve with a centrally aligned jet (jet angle 89 degrees). Orange lines: Site of phase contrast planes; Red arrows: direction of flow Fig. 2 Assessment of jet eccentricity. A, B double-oblique long-axis of a bicuspid aortic valve with an eccentric jet (jet angle 65 degrees). C, D
trileaflet aortic valve with a centrally aligned jet (jet angle 89 degrees). Orange lines: Site of phase contrast planes; Red arrows: direction of flow Fig. 2 Assessment of jet eccentricity. A, B double-oblique long-axis of a bicuspid aortic valve with an eccentric jet (jet angle 65 degrees). C, D
trileaflet aortic valve with a centrally aligned jet (jet angle 89 degrees). Orange lines: Site of phase contrast planes; Red arrows: direction of flow mmHg]. Demographic, echocardiographic and CMR
characteristics are depicted in Table 1. A bicuspid aortic
valve was present in 36 (41%) patients. No patient had
more than trace mitral regurgitation. Aortic diameter
was higher in bicuspid vs tricuspid patients (3.6 ± 0.5 vs
3.4 ± 0.4 cm respectively, p = 0.037). Thirty-three (38%)
patients were scanned at 1.5T and 55 (62%) at 3T. and any difference exceeding this limit was considered a
significant underestimation or overestimation. Receiver
operating curves (ROC) were performed to derive the
best thresholds for each parameter associated with dis-
cordance. Variability of measurements for SVLVOT and
SVAA vs SVVM were stratified according to aortic ste-
nosis severity, aortic valve morphology and eccentric-
ity of the jet. Statistical analyses were performed with
STATA (version 15.3, Stata Corporation, College Station,
Texas, USA). A two-sided p value < 0.05 was considered
significant. Forward stroke volume estimation according to different
methodsf LV SVs by different methods are shown in Table 1. Over-
all, correlation between SVAAo and SVLVOT was excellent
(r = 0.89, p < 0.001, Additional file 1: Figure S2). However,
SVAAo lead to lower SV values than SVLVOT, while SVVM
was statistically higher than both SVLVOT and SVAAo (both
p < 0.001, Additional file 1: Table S1). Exclusion of the Factors associated with discordance between SVAAo
and SVVMh There was no relationship between the degree of underes-
timation of SV estimated by SVAAo as compared to SVVM
and peak aortic velocity (r = − 0.14, p = 0.19). Difference
between SVAAo and SVVM was related to the jet angle
(more discordance in more eccentric jets, Additional file 1:
Figure S3). ROC analysis suggested an angle of 85 degrees
as the best threshold to predict SVAAo vs SVVM discord-
ance (Additional file 1: Figure S4). A jet angle < 85º was
present in 45 (51%) patients and was more frequent as
aortic stenosis severity increases [aortic sclerosis: 3 (10%);
mild aortic stenosis: 22 (58%); moderate aortic stenosis 10
(77%); severe aortic stenosis: 5 (83%), p < 0.001]. In central
jets (angle 85–90°), bias between SVAAo and SVVM was
lower than for eccentric jets (absolute difference 6 ± 6 ml
vs 12 ± 9 ml respectively, p < 0.001). Concordance of SVAAo
was significantly higher in central vs eccentric jets (Fig. 4). Mathematical correction for the eccentricity angle how-
ever restored the concordance with SVVM (91% concord-
ance after correction) with lower overall bias (Table 1).hi There was a non-significant trend for better concord-
ance in patients with trileaflet vs bicuspid valves (Fig. 4). Patients with a bicuspid valve had however more frequently
eccentric jets than those with trileaflet morphology (65%
vs 35%, p < 0.001) and had higher peak aortic velocities [2.7
(2.4–3.0) m/s vs 2.2 (1.9–2.4 m/s), p < 0.01]. After multivari-
ate adjustment for valve morphology, eccentricity and peak
aortic velocity, the only variable that remained associated
with discordance between SVAAo and SVVM was eccentric-
ity of the jet, either as a continuous variable (jet angle) or
dichotomized as central jet/eccentric jet (Table 2). There
was no association between the aortic diameter and the
degree of discordance between SVAAo and SVVM (p = 0.26). *By Echocardiography. Data presented as count (%), mean ± standard deviation
or median [interquartile range] according to variable distribution. LV left
ventricular, RV right ventricular, EDV end-diastolic volume, ESV end-systolic
volume, EF ejection fraction, PC phase-contrast, PM papillary muscles, SV stroke
volume *By Echocardiography. Data presented as count (%), mean ± standard deviation
or median [interquartile range] according to variable distribution. Study population Aortic stenosis severity ranged from none to severe [peak
velocity 2.3 (1.7–3.0) m/s, mean gradient 13 (4, 7–19) Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 5 of 10 Table 1 Baseline characteristics
*By Echocardiography. Data presented as count (%), mean ± standard deviation
or median [interquartile range] according to variable distribution. LV left
ventricular, RV right ventricular, EDV end-diastolic volume, ESV end-systolic
volume, EF ejection fraction, PC phase-contrast, PM papillary muscles, SV stroke
volume
Clinical data
All patients (n = 88)
Age, years
55 [31–69]
Male sex, n (%)
60 (68%)
Bicuspid aortic valve, n (%)
36 (41%)
Echocardiographic data
Peak aortic valve velocity, m/s*
2.3 [1.7–3.0]
Mean gradient*
13 [4, 7–19]
Aortic stenosis severity*
Aortic sclerosis (Vmax < 200 cm/s)
31 (35%)
Mild (Vmax 200–300 cm/s)
38 (43%)
Moderate (Vmax 300–400 cm/s)
13 (15%)
Severe (Vmax > 400 cm/s)
6 (7%)
Aortic regurgitation*
None/trace
66 (75)
Mild
16 (18)
Moderate
6 (7)
Cardiovascular magnetic resonance data
Eccentric jet (angle < 85°), n (%)
45 (51%)
Among trileaflet valve
19 (36%)
Among bicuspid valve
26 (72%)
LVEDV, ml
155 ± 40
LVEDV (excluding PM), ml
138 ± 35
LVESV, ml
67 ± 24
LVESV (excluding PM), ml
52 ± 19
LVEF, %
57 ± 6
LVEF (excluding PM), %
63 ± 7
RVEDV, ml
159 ± 41
RVESV, ml
74 ± 25
RVEF, %
54 ± 6
Aortic diameter, cm
3.5 ± 0.4
Among trileaflet valve
3.4 ± 0.4
Among bicuspid valve
3.6 ± 0.5
Stroke volume estimations
LV stroke volume (volumetric), ml
87 ± 20
LV stroke volume (volumetric, excluding PM), ml
86 ± 20
LV stroke volume (PCAA), ml
80 ± 20
LV stroke volume (PCLVOT), ml
84 ± 20
RV stroke volume (volumetric), ml
85 ± 19 cases, vs 59% for SVAAo (p < 0.001, Fig. 3). Similar results
were obtained when papillary muscles were excluded from
blood pool (93% vs 59% concordance for SVLVOT vs SVAAo
respectively, p < 0.001). CMR field strength had no impact
on the discordance between SVAAo and SVLVOT compared
to SVVM (p = 0.12). The use of background static tissue cor-
rection in discordant cases did change the SV by an average
of 1 ± 1 ml in the LVOT and 1 ± 1 ml in the aorta, without
changing the concordant/discordant status in any case. Factors associated with discordance between SVAAo
and SVVMh LV left
ventricular, RV right ventricular, EDV end-diastolic volume, ESV end-systolic
volume, EF ejection fraction, PC phase-contrast, PM papillary muscles, SV stroke
volume papillary muscles from the blood pool led to significantly
lower end-diastolic and end-systolic volumes and signifi-
cantly higher LVEF (Table 1). SV, albeit with a statistically
significant difference (86 ± 20 ml vs 87 ± 20 ml excluding
and including papillary muscles within LV mass respec-
tively), was clinically comparable (average difference
1 ± 5 ml). SVLVOT was concordant with SVVM in 90% of the Factors associated with discordance between SVLVOT
and SVVMhi There was no significant predictor of SVLVOT/SVVM dis-
cordance for the studied variables (peak aortic velocity:
p = 0.22, jet angle: p = 0.21, aortic diameter: p = 0.58,
valve morphology: p = 0.54). Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 6 of 10 Fig. 3 Agreement between PCAA, PCLVOT and volumetric method. Upper Panels: Bland–Altman plots comparing stroke volume (SV) estimated
by phase contrast (PC) at the LVOT, AAo and corrected AAo flow respectively as compared to the reference (volumetric method). Data presented
included the papillary muscles in the blood pool (similar results obtained by excluding them). Solid red lines: mean bias ± 2 standard deviations. Dashed green line: level of zero bias. Pie charts show the proportion of concordance, over- and under-estimation of SV for 3 methods Fig. 3 Agreement between PCAA, PCLVOT and volumetric method. Upper Panels: Bland–Altman plots comparing stroke volume (SV) estimated
by phase contrast (PC) at the LVOT, AAo and corrected AAo flow respectively as compared to the reference (volumetric method). Data presented
included the papillary muscles in the blood pool (similar results obtained by excluding them). Solid red lines: mean bias ± 2 standard deviations. Dashed green line: level of zero bias. Pie charts show the proportion of concordance, over- and under-estimation of SV for 3 methods Aortic stenosis, turbulent jets and phase‑contrast CMR Aortic stenosis, turbulent jets and phase‑contrast CMR
PC-CMR is a powerful, accurate and reproducible non-
invasive tool to assess blood flow [1, 18]. However,
some caveats should be considered: (1) Acquisition
plane should be reasonably perpendicular to the direc-
tion of flow; this direction is not always in line with
the anatomic orientation of the cavity/vessel in which
the flow is measured. In some cases, 2 or more jets dif-
ferentially oriented may co-exist, and can also change
their direction throughout the cardiac cycle. Also, high
velocity jets may provoke signal loss due to flow accel-
eration and intravoxel dephasing [10]. Therefore, aortic
stenosis is challenging as it presents both problems: Assessment of aortic regurgitationh and (4) consistent with previous studies and current rec-
ommendation, aortic regurgitant volume is likely under-
estimated when assessed in the LVOT. To the best of our
knowledge this is the first study to formally explore the
validity of PC CMR measurement site in a population of
this size with various degrees of aortic stenosis severity. The grade of aortic regurgitation as determined by echo-
cardiography was none/trace in 66 (75%) patients, mild
in 16 (18%) and moderate in 6 (7%) patients. No patients
had severe aortic regurgitation as per exclusion criteria. Regurgitant volume by PC in the LVOT was 30% smaller
vs the values obtained in the aorta in the whole cohort
(3 ± 3 ml vs 5 ± 4 ml, p < 0.01) and a similar numerical
trend was observed in the 6 patients with moderate AR
(10 ± 8 vs 14 ± 7 ml, p = 0.25). Regurgitant volume esti-
mated in the AAo correlated better with the difference
between RV and LV SVs (Additional file 1: Figure S5). Discussionhi Also, PC-CMR imaging has made advances
since its first implementation, with shorter echo-times
minimizing the impact of accelerating flow. SVAAo was
acquired at a plane in the ascending aorta approximately
10 mm from the valve, which is distal to the vena con-
tracta [16]. Previous works showed that shorter echo-
times and distance from the stenosis can reduce the error
in PC assessment of flow [6, 10, 16]. While we did not
acquire flow data more distally in the AAo, it is likely that
the discordance would decrease as the measurement site
moves away from the stenosis. aortic regurgitation and is limited by a small number of
patients with significant aortic regurgitation. Neverthe-
less, our results show 30% difference between LVOT
and AAo, while the AAo correlated better with RV-LV
SV differences. This is consistent with previous studies
[11] and suggest that in the case of mixed aortic dis-
ease with both stenosis and regurgitation, using 2 sites
(LVOT for forward flow; AAo for regurgitant flow)
might provide the best assessment. Despite its good performance, the use of LVOT can be
limited in case of subvalvular flow acceleration (hyper-
trophic cardiomyopathy/sub-aortic membrane). In this
case, the selection of an alternative site to confirm for-
ward SV is advisable. The use of either an aortic plane as
distal as possible from the flow turbulence, right sided PC
planes or combination of both can be considered. Sug-
gested approaches for PC planning in different clinical
scenarios are presented in Table 3. The only factor independently associated with the
degree of discordance between SVAAo and SVVM in our
cohort was jet eccentricity. It is known that stroke vol-
ume measurement requires an imaging plane positioned
orthogonal to the main direction of flow [1, 3, 18]. How-
ever, in cases of eccentric jets (which are misaligned to
the main longitudinal axis of the aorta), optimal PC plan-
ning can be extremely challenging and time-consuming. Measurement of SV at the LVOT showed improved accu-
racy compared to the AAo. Interestingly, mathematical
correction for the eccentricity angle improved the con-
cordance which became similar to what is observed with
the LVOT measures. Regarding valve morphology, pre-
vious studies have shown that in bicuspid patients, flow
measurement at the AAo lead to underestimation of for-
ward flow [19]. However, bicuspid valves are frequently
associated with complex flow patterns. Discussionhi The main findings of this study are: (1) in patients with
aortic stenosis, SVLVOT has better overall agreement to
volumetric measurements than SVAAo; (2) jet eccentricity
is the main factor associated with discordant SVAAo; (3)
mathematical correction using measured SV and eccen-
tricity angle corrected the discordance in our population Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 7 of 10 Fig. 4 Concordance according to jet eccentricity and valve morphology. Bar charts representing the proportion of concordance, under and
overestimation according to jet eccentricity (A, B) and valve morphology (C, D) for SVAAo (left panels) and SVLVOT (right panels) Fig. 4 Concordance according to jet eccentricity and valve morphology. Bar charts representing the proportion of concordance, under and
overestimation according to jet eccentricity (A, B) and valve morphology (C, D) for SVAAo (left panels) and SVLVOT (right panels) Fig. 4 Concordance according to jet eccentricity and valve morphology. Bar cha
overestimation according to jet eccentricity (A, B) and valve morphology (C, D) fo Bar charts representing the proportion of concordance, under and
C D) for SV
(left panels) and SV
(right panels) Table 2 Univariate and multivariate analyses of correlates with absolute discordance between PCAAo and SVVM
PC phase contrast, SE standard error
Univariate
Multivariate
Model 1
Model 2
Standardized β
coefficient ± SE
P value
Standardized β
coefficient ± SE
P value
Standardized β
coefficient ± SE
P value
Peak aortic velocity
0.17 ± 0.01
0.11
0.04 ± 0.01
0.72
0.05 ± 0.01
0.69
Bicuspid valve
0.18 ± 1.71
0.10
0.05 ± 1.79
0.63
0.04 ± 1.88
0.76
Eccentric jet
0.35 ± 1.60
0.001
0.32 ± 1.80
0.007
Jet angle (°)
− 0.32 ± 0.11
0.003
− 0.28 ± 0.13
0.02 Table 2 Univariate and multivariate analyses of correlates with absolute discordance between PCAAo and SVVM suggest that aortic stenosis hemodynamic severity might
not be by itself a reason to use another site to measure
SV. Importantly, the most severe spectrum of aortic ste-
nosis is underrepresented in the current cohort, and
this absence of relation could be related to low statistical high velocity jets which are frequently eccentric. Use of
a plane upstream of the stenotic lesion (LVOT) might
circumvent these problems. Interestingly, there was no relation between SVAAo
discordance and peak aortic velocity in our cohort. This Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Page 8 of 10 power. Limitations
O
d Our data are from a single center study, and the popula-
tion limited to the inclusion criteria of the PROGRESSA
study with a low prevalence of severe aortic stenosis. We
cannot exclude that very high maximal velocity aortic
stenosis can influence SV measurement in the aorta, even
in central jets. AAo dilatation is a potential cause of flow
turbulence; while the association with discordance was
not significant, we had few patients with dilated AAo. Also, few patients had significant aortic regurgitation
because of the specific nature of the cohort study which
includes mainly patients with aortic stenosis. There was
no phantom correction for the flow acquisitions. The aor-
tic phase contrast sequences were planned relatively close
to the valve (10 mm): a plane closer to the aortic arch was
not assessed but could have decreased discordance. The
mathematical correction used in this study does not take
into account the whole complexity of flow turbulence and
will require validation in other cohorts. PC planning in
the LVOT can be potentially challenging, however imag-
ing quality was excellent in all cases with good concord-
ance with SVVM. The assessment of other sites (right
sided PC, combination of descending aorta/superior vena
cava) has not been explored. Coronary flow can explain
in part the difference between SV measured in AA vs
LVOT—however as coronary perfusion is in diastole and
we have measured systolic forward flow, this component
has likely a minor impact. Finally, time-resolved 3D PC
(4D-flow) was not performed. 4D flow has been shown to
overcome some of the 2D PC-CMR limitations and is an
extremely promising tool in this field [26]. Discussionhi Our results sug-
gest that bicuspid valve morphology is more often asso-
ciated with eccentric jets, but otherwise not directly
associated with the degree of discordance. Clinical relevance for CMR and aortic regurgitation
quantification. CMR is increasingly suggested to assess the sever-
ity of mitral regurgitation and aortic regurgitation. Several studies have evaluated the reliability of mitral
regurgitation quantification by CMR, including sys-
tematic review of more than 30 studies [20–22]. The
PC plane used to derive aortic forward flow was mostly
the AAo at sinotubular junction. Most studies do not
mention the use of SVLVOT or another site as an alter-
native to SVAAo, and did not include patients with con-
comitant aortic stenosis; however coexisting aortic
and mitral diseases are frequent in real life practice. It is estimated that up to 20% of patients have at least
two moderate valvular pathologies and this will likely
expand in the future as the prevalence is constantly
increasing [23, 24]. Regarding aortic regurgitation
quantification, potential limitations of PC have been
discussed in the presence of non-laminar flow [25]. Our
study was not designed to assess the best PC plane for Page 9 of 10 Guzzetti et al. J Cardiovasc Magn Reson (2021) 23:124 Table 3 Phase contrast suggested plane locations in patients with valvular disease, for which left heart stroke volume is needed to
compute mitral regurgitation or shunt
Aortic valve status
Best plane for phase contrast forward stroke
volume
Comments
No evidence of aortic disease
Ascending aorta
Ascending aorta is the easiest plane to acquire
Mitral regurgitation volume = SVVM − systolic
SVAAo
Presence of aortic stenosis without aortic
regurgitation
Left ventricular outflow tract
Alternatives:
1. Ascending aorta: if the flow is aligned with
the vessel. Consider angle correction in case of
eccentric jets
2. Right-sided phase contrast is reasonable in
absence of shunt or aortic/pulmonary regurgita‑
tion
Mitral regurgitation volume = SVVM − systolic
SVLVOT
Limitation: aortic regurgitation cannot be
assessed accurately using the LVOT flow
If SVAAo is used, a plane in the distal ascend‑
ing aorta is preferable to minimize the effect of
turbulent flow
Presence of aortic regurgitation or mixed aortic
disease
Need for aortic regurgitation quantification
Left ventricular outflow tract
AND
Ascending aorta
Alternative: Single plane in the ascending
aorta if the flow is aligned with the vessel. AAo: Ascending aorta; CMR: Cardiovascular magnetic resonance; LV: Left
ventricle/left ventricular; LVEDV: Left ventricular end-diastolic volume; LVEF:
Left ventricular ejection fraction; LVESV: Left ventricular end-systolic volume;
LVOT: Left ventricular outflow tract; PC: Phase-contrast; RV: Right ventricle/
right ventricular; RVEDV: Right ventricular end-diastolic volume; RVEF: Right
ventricular ejection fraction; RVESV: Right ventricular end-systolic volume; SV:
Stroke volume; SVAAo: Stroke volume measured at the ascending aorta (phase-
contrast); SVLVOT: Stroke volume measured at the LVOT (phase-contrast); Conclusion Aortic stenosis can negatively influence the PC SVAAo. SVLVOT has overall better agreement with SVVM than
SVAAo, especially in patients with eccentric jets. There-
fore, flow jet direction rather than aortic stenosis sever-
ity alone should be assessed to select the best plane
for SV measurement. However, LVOT plane under-
performs for aortic regurgitation quantification. Thus,
the use of an additional PC-CMR plane at the LVOT in
addition to -but not instead of- the conventional plane
at the AAo might be preferable in patients with mixed
aortic disease. Mathematical correction of SVAAo for
eccentric jets should be explored in future studies. Presence of LVOT obstruction (mitral systolic
anterior motion, septal hypertrophy, sub-aortic
membrane) Clinical relevance for CMR and aortic regurgitation
quantification. Con‑
sider angle correction in case of eccentric jets
Mitral regurgitation volume = SVVM − systolic
SVLVOT
Forward aortic SV: systolic SVLVOT
Aortic regurgitation volume: diastolic flow in the
AAo
If SVAAo is used, a plane in the distal ascend‑
ing aorta is preferable to minimize the effect of
turbulent flow
Presence of LVOT obstruction (mitral systolic
anterior motion, septal hypertrophy, sub-aortic
membrane)
Ascending aorta, especially for aortic regurgita‑
tion volume
Can be combined to right-sided phase contrast
to confirm determine total effective SV in the
absence of shunt
LVOT is likely not valid for either forward or
regurgitant flow
Aortic regurgitation volume: diastolic flow in the
AAo
Mitral regurgitation volume:
SVVM—SVright side—AR volume (if no shunt); or
SVVM—systolic SVAAo (consider angle correction if
eccentric jet)
If systolic SVAAo is used, a plane in the distal
ascending aorta is preferable to minimize the
effect of turbulent flow Table 3 Phase contrast suggested plane locations in patients with valvular disease, for which left heart stroke volume is needed to
compute mitral regurgitation or shunt Best plane for phase contrast forward stroke
volume
Comments Presence of LVOT obstruction (mitral systolic
anterior motion, septal hypertrophy, sub-aortic
membrane) Mitral regurgitation volume: Mitral regurgitation volume: SVVM—SVright side—AR volume (if no shunt); or
SVVM—systolic SVAAo (consider angle correction if
eccentric jet) If systolic SVAAo is used, a plane in the distal
ascending aorta is preferable to minimize the
effect of turbulent flow Potential limitation should be acknowledged
when using multiple PC planes as the risk of error
increases with the number of measurements SVVM: Stroke volume measured using volumetric method; TTE: Transthoracic
echocardiography. The online version contains supplementary material available at https://doi.
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the manuscript; MS, LT, EL, PB and MAC have contributed to data acquisition
and performed important revisions to the manuscript. All authors read and
approved the final manuscript. Supplementary Information The online version contains supplementary material available at https://doi.
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Insecticide resistance status of malaria vectors Anopheles gambiae (s.l.) of southwest Burkina Faso and residual efficacy of indoor residual spraying with microencapsulated pirimiphos-methyl insecticide
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Insecticide resistance status of malaria vectors
Anopheles gambiae (s.l.) of southwest Burkina Faso and
residual efficacy of indoor residual spraying with
microencapsulated pirimiphos-methyl insecticide Insecticide resistance status of malaria vectors
Anopheles gambiae (s.l.) of southwest Burkina Faso and
residual efficacy of indoor residual spraying with
microencapsulated pirimiphos-methyl insecticide
Dieudonné Diloma Soma, Barnabas Zogo, Domonbabele François de Sales
Hien, Aristide Sawdetuo Hien, Didier Alexandre Kaboré, Mahamadi Kientega,
Anicet Georges Ouédraogo, Cedric Pennetier, Alphonsine Amanan Koffi,
Nicolas Moiroux, et al. To cite this version: Dieudonné Diloma Soma, Barnabas Zogo, Domonbabele François de Sales Hien, Aristide Sawdetuo
Hien, Didier Alexandre Kaboré, et al.. Insecticide resistance status of malaria vectors Anopheles
gambiae (s.l.) of southwest Burkina Faso and residual efficacy of indoor residual spraying with mi-
croencapsulated pirimiphos-methyl insecticide. Parasites & Vectors, 2021, 14 (1), 10.1186/s13071-
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abroad, or from public or private research centers. Soma et al. Parasites Vectors (2021) 14:58
https://doi.org/10.1186/s13071-020-04563-8 Parasites & Vectors Abstract Keywords: Vector control, Resistance, Chemical analysis, Burkina Faso, IRS Keywords: Vector control, Resistance, Chemical analysis, Burkina Faso, IRS Insecticide resistance status of malaria
vectors Anopheles gambiae (s.l.) of southwest
Burkina Faso and residual efficacy of indoor
residual spraying with microencapsulated
pirimiphos‑methyl insecticide Dieudonné Diloma Soma1,2,3*† , Barnabas Zogo3,4†, Domonbabele François de Sales Hien1,
Aristide Sawdetuo Hien1,2, Didier Alexandre Kaboré1,2, Mahamadi Kientega1,2, Anicet Georges Ouédraogo2,
Cédric Pennetier3,4, Alphonsine Amanan Koffi4, Nicolas Moiroux1,3† and Roch Kounbobr Dabiré1† Abstract Background: The rapid spread of insecticide resistance in malaria vectors and the rebound in malaria cases observed
recently in some endemic areas underscore the urgent need to evaluate and deploy new effective control interven-
tions. A randomized control trial (RCT) was conducted with the aim to investigate the benefit of deploying com-
plementary strategies, including indoor residual spraying (IRS) with pirimiphos-methyl in addition to long-lasting
insecticidal nets (LLINs) in Diébougou, southwest Burkina Faso. Methods: We measured the susceptibility of the Anopheles gambiae (s.l.) population from Diébougou to conven-
tional insecticides. We further monitored the efficacy and residual activity of pirimiphos-methyl on both cement and
mud walls using a laboratory susceptible strain (Kisumu) and the local An. gambiae (s.l.) population. Methods: We measured the susceptibility of the Anopheles gambiae (s.l.) population from Diébougou to conven-
tional insecticides. We further monitored the efficacy and residual activity of pirimiphos-methyl on both cement and
mud walls using a laboratory susceptible strain (Kisumu) and the local An. gambiae (s.l.) population. Results: An. gambiae (s.l.) from Diébougou was resistant to DDT, pyrethroids (deltamethrin, permethrin and alpha-
cypermethrin) and bendiocarb but showed susceptibility to organophosphates (pirimiphos-methyl and chlorpyrimi-
phos-methyl). A mixed-effect generalized linear model predicted that pirimiphos-methyl applied on cement or mud
walls was effective for 210 days against the laboratory susceptible strain and 247 days against the local population. The residual efficacy of pirimiphos-methyl against the local population on walls made of mud was similar to that of
cement (OR = 0.792, [0.55–1.12], Tukey’s test p-value = 0.19). Results: An. gambiae (s.l.) from Diébougou was resistant to DDT, pyrethroids (deltamethrin, permethrin and alpha-
cypermethrin) and bendiocarb but showed susceptibility to organophosphates (pirimiphos-methyl and chlorpyrimi-
phos-methyl). A mixed-effect generalized linear model predicted that pirimiphos-methyl applied on cement or mud
walls was effective for 210 days against the laboratory susceptible strain and 247 days against the local population. The residual efficacy of pirimiphos-methyl against the local population on walls made of mud was similar to that of
cement (OR = 0.792, [0.55–1.12], Tukey’s test p-value = 0.19). Conclusions: If data on malaria transmission and malaria cases (as measured trough the RCT) are consistent with
data on residual activity of pirimiphos-methyl regardless of the type of wall, one round of IRS with pirimiphos-methyl
would have the potential to control malaria in a context of multi-resistant An. gambiae (s.l.) for at least 7 months. Background *Correspondence: dieusoma@yahoo.fr *Correspondence: dieusoma@yahoo.fr
†Dieudonné Diloma Soma, Barnabas Zogo, Nicolas Moiroux and Roch
Kounbobr Dabiré contributed equally to this work
1 Institut de Recherche en Sciences de la Santé, Bobo‑Dioulasso, Burkina
Faso Long Lasting Insecticidal mosquito Nets (LLINs) and
indoor residual spraying (IRS) are major malaria vec-
tor control strategies [1]. Both strategies have had
substantial impacts on the malaria burden over the Full list of author information is available at the end of the article © The Author(s) 2021. 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://creativeco
mmons.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. However, options avail-
able for continued insecticide rotation are very lim-
ited in many endemic countries because resistance to
multiple insecticide classes is very common in vector
populations. According to WHO, resistance to organo-
chlorines and carbamates was confirmed, respectively,
in 62.4% and 30.6% of the sites tested in Africa between
2010 and 2016 [6]. Resistance to organophosphate was
less common, with 14.1% of the sites tested in Africa
confirming its occurrence [6]. (including those of LLINs and IRS used in the RCT). Furthermore, we assessed the residual bio-efficacy of
pirimiphos-methyl on mud and cement walls treated
during the trial using a susceptible strain of Anopheles
gambiae (s.s.) (Kisumu) and a wild An. gambiae (s.l.)
population. House spraying
A t lli ®300CS p
y
g
Actellic®300CS (Syngenta AG, Basel, Switzerland)
was applied at a target dosage of 1 g of active ingredi-
ent (pirimiphos-methyl) per square meter (1g a.i./m2) in
all houses of both villages in September 2017. We per-
formed IRS using Hudson® X-pert spray pumps (H.D. Hudson Manufacturing Co., Chicago, IL). The spray
pumps (15 l) were fitted with a 1.5 bar control flow
valve on the lance pressure and equipped with a ceramic
8002E nozzle according to the WHO guidelines [16]. The
spraying was performed by volunteers from the local
communities who were trained by the National Malaria
Control Program (NMCP) staff on a previous IRS cam-
paign in Diébougou in 2012. We re-trained the spray
operators and supervisors prior to IRS operations in the
villages. i
In addition to the insecticide physiological resistance,
a variety of factors can affect the effectiveness of IRS. Indeed, the residual life and efficacy of the insecticide
used can vary according to the formulation, the quality
of sprays and the type of walls (cements, mud, wood)
[10, 11]. This study was part of a randomized-controlled trial
(RCT) in the rural area of Diébougou, Southwest Bur-
kina Faso, aiming at investigating whether the use of
complementary strategies together with LLINs affords
additional reduction in malaria transmission and cases. One of the strategies evaluated was IRS with microen-
capsulated formulation of pirimiphos-methyl. Micro-
encapsulation is a technology that allows insecticides
to last longer on substrates than usual [12]. In the pre-
sent study, we tested the susceptibility of the An. gam-
biae (s.l.) population from the rural area of Diébougou
(southwest Burkina Faso) to conventional insecticides © The Author(s) 2021. 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://creativeco
mmons.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. Soma et al. Parasites Vectors (2021) 14:58 Page 2 of 9 past 15 years. Indeed, LLIN and IRS accounted for an
estimated 68 and 11% of the malaria averted cases,
respectively, between 2000 and 2015 [2]. Historically,
IRS based on DDT was the cornerstone of the global
malaria eradication campaign that led to the elimina-
tion of malaria in 15 countries in Europe and America
during the 1950s and 1960s. In Africa, however, these
campaigns were not widely implemented because of
a number of reasons including limited resources [3]. Subsequently, the coverage of IRS has dropped con-
siderably in favor of LLINs. Until 2014, very few Afri-
can countries still considered IRS as a prior action in
malaria vector control [4]. More recently in 2017, IRS
was implemented, either alone or in combination with
LLINs, in 40 African countries [5]. Interest in combin-
ing IRS with LLIN seems to have increased in recent
years across Africa because of the raise of pyrethroids
resistance within the main major malaria vectors [6]. As
of 2017, the arsenal of insecticides recommended for
IRS has been improved considerably, making available
five classes of insecticides including organochlorines,
carbamates, organophosphates, pyrethroids and neoni-
cotinoids [7, 8]. The Global Plan for Insecticide Resist-
ance Management (GPIRM) recommends rotation of
non-pyrethroid insecticides for IRS in areas where IRS
and LLIN are combined [9]. Methods
Study areah This study was carried out in two villages, Dangbara
(− 3.284°; 10.766°) and Nipodja (− 3.383°; 10.988°),
located in the Diébougou health district in southwest
Burkina Faso (Fig. 1). These villages were selected
(based on their accessibility and the presence of both
banco- and cement-made houses) among the five vil-
lages which received a pirimiphos-methyl IRS interven-
tion in a randomized control trial run in Diébougou,
southwest Burkina Faso [13]. The Diébougou area is
characterized by an average annual rainfall of 1200 mm. The climate is tropical with two seasons: one dry season
from October to May and one rainy season from June
to September. Average daily temperature amplitudes
are 18–36 °C, 25–39 °C and 23–33 °C in the dry cold
(November to February), dry hot (March to May) and
rainy season (June to October), respectively. Agricul-
ture is the main economic activity in the area, followed
by artisanal gold mining and coal and wood produc-
tions [14, 15]. Safety precautions We took standard safety precautions with regard to mix-
ing, handling and spraying insecticides [16, 17]. Spray
operators and supervisors used appropriate protective
equipment (gloves, hats, overalls, boots and facemasks). Spray operators, supervisors and householders were pro-
vided with an illustrated information sheet on the study, Soma et al. Parasites Vectors (2021) 14:58 Soma et al. Parasites Vectors (2021) 14:58 Page 3 of 9 the possible adverse events in case of inappropriate
spraying and safety precautions. We properly disposed of
the leftover insecticides and bottles according to stand-
ard procedures [16]. The householders were advised by
IRS operation teams about safety precautions to avoid
possible risks during and after spraying. They were
advised to remain outside the rooms during spraying
and until 3 h after spraying. Adult heads of households
were advised to ask their children not to intentionally
touch the sprayed walls for at least 1 day after spraying,
as the walls remained wet for about 1 day. We advised the
householders, if possible, to not scrub, mutilate or plaster
the walls until the end of the study. The medical team of
the Diébougou health district participated in the trial to
attend to any medical illnesses of the inhabitants or IRS
operation team members. three made of concrete walls) per village. On each four
inner walls, two types of filter papers (one plasticized
and one classical) were fixed to test for a possible migra-
tion of the insecticide from the filter papers to the wall
as hypothesized by Moiroux et al. [18]. Plasticized and
classical papers were fixed in areas where spray overlap
was unlikely to ensure that the quantity of insecticide was
constant. We also marked positions of filter papers on the
wall to avoid carrying out subsequent cone bioassays at
such surfaces. The filter papers were removed 24 h after
spraying and placed individually in aluminum foil with
appropriate labels (village code, house number, type of
surface and date of spraying). We stored the packed sam-
ples in a refrigerator at + 4 °C before sending them to the
WHO collaborating center, Gembloux, Belgium, for anal-
ysis of the pirimiphos-methyl content. Chemical analysis Before spraying, we attached Whatman No. 1 filter
papers (10 × 10 cm) to the four inner walls of six ran-
domly selected houses (three made of mud walls and Both wild An. gambiae (s.l.) from the study area and the
susceptible An. gambiae (s.s.) Kisumu strain (KISUMU1, Fig. 1 Location of the study areas 1 Location of the study are Soma et al. Parasites Vectors (2021) 14:58 Page 4 of 9 MRA-762, VectorBase stable ID VBS0000026 on vector-
base.org) were used in the following bioassay. We col-
lected Anopheles sp. larvae in Bagane (3.150°; 10.575°,
unsprayed village). Larvae were reared in the insectary of
IRSS (temperature 27 ± 2 °C; relative humidity: 70 ± 5%;
12 h:12 h light:dark regimen) to adulthood. We fed lar-
vae every day with Tetramin® baby fish food. After emer-
gence, mosquitoes were identified to species level using
morphological keys [19]. Adult Anopheles mosquitoes
belonging to An. gambiae (s.l.) were provided with a
sugar solution (10%), until their use for bioassays. improved frequentist properties (bias, mean squared
error) that are always finite even in cases where the maxi-
mum likelihood estimates are infinite (data separation). We used the ‘emmeans’ function of the ‘emmeans’ pack-
age to calculate estimated marginal means (EMM) of
mortality for each insecticide and 95% confidence inter-
vals [24].i We compared pirimiphos-methyl concentrations on fil-
ter papers using a linear (Gaussian) mixed effect model
(LMM) with the wall surface (mud or cement), type of
filter paper (classical or plasticized) and interaction as
fixed effects. The house and the wall in the house were
set as nested random intercept. Tukey’s post-hoc method
was used to do multiple comparisons among modalities
of the fixed terms (wall surface and paper type) using the
‘emmeans’ function of the ‘emmeans’ package [24]. Mean
differences (MD) and their 95% confidence interval were
calculated. We tested the susceptibility of an An. gambiae (s.l.)
population from Bagane (F0 derived from larval collec-
tions) to six insecticides using the standard WHO pro-
tocol [20]. We exposed four replicate samples of 20–25
non-blood-fed females, 3–5 days old, An. gambiae
(s.l.), for 60 min to each insecticide. We recorded mor-
tality after 24 h. Four insecticide classes were tested:
carbamates (bendiocarb 0.1%), pyrethroids (alphacy-
permethrin 0.05%, permethrin 0.75% and deltamethrin
0.05%), organochlorine (DDT 4%) and organophosphates
(chlorpyrifos-methyl 0.4% and pirimiphos-methyl 0.25%)
[20]. Insecticide susceptibility bioassaysh The An. gambiae (s.l.) population from Diébougou was
highly resistant to DDT and pyrethroids (alphacyperme-
thrin, permethrin and deltamethrin), with mortality rates
< 15%, as recorded in WHO susceptibility bio-assays
(Fig. 2). This population was also resistant to bendiocarb
(mortality rate = 67%, 95% CI [57; 75]). However, it was
fully susceptible (100% mortality) to both organophos-
phate insecticides tested (pirimiphos-methyl and chlor-
pyriphos-methyl). No mortality (0%) was observed in the
negative control tubes (silicon oil). Mortality rate of the
susceptible An. gambiae (s.s.) Kisumu mosquitoes for all
the insecticides tested was 100%. Chemical analysis As a negative control, two replicates of the same
batch of mosquitoes were exposed to silicon oil-impreg-
nated papers. As a positive control, four replicates of
susceptible An. gambiae (s.s.) Kisumu mosquitoes were
tested with all insecticides. For each strain, we analyzed the mortality rate recorded
in cone bioassays using a binomial response mixed effect
model. We set the wall surface (cement or mud), time
after spraying (log-transformed) and interactions as fixed
effects. The house was set as a random intercept. Odds
ratio (OR) and their 95% confidence intervals (CI) were
computed. The ‘predict’ function in R applied on the bio-
assay mortality models was used to predict the time at
which mortality fell under the 80% mortality threshold. We computed 95% confidence intervals of predictions. In the same 12 houses randomly selected for chemi-
cal analysis, WHO cone tests were performed on days
2, 30, 60, 90, 120, 150, 180 and 210 post-spraying using
both susceptible An. gambiae (s.s.) and wild An. gambiae
(s.l.). Bioassays were further performed on day 360, but
only using the susceptible An. gambiae (s.s.) Kisumu. In
each house, we performed WHO cone tests on the four
inner walls according to WHO guidelines [20]. A WHO
cone test consists of introducing 10 to 15 unfed mosqui-
toes (3–5 days old) into a standard WHO cone for 30 min
of exposure to the wall. As a control, four cone tests were
performed on four unsprayed blocks. After exposure
time, mosquitoes were placed in 150 ml plastic cups (1
replicate per cup) with 10% sucrose solution. All mosqui-
toes were held for 24 h in the laboratory (27 °C ± 2 °C and
70% ± 5% relative humidity) to assess mortality. Discussion Insecticide resistance management has remained a major
challenge for malaria control and elimination for years
[6, 9]. This is, in large part, because malaria vectors are
developing resistance to most of the insecticides cur-
rently used in public health [6]. Insecticide susceptibil-
ity assays showed high resistance of An. gambiae (s.l.)
from Diébougou to all pyrethroids tested (deltamethrin,
permethrin and alphacypermethrin). Our results are
consistent with that of a recent investigation conducted
in an area of southwest Burkina Faso in 2016 [25]. How-
ever, compared with data collected pre-2010, this study
suggests that the prevalence of pyrethroid resistance has
increased considerably over time [26–28]. In addition,
DDT and Bendiocarb induced respectively 4% and 67%
mortality rates, indicating a multi-resistance of the wild
An. gambiae (s.l.) populations to pyrethroids, organo-
chlorines and carbamates. Chemical analysis
ll We analyzed insecticide susceptibility of the wild An. gambiae (s.l.) using a binomial generalized model with
the mortality recorded in each tube as the response and
the insecticide as fixed effect. The ‘brglm’ function of the
‘brglm’ package [21] in the software ‘R’ [22] was used for
this analysis. It allows to fit binomial-response regres-
sion models using the bias-reduction method developed
by Firth [23]. These procedures return estimates with On cement walls, chemical analysis indicated that the
mean concentrations of pirimiphos-methyl on classi-
cal and plasticized filter papers were 1428 mg/m2 (95%
CI [719; 2136]) and 1421 mg/m2 (95% CI [713; 2130]),
respectively (Fig. 3a). We did not find a difference in
pirimiphos-methyl concentration between the classical
and plasticized papers applied on cement walls: mean Soma et al. Parasites Vectors (2021) 14:58 Page 5 of 9 difference (MD) = 6.13 mg/m2 (95% CI [− 309; 322]),
Tukey’s test p-value = 0.96. show that pirimiphos-methyl treatment was effective
(mortality > 80%) until the 247th day post-spraying,
on both cement and mud walls (Fig. 4b). The residual
efficacy of pirimiphos-methyl was lower on mud than
on cement walls (OR = 0.257, 95% CI [0.07; 0.86],
p-value = 0.02). difference (MD) = 6.13 mg/m2 (95% CI [− 309; 322]),
Tukey’s test p-value = 0.96. On mud walls, chemical analysis indicates that the
mean concentrations of pirimiphos-methyl on classi-
cal and plasticized papers from sprayed houses were
1569 mg/m2 (95% CI [861; 2278]) and 1665 mg/m2 (95%
CI [957; 2373]), respectively (Fig. 3b). We were not able
to find a difference in pirimiphos-methyl concentra-
tion between classical and plasticized papers applied
on mud walls (MD = − 95.76, 95% CI [− 411; 220],
Tukey’s test p-value = 0.54). Moreover, the pirimiphos-
methyl concentration on papers placed on cement did
not differ from that on mud walls (MD = − 193, 95% CI
[− 1039; 653], Tukey’s test p-value = 0.65). Insecticide residual efficacy 3 Applied doses of pirimiphos-methyl on cement (a) and mud (b) walls. Red diamonds show the mean concentrations of pirimiphos-methyl
on filter papers. Boxes show first and third quartiles as well as the median concentration. The whiskers extend to the largest and lowest values
that are no further than 1.5 * IQR (where IQR is the interquartile range or distance between the first and third quartiles). Black dots represent
concentration measured for all filter papers Fig. 3 Applied doses of pirimiphos-methyl on cement (a) and mud (b) walls. Red diamonds show the mean concentrations of pirimiphos-methyl
on filter papers. Boxes show first and third quartiles as well as the median concentration. The whiskers extend to the largest and lowest values
that are no further than 1.5 * IQR (where IQR is the interquartile range or distance between the first and third quartiles). Black dots represent
concentration measured for all filter papers the precise effective duration because further testing was
not performed beyond 7 months.hfi pyrethroids were recorded in a neighboring population
from Diébougou [25]. The same authors also describe
the presence at low frequency of the ace-1 mutation that
confers cross-resistance to both carbamates and organo-
phosphorous and evidenced the presence of metabolic
resistance mechanisms (esterase and GST) that may con-
fer resistance to all tested families of insecticides [6]. The
large-scale use of LLINs across the country [29] might
have contributed to the selection of these resistance
mechanisms, particularly those involved in pyrethroid
resistance, as well as the intense use of insecticides in
agriculture [30–32]. Nevertheless, the wild population
of An. gambiae (s.l.) from our study area was found to
be fully susceptible to organophosphorus compounds
(chlorpyrifos-methyl and pirimiphos-methyl). These data
were strengthened by the results of the WHO cone bioas-
say done in houses of two villages sprayed with pirimi-
phos-methyl CS. Indeed, the duration of residual efficacy
(mortality > 80%) of pirimiphos-methyl IRS on mud and
cement walls was > 7 months against wild strains of An. gambiae. Unfortunately, we were not able to determine The mortality model predicted that the residual effi-
cacy of pirimiphos-methyl IRS lasted for 247 days
(8–9 months) against the susceptible An. gambiae
Kisumu strain. In Benin, pirimiphos-methyl sprayed in
experimental huts has shown 9 and 6 months of effec-
tive residual efficacy on cement and mud substrates,
respectively, against susceptible An. gambiae Kisumu
[33]. Insecticide residual efficacy Predictions from the mortality model of An. gambiae
(s.l.) wild strain showed that pirimiphos-methyl efficacy
remained > 80% until the last test (i.e. 210 days after
spraying) on both cement and mud walls (Fig. 4a). We
were not able to find a difference in residual efficacy
of pirimiphos-methyl between cement and mud walls
with An. gambiae (s.l.) wild strain (OR = 0.792, 95% CI
[0.55; 1.12], p-value = 0.19). Many mechanisms might be involved in this multiple
resistance. Indeed, both L1014F and L1014S kdr muta-
tions that confer cross-resistance to organochlorine and With the susceptible An. gambiae (s.s.) Kisumu strain,
for which a supplementary test was done on day 360
post-spraying, predictions from the mortality model Fig. 2 Susceptibility of wild An. gambiae (s.l.) from southwest Burkina Faso to seven insecticides used for malaria control. Bars indicate estimated
marginal means (EMM) of mortality as predicted by a generalized linear model. Error bars represent the 95% confidence intervals of the EMMs. If
mortality falls under 90% (red dashed line), the mosquito population is considered resistant to the tested insecticide Fig. 2 Susceptibility of wild An. gambiae (s.l.) from southwest Burkina Faso to seven insecticides used for malaria control. Bars indicate estimated
marginal means (EMM) of mortality as predicted by a generalized linear model. Error bars represent the 95% confidence intervals of the EMMs. If
mortality falls under 90% (red dashed line), the mosquito population is considered resistant to the tested insecticide Fig. 2 Susceptibility of wild An. gambiae (s.l.) from southwest Burkina Faso to seven insecticides used for malaria control. Bars indicate estimated
marginal means (EMM) of mortality as predicted by a generalized linear model. Error bars represent the 95% confidence intervals of the EMMs. If
mortality falls under 90% (red dashed line), the mosquito population is considered resistant to the tested insecticide Soma et al. Parasites Vectors (2021) 14:58 Soma et al. Parasites Vectors Page 6 of 9 Fig. 3 Applied doses of pirimiphos-methyl on cement (a) and mud (b) walls. Red diamonds show the mean concentrations of pirimiphos-methyl
on filter papers. Boxes show first and third quartiles as well as the median concentration. The whiskers extend to the largest and lowest values
that are no further than 1.5 * IQR (where IQR is the interquartile range or distance between the first and third quartiles). Black dots represent
concentration measured for all filter papers Fig. Insecticide residual efficacy However, these durations dropped to 5 months on
both substrates in houses of northern Benin with suscep-
tible An. gambiae Kisumu [34]. In Ivory Coast, 5 and 7
months of residual efficacy were observed on mud and
cement walls, respectively, against An. gambiae Kisumu
[35]. In Ethiopia, Yewhalaw and colleagues observed a
6-month residual efficacy against a susceptible strain of
An. arabiensis on mud substrates [36]. In Tanzania, piri-
miphos-methyl displayed 3 to 6 months of residual effi-
cacy depending on the substrate [37]. In a multi-country
study [38], pirimiphos-methyl CS duration of residual
efficacy ranged from 2 to 9 months. Many factors such as Soma et al. Parasites Vectors (2021) 14:58 Page 7 of 9 Soma et al. Parasites Vectors Fig. 4 Efficacy (mortality) over time of indoor residual spraying of pirimiphos-methyl against wild An. gambiae (s.l.) (a) and susceptible An. gambiae
s.s (b). Mortality rates were predicted from a binomial-response mixed effect model. Pirimiphos-methyl at 4 ml/m2 targeted dose applied on mud
(dashed lines) or cement (solid lines) walls are compared. Gray areas are 95% confidence interval of predicted means. Mortality values measured on
the field are shown as blue circles (cement) and black squares (mud) of size proportional to the number of values (maximum = 20) Fig. 4 Efficacy (mortality) over time of indoor residual spraying of pirimiphos-methyl against wild An. gambiae (s.l.) (a) and susceptible An. gambiae
s.s (b). Mortality rates were predicted from a binomial-response mixed effect model. Pirimiphos-methyl at 4 ml/m2 targeted dose applied on mud
(dashed lines) or cement (solid lines) walls are compared. Gray areas are 95% confidence interval of predicted means. Mortality values measured on
the field are shown as blue circles (cement) and black squares (mud) of size proportional to the number of values (maximum = 20) Fig. 4 Efficacy (mortality) over time of indoor residual spraying of pirimiphos-methyl against wild An. gambiae (s.l.) (a) and susceptible An. gambiae
s.s (b). Mortality rates were predicted from a binomial-response mixed effect model. Pirimiphos-methyl at 4 ml/m2 targeted dose applied on mud
(dashed lines) or cement (solid lines) walls are compared. Gray areas are 95% confidence interval of predicted means. Insecticide residual efficacy Mortality values measured on
the field are shown as blue circles (cement) and black squares (mud) of size proportional to the number of values (maximum = 20) of alpha-cypermethrin on filter paper placed on mud
walls was lower than on filter papers placed on cement
walls. They hypothesized that a quantity of the insecti-
cide applied on mud surfaces may migrate from the filter
papers to the wall, diminishing the insecticide content on
classical papers placed on mud surfaces. Consequently,
they suggest the use of plasticized papers to address the
issue [18]. Here, we did not find any differences in con-
centration between papers placed on mud or cement
surfaces. Further replications of this experiment are
nevertheless recommended as we did not use the same
insecticide as Moiroux et al. quality of spraying [39], vector resistance [6, 40], season/
climate [41] and wall modifications post-application [42]
can explain the observed differences in residual efficacy
between sites and studies.if In this study, we did not find any difference in the resid-
ual efficacy between cement and mud surfaces. However,
according to the results of the above-mentioned studies,
insecticides often performed poorly on mud surfaces,
probably because these surfaces are porous and hence
absorb a quantity of the applied insecticide. Our results
suggest that mud walls in the rural area of Diébougou
might be less porous. The absence of difference in the
residual efficacy between cement and mud surfaces could
also be explained by the quantity of insecticides sprayed
on the walls, which exceeds the recommended dose. Indeed, chemical analysis of classical filter papers showed
that applied target dose ratios were 1.43 [0.81; 2.05] on
cement surfaces and 1.57 [0.95; 2.19] on mud surfaces. This would indicate that the residual efficacy of pirimi-
phos-methyl CS would be reduced if the applied dose was
within the recommended ± 25% limit of the target dose. In this study, we have tested two filter papers: classical
and plasticized papers. Plasticized papers were tested
because Moiroux et al. [18] found that the concentration To date, only microencapsulated formulation of pirimi-
phos-methyl and new formulations of clothianidin (neo-
nicotinoids) (SumiShield® 50WG and Fludora Fusion®
WP-SB) have the potential to be effective for > 6 months
[4, 7]. This was confirmed in this study for pirimiphos-
methyl. Abbreviations
IRS I d
id 6. WHO. Global report on insecticide resistance in malaria vectors
2010–2016. Geneva: World Health Organisation; 2018. p. 1–72. IRS: Indoor residual spraying; LLIN: Long-lasting insecticidal net; WHO: World
Health Organization; GPIRM: Global Plan for Insecticide Resistance Manage-
ment; INSD: Institut National de la Statistique et de la Démographie; NMCP:
National Malaria Control Program; IRSS: Institut de Recherche en Sciences de
la Santé. 7. WHO. List of WHO prequalification vector co
World Health Organisation; 2019. p. 1–5. 7. WHO. List of WHO prequalification vector control products. Genev
World Health Organisation; 2019. p. 1–5. 8. WHO. The use of DDT in malaria vector control. Global malaria pro-
gramme. Geneva: World Health Organisation; 2011. p. 1–9. 9. WHO. Global plan for insecticide resistance management in malaria vec-
tors. Geneva: World Health Organisation; 2012. p. 1–130. 9. WHO. Global plan for insecticide resistance management in malaria vec-
tors. Geneva: World Health Organisation; 2012. p. 1–130. Insecticide residual efficacy For clothianidin, no field trial was carried out
to evaluate its residual efficacy as IRS in southwest Bur-
kina Faso but local vector populations were shown to be
susceptible [43]. Therefore, pirimiphos-methyl and clo-
thianidin used in rotation or mosaic might constitute an
effective insecticide resistance management strategy in
southwest Burkina Faso. Soma et al. Parasites Vectors (2021) 14:58 Soma et al. Parasites Vectors (2021) 14:58 Page 8 of 9 Page 8 of 9 Funding This work was part of the REACT project, funded by the French Initiative
5%—Expertise France (no. 15SANIN213). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript. 17. WHO. Pesticides and their application for the control of vectors and pests
of public health importance. Geneva: World Health Organisation; 2006. p. 1–104. 18. Moiroux N, Djènontin A, Zogo B, Bouraima A, Sidick I, Pigeon O. Small-
scale field testing of alpha-cypermethrin water-dispersible granules in
comparison with the recommended wettable powder formulation for
indoor residual spraying against malaria vectors in Benin. Parasites Vec-
tors. 2018;11:1–8. Acknowledgements We are grateful to the Burkina Faso Ministry of Health and local authorities for
their participation in the study. We acknowledge the logistic support (spray
pumps) provided to the project by National Malaria Control Programs. We
thank the Diébougou district medical team, particularly Dr. Dembélé Henri
and Mr. Kaboré Adama, for field support during insecticide application. We
thank the technical staff of IRSS-DRO, especially to Ouari Ali, Meda G. Benson
and Millogo S. Abel, for their assistance. We also thank to Ouattara Adama and
Dahounto Amal for their support. We extend our sincere appreciation to the
populations from all selected villages, spray operators and all volunteers for
the support and kind cooperation during fieldwork implementation. 10. Sibanda MM, Focke WW, Labuschagne FJWJ, Moyo L, Nhlapo NS, Maity
A, et al. Degradation of insecticides used for indoor spraying in malaria
control and possible solutions. Malar J. 2011;10:1–12. 11. Djènontin A, Aïmihouè O, Sèzonlin M, Damien GB, Ossè R, Soukou B, et al. The residual life of bendiocarb on different substrates under laboratory
and field conditions in Benin, Western Africa. BMC Res Notes. 2013;6:1–6. 12. Poshadri A, Kuna A. Microencapsulation technology: a review. J Res
ANGRAU. 2010;38:86–102. 13. Soma DD, Zogo BM, Somé A, Tchiekoi BN, Hien DFDS, Pooda HS, et al. Anopheles bionomics, insecticide resistance and malaria transmis-
sion in southwest Burkina Faso: a pre-intervention study. PLoS ONE. 2020;15:1–21. Availability of data and materials All relevant data are within the manuscript and its Additional files. The data-
sets used during this article are fully available without restriction. Consent for publication
Not applicable. 21. Kosmidis I. Bias reduction in binomial-response generalized linear mod-
els. 2019. https://github.com/ikosmidis/brglm. BugReports. p. 1–24. 22. The R Development Core Team. R : a language and environment for
statistical computing. 2008. http://www.gnu.org/copyleft/gpl.html. p. 1–2547. References 1. WHO. World malaria report 2019. Geneva: World Health Organisation;
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CNRS, Univ. Montpellier, Montpellier, France. 4 Institut Pierre Richet, Institut
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ticide pirimiphos methyl for prolonged control of pyrethroid-resistant
mosquitoes: an experimental hut trial in Benin. PLoS ONE. 2013;8:1–10. 34. Salako AS, Dagnon F, Sovi A, Padonou GG, Aïkpon R, Ahogni I, et al. Publisher’s Note 34. Salako AS, Dagnon F, Sovi A, Padonou GG, Aïkpon R, Ahogni I, et al. Efficacy of Actellic 300 CS-based indoor residual spraying on key ento-
mological indicators of malaria transmission in Alibori and Donga, two
regions of northern Benin. Parasites Vectors. 2019;12:1–14. Springer Nature remains neutral with regard to jurisdictional claims in pub-
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https://cyberleninka.ru/article/n/the-study-of-biochemical-parameters-of-liver-mitochondria-as-markers-of-hypoxia-in-burn-regeneration-after-experimental-thermal-injury-1/pdf
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The Study of Biochemical Parameters of Liver Mitochondria as Markers of Hypoxia in Burn Regeneration after Experimental Thermal Injury
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Sovremennye tehnologii v medicine
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cc-by
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biophotonics in regenerative medicine
The Study of Biochemical Parameters of Liver Mitochondria
as Markers of Hypoxia in Burn Regeneration
after Experimental Thermal Injury
DOI: 10.17691/stm2018.10.1.10
Received October 23, 2017
A.G. Soloveva, PhD, Senior Researcher, Head of the Department of Experimental Medicine,
Institute of Traumatology and Orthopedics;
K.V. Kulakova, PhD, Research Assistant, Department of Tissue Preservation,
Institute of Traumatology and Orthopedics;
N.Yu. Orlinskaya, DSc, Professor, Head of the Department of Pathology and Tissue Preservation,
Institute of Traumatology and Orthopedics;
E.A. Galova, MD, PhD, Scientific Director, University Clinic
Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod,
603005, Russia
The aim of this work was to study pro- and antioxidant systems and energy-generating functions of mitochondria in case of combined
thermal injury.
Materials and Methods. The experiment was carried out on male rats of Wistar line. Two groups were formed: control group 1 (n=10)
of intact healthy animals; experimental group 2 (n=10) of animals with combined thermal injury (20% contact burns and thermal inhalation
impact of hot air and combustion products). Animals were taken out of the experiment on days 1, 7, and 14 post-injury by decapitation under
anesthesia (Zoletil 100 + XylaVET).
Mitochondria were obtained by differential centrifugation. For the mitochondria identification, an electron microscopic study was
conducted. In the liver mitochondria, the intensity of free radical oxidation, the activity of catalase, superoxide dismutase, succinate
dehydrogenase, and cytochrome c oxidase were evaluated. The research results were processed using Statistica 6.0 (StatSoft Inc., USA).
Results. An increase of intensity of free radical oxidation in the liver mitochondria on days 7 and 14 post-injury was registered.
Meanwhile, total antioxidant activity of blood plasma and catalase activity in erythrocytes in case of thermal injury were decreasing on all
test days after the burn as compared to the control group.
Towards days 7 and 14, superoxide dismutase activity significantly decreased in comparison with the healthy animals. The study of
succinate dehydrogenase and cytochrome c oxidase showed a decrease in specific activity of enzymes in the liver mitochondria on days
1, 7, and 14 after combined thermal injury. The most pronounced decrease in the activity of succinate dehydrogenase and cytochrome c
oxidase was observed on day 14 after the burn.
Conclusion. A presence of oxidative stress during combined thermal injury was revealed, as well as a comprehensive mechanism
of its formation, implicating both activation of free radical oxidation and decrease in antioxidative capacity imbalance in the functioning of
prooxidant and antioxidant systems of the body. Inhibition of energy supply of cells, reduction in the cell aerobic and increased anaerobic
oxidation were revealed.
Key words: mitochondria; combined thermal trauma; free radical oxidation; antioxidant system; succinate dehydrogenase; cytochrome
c oxidase.
Hоw to cite: Soloveva A.G., Kulakova K.V., Orlinskaya N.Yu., Galova E.A. The study of biochemical parameters of liver mitochondria
as markers of hypoxia in burn regeneration after experimental thermal injury. Sovremennye tehnologii v medicine 2018; 10(1): 81–87, https://
doi.org/10.17691/stm2018.10.1.10
Russian
Исследование биохимических показателей митохондрий печени
как маркеров гипоксии при регенерации ожоговой раны
после экспериментальной термической травмы
Цель исследования — изучение про- и антиоксидантной систем, а также энергообразующей функции митохондрий при комбинированной термической травме.
Corresponding author: Anna G. Soloveva, e-mail: sannag5@mail.ru
The Biochemical Markers of Hypoxia in Burn Regeneration
СТМ ∫ 2018 — vol. 10, No.1 81
biophotonics in regenerative medicine
Материалы и методы. Эксперимент выполнен на крысах-самцах линии Wistar. Сформировано две группы: 1-я, контрольная
(n=10), — интактные здоровые животные; 2-я, опытная (n=10), — животные с комбинированной термической травмой (контактный
ожог на площади 20% поверхности тела и термоингаляционное воздействие горячим воздухом и продуктами горения). Животных
выводили из эксперимента на 1, 7 и 14-е сутки после травмы путем декапитации под наркозом (Золетил + Ксила).
Митохондрии получали путем дифференциального центрифугирования. Для их идентификации проведено электронно-микроскопическое исследование. В митохондриях печени оценивали интенсивность свободнорадикального окисления, активность каталазы, супероксиддисмутазы, сукцинатдегидрогеназы, цитохром с-оксидазы. Результаты исследований обрабатывали с использованием программы Statistica 6.0 (StatSoft Inc., USA).
Результаты. Отмечено повышение интенсивности свободнорадикального окисления в митохондриях печени на 7-е и 14-е
сутки после травмы. При этом происходило снижение общей антиоксидантной активности плазмы крови и активности каталазы в
эритроцитах при термической травме во все исследуемые сутки после ожога по сравнению с контролем.
К 7-м и 14-м суткам активность супероксиддисмутазы статистически значимо уменьшилась по сравнению со здоровыми животными. Исследование сукцинатдегидрогеназы и цитохром с-оксидазы показало снижение удельной активности ферментов в митохондриях печени на 1, 7 и 14-е сутки после комбинированной термической травмы. Наиболее выраженное снижение активности
сукцинатдегидрогеназы и цитохром с-оксидазы отмечено на 14-е сутки после ожога.
Заключение. Выявлены наличие окислительного стресса при комбинированной термической травме, а также комплексный
механизм его формирования, подразумевающий как активацию свободнорадикального окисления, так и снижение антиокислительного потенциала с нарушением баланса в функционировании про- и антиоксидантных систем организма. Установлено угнетение
энергетического обеспечения клетки, снижение в ней аэробного и усиление анаэробного окисления.
Ключевые слова: митохондрии; комбинированная термическая травма; свободнорадикальное окисление; антиоксидантная
система; сукцинатдегидрогеназа; цитохром с-оксидаза.
Introduction
The study of structural and functional features of
mitochondria enhances understanding about the origins
and development of many pathological processes at the
cellular level and in the organism. Damages associated
with characteristic abnormalities of the biochemical
processes of the cell form the basis of specific clinical
manifestations of human diseases. Burns are one of the
most important problems of modern medicine as well as
of surgical practices. Pathological changes developing
in the liver in burn disease lead to the hypoxia and the
disruption of many organism functions. A comprehensive
study of the processes occurring in the conditions of
oxygen lack is vital in the burn disease. In this situation,
the metabolic disorder is one of the most characteristic
hypoxia manifestations [1].
Increase of free radical oxidation (FRO) and the
disruption of the antioxidant defense system are the
most pathological changes induced in organs and tissues
during hypoxia and lead to oxidative stress [2]. Oxidative
stress causes an increase in intracellular generation of
reactive oxygen species (ROS) and oxidative damage of
molecular structures in cells [3].
Mitochondria accumulate a lot of the oxidative
metabolic pathways and contain numerous redox vectors
and sites potentially capable to one-electron reduction
of oxygen into superoxide anion (O2•–) — the precursor
of other ROS [4]. Generated O2•– may be involved
in the regulation of cell metabolism or eliminated by
mitochondrial superoxide dismutase [5]. The energy and
oxidative metabolism of hepatocytes are determined by
the state of the respiratory chain of mitochondria, which
depends on the functioning of the liver.
82 СТМ ∫ 2018 — vol. 10, No.1
The level of production of ROS in the mitochondria of
hepatocytes significantly increases in pathologies of the
liver [6]. However, the mechanism of increased production
of O2•− in the respiratory chain of mitochondria in thermal
injury is practically not studied. In addition, a study of
the metabolic state of rat liver mitochondria could help
in the development of adequate tactics of burn disease
treatment.
Evaluation of the activity of succinate dehydrogenase
(SDH) can serve as one of the criteria of hypoxia. SDH
largely determines the rate of consumption of oxygen
and formation of adenosine triphosphate (ATP) in the
respiratory chain of mitochondria [7].
Thus, the study of regulation of mitochondrial functions
is one of the most important problems, the solution
to which will contribute to the normalization of energy
processes playing a critical role in the metabolism of cells
in hypoxia after burn disease.
The aim of this work was to study prooxidant and
antioxidant systems, energy-generating functions of
mitochondria in case of combined thermal injury.
Materials and Methods
The experiment was carried out on 20 white male rats
of Wistar line weighing among 200–250 g received from
the Stolbovaja farm (Moscow, Russia). All animals were
kept in standard vivarium conditions in cages with free
access to food and water. Research work was carried out
according to the rules of the European Convention for the
Protection of Vertebrate Animals used for Experimental
and Other Scientific Purposes (Strasbourg, 2006). After
the quarantine (14 days) two groups of animals were
formed: control group 1 (n=10) of intact healthy animals;
A.G. Soloveva, K.V. Kulakova, N.Yu. Orlinskaya, E.A. Galova
biophotonics in regenerative medicine
experimental group 2 (n=10) of animals with combined
thermal injury (CTI).
Combined thermal injury including contact burns on
20% of the body surface and thermo-inhalation exposure
to hot air and combustion products for 20–30 s in the
inhalation chamber was performed under anesthesia
(Zoletil 100, VIRBAC, France (60 mg/kg) + XylaVET,
Pharmamagist Ltd., Hungary (6 mg/kg)) [8]. Animals
were taken out of the experiment on days 1, 7, and 14 by
decapitation with preliminary carotid artery cut performed
under anesthesia (Zoletil (60 mg/kg) + XylaVET
(6 mg/kg)).
Mitochondria
were
obtained
by
differential
centrifugation. For the mitochondria identification, the
electron microscopic study was conducted. For electron
microscopic study, the material was preliminarily
fixed according to the standard procedure in 2.5%
glutaraldehyde in phosphate buffer followed by additional
fixation with tetroxide of osmium. After washing with buffer
solution, transaction in alcohols of the increasing strength,
and impregnation, the material was enclosed in epoxy
resin. Preparation of ultrathin sections of 100 nm from the
polymerized Epon-Araldite blocks was carried out on the
PC Power Tome ultramicrotome (RMC Products, USA).
For the contrast enhancement, we used a double staining
of ultrathin sections with uranyl acetate and lead citrate.
Electron microscopic study of the mitochondria fraction
was performed on the transmission electron microscope
HT7700 (Hitachi, Japan).
Prooxidant and antioxidant status in the liver
mitochondria
was
assessed. The
activity
of
FRO was studied using the method of induced
biochemiluminescence
on
the
BHL-06
device
(Medozons, Russia). The following parameters
of biochemiluminescence were evaluated: tg 2α,
characterizes the speed of reduction of FRO processes
in the plasma and indicates total antioxidant activity;
S, the light sum of chemiluminescence in 30 s, reflects
the potential ability of a biological object to FRO. The
concentration of malonic dialdehyde was evaluated by
Mihara, Uchiyama and Fukuzawa [9]. For the evaluation of
catalase activity, we used the spectrophotometric method
based on the determination of the rate of decomposition of
hydrogen peroxide by catalase from the sample [10]. The
activity of superoxide dismutase (SOD) was determined
by inhibition of formation of adrenaline oxidation product
[11]. The activity of SDH was determined by a method
based on the use of artificial electron acceptors [12]. The
catalytic activity of cytochrome c oxidase was recorded
according to the rate of oxidation of cytochrome c by the
method Schwitzguebel and Siegenthaler [13]. Protein
concentration was calculated by the modified method of
Lowry [14].
Statistical data processing was performed by the
software (Statistica 6.0 (StatSoft Inc., USA). The
significance of differences between groups was assessed
using Student’s t-test. The differences were considered
statistically significant at p<0.05.
The Biochemical Markers of Hypoxia in Burn Regeneration
Results
In the first phase of the study, the quality of the
mitochondrial fractions obtained with the chosen method
was assessed and evaluated. The obtained samples
are informative and suitable for high-quality electron
microscopic photographs (Figure 1).
The study revealed the polymorphism of mitochondrial
fractions: small and large mitochondria with intact
membranes were present in the examined samples.
A part of the mitochondria had a dense matrix and clearcut cristae (Figure 2 (a)).
In addition to mitochondria with preserved structure,
swollen mitochondria with violation of intra-mitochondrial
architectonics were found. In some mitochondria a
change of the electron density of the mitochondrial
matrix, its focal lysis, clarification as well as shortening,
fragmentation, and reduction of cristae (Figure 2 (b)–(d))
were observed. A part of the mitochondria, in contrast,
had a dense granular matrix, masking the cristae.
Also, electron-dense rosettes of glycogen granules,
inclusions of cisterns of the endoplasmic reticulum and
multiple ribosomes were present in the studied samples,
including in the form of clusters on the mitochondrial
membranes (Figure 3).
All of the aforementioned ultrastructural changes
in the form of the degeneration of the elements in the
mitochondrial fraction may represent a consequence
of the peculiarities of sample preparation. The quality of
the material appears to be satisfactory and sufficient for
further evaluation of metabolic changes with the selected
methods.
Mitochondria are the indicators of the functional state
of cells involved in metabolism via the Krebs cycle and
Figure 1. Electron microscopic image of the mitochondria
fraction from rat liver, transmission electron microscope
HT7700 Hitachi (38,000×)
СТМ ∫ 2018 — vol. 10, No.1 83
biophotonics in regenerative medicine
The intensity of free radical oxidation
in liver mitochondria of rats in the experiment
with combined thermal injury
S (RU)
tg 2α (RU)
Malonic
dialdehyde
(µmol/l)
Control
9.66±0.12
0.929±0.011
4.579±0.045
Burn, day 1
9.64±0.23
0.534±0.015*
4.494±0.051
Burn, day 7
11.93±0.17*
0.755±0.022*
4.984±0.028*
Burn, day 14
13.66±0.34*
0.534±0.030*
6.476±0.018*
The conditions
of the experiment
а
b
* differences were statistically significant compared with the
control (p<0.05).
c
d
Figure 2. Heterogeneity of ultrastructure of the
mitochondrial fraction from rat liver, transmission electron
microscope HT7700 Hitachi (22,000×):
(a) a dense matrix and clear-cut cristae of the mitochondria;
(b)–(d) a change of the electron density of the mitochondrial
matrix, its focal lysis, clarification as well as shortening,
fragmentation, and reduction of cristae
Figure 3. Electron microscopic image of the mitochondria
fraction from rat liver, transmission electron microscope
HT7700 Hitachi (10,000×)
transport of electrons in the respiratory chain. The energy
produced by mitochondria is converted and stored within
the molecules of ATP as the result of oxygen-dependent
free radical reactions related to the oxidation in the
respiratory chain of mitochondria [6].
84 СТМ ∫ 2018 — vol. 10, No.1
According to biochemiluminescence (S) and content
of malonic dialdehyde in liver mitochondria no statistically
significant differences between control and experimental
groups (day 1 after the trauma) were revealed. This was
probably caused by an increase of consumption of fatty
acids, mainly polyunsaturated [15]. An increase in the
intensity of FRO was observed in the liver mitochondria
on day 7 (by 1.2 times, p=0.017) and day 14 (by 1.4
times, p=0.023) after injury (see the Table). Similar
results were obtained in the study of the concentration of
the intermediate FRO product. The malonic dialdehyde
concentration in the liver mitochondria increased on day
7 after CTI by 1.1 times (p=0.035), on day 14 — by 1.4
times (p=0.041) in comparison with that of the intact rats.
The growth of FRO during the burn was not accompanied
by an increase in the total antioxidant protection. While
there was a decrease in total antioxidant activity on all
tested days after thermal injury: on day 1 after CTI — by
1.7 times (p=0.018), on day 7 — by 1.2 times (p=0.009),
on day 14 — by 1.7 times (p=0.011) in comparison with
the control.
A compensatory increase of the specific activity of
a key enzyme of the antioxidant system SOD by 1.1
times (p=0.037) was observed on day 1 after CTI in
comparison to healthy animals. On days 7 and 14 SOD
activity decreased by 1.8 times (p=0.036 and p=0.041,
respectively) in comparison to healthy animals (Figure 4).
Catalase activity decreased on day 1 after CTI by 4.1
times (p=0.001), on day 7 — by 2.5 times (p=0.001),
on day 14 after injury — by 3.7 times (p=0.003) in
comparison to control (Figure 5).
The study of one of the key enzymes of the citric
acid cycle of Krebs, SDH, involved in the reaction of
dehydrogenation of succinic acid to fumaric acid showed
a decrease in the specific activity of SDH (complex II) in
mitochondria of the liver on days 1, 7, and 14 after CTI
by 1.1 times (p=0.016), by 2.6 times (p=0.017), by 10.2
times (p=0.008), respectively in comparison with healthy
rats (Figure 6).
A decrease in the activity of cytochrome c oxidase
(complex IV of the respiratory chain of mitochondria)
A.G. Soloveva, K.V. Kulakova, N.Yu. Orlinskaya, E.A. Galova
biophotonics in regenerative medicine
1800
Activity (RU/min·mg)
700
*
1600
1400
1200
*
1000
*
800
600
400
200
0
Сontrol
Burn, day 1 Burn, day 7 Burn, day 14
Figure 4. Superoxide dismutase activity in the liver
mitochondria of rats in the experiment with combined
thermal injury
* Differences were statistically significant compared with the
control (p<0.05)
Activity (RU/min·mg)
120
100
80
60
*
40
*
20
0
*
Control
Burn, day 1
Burn, day 7 Burn, day 14
600
Activity (μmol/L·min·mg)
600
500
*
400
*
300
*
200
100
0
Сontrol
Burn, day 1
Burn, day 7
Burn, day 14
Figure 7. Cytochrome c oxidase activity in the liver
mitochondria of rats in the experiment with combined
thermal injury
* Differences were statistically significant compared with the
control (p<0.05)
transferring electrons from cytochrome c to molecular
oxygen was revealed on days 1, 7, and 14 after burn by
1.5 times (p=0.002), by 1.9 times (p=0.001), and by 2.4
times (p=0.001), respectively in comparison to healthy
animals (Figure 7).
Therefore, the activity of SDH and cytochrome c
oxidase demonstrated the maximum decrease on day 14
after CTI.
Discussion
Figure 5. Catalase activity in the liver mitochondria of rats
in the experiment with combined thermal injury
* Differences were statistically significant compared with the
control (p<0.05)
500
*
400
300
*
200
100
0
Activity (μmol/L·min·mg)
2000
*
Сontrol
Burn, day 1
Burn, day 7
Burn, day 14
Figure 6. Succinate dehydrogenase activity in the liver
mitochondria of rats in the experiment with combined
thermal injury
* Differences were statistically significant compared with the
control (p<0.05)
The Biochemical Markers of Hypoxia in Burn Regeneration
The reduction of catalytic properties of SOD in a burn
was probably the result of the intensification of redox
processes and reduction of energy metabolism in the
mitochondria. The increased formation of superoxide
radical during the activation of FRO leads to substrate
inhibition of superoxide dismutase in liver mitochondria of
rats with CTI.
There is also evidence of an increase of FRO
processes of lipids and proteins with inhibition of the
antioxidant systems in patients with thermal injury in the
literature [16]. Reasons for the imbalance in the system
of prooxidants–antioxidants may be the impairment of
lipid and protein profile of mitochondria in thermal injury
[17]. On the other hand, the activation of the FRO in the
mitochondria during the burn can be one of the causes of
damage of the liver tissue.
One of the possible mechanisms of changes in
the activity of catalase in burn disease may be a
conformational modification of the enzyme molecule
under conditions of oxidative stress.
Thus, the obtained results confirm information
about the presence of oxidative stress manifested in
the enhancement of FRO and the decrease of general
and enzymatic activity of antioxidant defense system
in thermal injury. This leads to the development of
systemic disorders during the burn. Causes of oxidative
СТМ ∫ 2018 — vol. 10, No.1 85
biophotonics in regenerative medicine
stress under CTI may be excessive ROS production,
the activation of the FRO and the negative response
of reserves of the antioxidant protective system. The
development of oxidative stress leads to an increase of
destructive processes, which may be a pathogenetic
factor of burn disease.
The decrease in the specific activity of SDH in
mitochondria of the liver on days 1, 7, and 14 after CTI
indicates a decrease in succinate dependent respiration
in the mitochondria and reduction of activity of the citric
acid cycle. SDH is associated with a chain of electron
transfer and in contrast to other enzymes of the Krebs
cycle is an integral protein of the internal mitochondrial
membrane. Two electrons of flavin adenine dinucleotide
(FADH2) are directly transferred to the iron atoms of the
enzyme. Specific activators of SDH are ATP and restored
ubiquinone played a role as an acceptor of electrons from
succinate dehydrogenase complex (where SDH is the
first enzyme, and cytochrome c oxidase — the last) in the
mitochondria [7].
In addition, the decrease in the activity of SDH in the
conditions of O2 deficiency, developing in the cells in
thermal injury [18], is due to the activation of fumarate
reductase reaction. Fumarate reductase reaction is
the reverse of the reaction catalyzed by SDH in the
composition of the complex II system of tissue respiration.
In this process, free oxygen radicals that can directly
inactivate SDH are quickly formed [19]. Therefore, the
increase of their formation in conditions of lack of oxygen
led to a decrease of SDH activity.
The decrease in activity of SDH and cytochrome
c oxidase at CTI indicates a reduction of aerobic
metabolism, enhancement of anaerobic oxidation in the
cell and decrease of energy supply of the cells. These
changes appear from the disruption of electron transport
in the mitochondrial membrane and deficiency of ATP
production.
Probably a large number of ROS generated in
thermal injury are associated with the metal atoms within
cytochrome c oxidase, inhibiting the enzyme.
However, according to the literature data, information
about the state of the respiratory chain in burn disease
is very divergent and insufficient. Some authors argue
that the components of the respiratory chain in the
pathology of the liver are seriously damaged therefore
ATP decreases [20]. However, detailed studies of the
mechanisms of these disorders have not been carried
out yet. Other authors consider that the components
of the respiratory chain are not damaged, but unable
to change their functional activity [21]. The obtained
results allow the evaluation of the energy metabolism
changes during hypoxia developing in burn disease.
The study obtained fundamental data on changes in
aerobic and anaerobic metabolism of mitochondria of the
liver, which will help to expand existing concepts on the
pathogenetic mechanisms of hypoxia during pregnancy
with the subsequent possibility of searching for ways of its
correction. A critical decline of oxygen tension in tissue in
86 СТМ ∫ 2018 — vol. 10, No.1
a thermal injury leads to activation of anaerobic formation
of energy in the cell.
Conclusion
The obtained data confirm, firstly, the presence of
mitochondrial oxidative stress during combined thermal
injury and, secondly, a comprehensive mechanism of its
formation, including activation of free radical oxidation
and decrease in antioxidative potential. The inhibition of
energy support of mitochondria was detected during the
burn and manifested itself through decreased activity of
succinate dehydrogenase and cytochrome c oxidase
in mitochondria, which is a sign of reduction of aerobic
oxidation and intensification of anaerobic metabolism in
the cell.
In the current study, it was demonstrated that
identification of the activities of antioxidant enzymes and
succinate dehydrogenase as well as of cytochrome c
oxidase can be used as markers of metabolic disturbances
in the liver during hypoxia due to thermal injury.
Acknowledgements. Electron microscopic study was
carried out at the Laboratory of Molecular Epidemiology
of Viral Infections in Blokhina Scientific Research Institute
of Epidemiology and Microbiology of Nizhny Novgorod
(Russia). The authors express their deep appreciation
to the Professor E.I. Efimov, MD, DSc, Director of the
Institute, Head of the Department of Microbiology and
Immunology, Professor N.A. Novikova, PhD, Head of the
Laboratory of Molecular Epidemiology of Viral Infections,
for providing access to the equipment; deep thanks also
go to A.Yu. Kashnikov, Research Officer, for his help in
performing the study.
Financial Support. The study was carried out as a
part of the state task No.056-00096-18-00.
Conflict interest. The authors declare that they have
no conflict interests.
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Sepsis may dissimulate a lymphoma? Case report
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* Correspondence: ol_mihai@yahoo.com
1National Institute for Infectious Diseases “Prof. Dr. Matei Balş”, Bucharest,
Romania
Full list of author information is available at the end of the article Conclusion In patients with severe infection who have a long evolution
we must search for a hematological disease. Sometimes
both hematological disease and sepsis may intertwine,
being difficult to make a plain distinction. Marrow aspiration sample showed 28% lymphoid cells. The patient was transferred in our clinic with the diagno-
sis of sepsis. From family history we remark that his
mother had died a few years back at 50 years old with
acute leukemia. After clinical exam (pallor, splenomegaly),
laboratory evaluation (severe pancytopenia: leukocytes
0.88x103/µL, neutrophils 0.49x103/µL, Hb 9.5 g/dL,
platelets 25x103/µL), LDH 1,704 IU/L, prothrombin time
16.5 s, fibrinogen 46 mg/dL, partial thromboplastin time
56.9 s, procalcitonin 3.95 ng/mL), abdominal CT scan, we
considered intra-abdominal sepsis and disseminated intra-
vascular coagulation (DIC). Consent Written informed consent was obtained from the patient
for publication of this Case report and any accompanying
images. A copy of the written consent is available for
review by the Editor of this journal. Background In aggressive lymphoma, systemic “B” symptoms of
fatigue, fever, night sweats may occur frequently, but
these symptoms may occur also in severe sepsis. Many
hematological disorders, especially lymphoid neo-
plasms, have a high risk for infection because of altered
humoral and cell-mediated immunity. After 5 days procalcitonin was 1.57 ng/mL and the clini-
cal status was ameliorated but pancytopenia persisted. The
histopathological exam and the immunohistochemistry
aspect sustained the diagnosis of diffuse large B-cell lym-
phoma and the patient was transferred to the Hematology
Clinic. Case report We present a case of a man 37 years old who was diag-
nosed in our clinic on July 2014 with diffuse large B-cell
lymphoma. On July 5 the patient was admitted to the
County Hospital Piteşti with fatigue, fever, night sweats
and epigastric pain. Laboratory evaluation remarks a
severe pancytopenia but without inflammation marks
and a positive procalcitonin. From The 10th Edition of the Scientific Days of the National Institute for Infectious Diseases “Prof Dr Matei
Bals” was completed with vancomycin 2g/day, caspofungin
50 mg/day, levofloxacin 1g/day, amikacin 1g/day and
ethambutol 1g/day and also we performed a bone marrow
biopsy. Olariu et al. BMC Infectious Diseases 2014, 14(Suppl 7):P60
http://www.biomedcentral.com/1471-2334/14/S7/P60 Olariu et al. BMC Infectious Diseases 2014, 14(Suppl 7):P60
http://www.biomedcentral.com/1471-2334/14/S7/P60 POSTER PRESENTATION Open Access © 2014 Olariu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Authors’ details
1 1National Institute for Infectious Diseases “Prof. Dr. Matei Balş”, Bucharest,
Romania. 2Carol Davila University of Medicine and Pharmacy, Bucharest,
Romania. 3Fundeni Clinical Institute, Bucharest, Romania. 4University
Emergency Hospital of Bucharest, Romania. Treatment for 7 days with meronem 3g/day, corticother-
apy and supportive therapy, fresh frozen plasma, packed
red blood cells, platelet concentrates did not ameliorate
the clinical status and surgery exam disproved our suspi-
cion. Because procalcitonin rose to 10.35 ng/mL treatment Published: 15 October 2014 doi:10.1186/1471-2334-14-S7-P60
Cite this article as: Olariu et al.: Sepsis may dissimulate a lymphoma? Case report. BMC Infectious Diseases 2014 14(Suppl 7):P60. doi:10.1186/1471-2334-14-S7-P60
Cite this article as: Olariu et al.: Sepsis may dissimulate a lymphoma? Case report. BMC Infectious Diseases 2014 14(Suppl 7):P60. © 2014 Olariu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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Numerical modeling of the impact of pump wavelength on Yb-doped fiber amplifier performance
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Optical and quantum electronics
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Cronfa - Swansea University Open Access Repository This is an author produced version of a paper published in :
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http://cronfa.swan.ac.uk/Record/cronfa30992 This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the
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http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Albalawi, A., Zhu, H., Taccheo, S., Chiasera, A., Ferrari, M., Al-Zubi, J. & Al-Zubi, O. (2016). Numerical modeling of
the impact of pump wavelength on Yb-doped fiber amplifier performance. Optical and Quantum Electronics, 48(11)
http://dx.doi.org/10.1007/s11082-016-0771-z Paper: Albalawi, A., Zhu, H., Taccheo, S., Chiasera, A., Ferrari, M., Al-Zubi, J. & Al-Zubi, O. (2016). Numerical modeling of
the impact of pump wavelength on Yb-doped fiber amplifier performance. Optical and Quantum Electronics, 48(11)
http://dx.doi.org/10.1007/s11082-016-0771-z This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the
terms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions. When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEO
database to judge whether or not it is copyright safe to add this version of the paper to this repository. http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Opt Quant Electron (2016) 48:500
DOI 10.1007/s11082-016-0771-z & Ali Albalawi
681836@swansea.ac.uk Numerical modeling of the impact of pump wavelength
on Yb-doped fiber amplifier performance Ali Albalawi1 • Hongna Zhu2 • S. Taccheo1 • A. Chiasera3 •
M. Ferrari3 • Jafar Al-Zubi4 • Omar Al-Zubi4 Received: 20 August 2016 / Accepted: 18 October 2016
The Author(s) 2016. This article is published with open access at Springerlink.com Received: 20 August 2016 / Accepted: 18 October 2016 g
p
The Author(s) 2016. This article is published with open access at Springerlink.com Abstract
Ytterbium-doped optical amplifiers have become common tools for industrial
applications due to their high efficiency, relatively low cost and potentially very high
output power level. The efficiency of an ytterbium-doped fiber amplifier depends mainly
on the absorption of pump radiation, and, therefore, optimum pump wavelengths have been
proposed such as 915 nm. However, the semiconductor pump diodes batch supplied by
manufacturers may exhibit a spread in the output wavelength. This paper theoretically
investigates the performance of Yb-doped amplifiers for different pump wavelengths and
defines the pump power penalty when the pump source does not emit at the optimum
wavelength. The penalty has been defined as normalized excess pump power required to
achieve the desired gain. Keywords Ytterbium-doped fiber amplifier Pumping efficiency Yb-doped germano-
silicate glasses 1
Laser and Photonics Group, College of Engineering, Bay Campus, Swansea University,
Swansea SA1 8EN, UK 2
School of Physical Science and Technology, Southwest Jiaotong University, Chengdu, China
3
i
di
i
l
i
l ,
2
School of Physical Science and Technology, Southwest Jiaotong University, Chengdu, China
3
Istituto di Fotonica e Nanotecnologie - CNR, Povo, Tn, Italy 4
Al-Balqa’ Applied University, Amman, Jordan 1
Laser and Photonics Group, College of Engineering, Bay Campus, Swansea University,
Swansea SA1 8EN, UK
2
School of Physical Science and Technology, Southwest Jiaotong University, Chengdu, China
3
Istituto di Fotonica e Nanotecnologie - CNR, Povo, Tn, Italy
4
Al-Balqa’ Applied University, Amman, Jordan 1 Introduction Ytterbium-doped fibre amplifiers (YDFA) provide a wide gain bandwidth, high output
power, and a high electrical to optical power conversion efficiency, making them suit-
able for medium and high power applications (Paschotta et al. 1997; Zervas 2014; Injeyan 3
Istituto di Fotonica e Nanotecnologie - CNR, Povo, Tn, Italy 4
Al-Balqa’ Applied University, Amman, Jordan 123 123 500
Page 2 of 7 500
Page 2 of 7 A. Albalawi et al. and Goodno 2011). This makes YDFAs one of the most common type of amplifiers in
numerous applications, ranging from medium to high-power amplification, such as mass,
manufacturing, fiber sensing, free-space laser communication, and ultra-short pulse
amplification (Paschotta et al. 1997; Pask et al. 1995; O’Neill 2008; Zhang et al. 2012a, b;
Zervas 2014; Nogee 2014). In case of device low price target for large market production
there is a sensitive economic-engineering issue related to supplied pump diodes. In fact the
semiconductor pump lasers are usually supplied, to reduce cost, without a specific
wavelength, and therefore the supplied batch may exhibit a significant wavelength spread. The non-optimum pump diode wavelength implies need of driving the diode with higher
current intensity to achieve the desired gain and this turns out into higher running costs as
well accelerated diode aging. The aim of this paper is to numerically explore the impact of
the diode pump wavelength on the amplifier efficiency and provide guidelines on tolerated
pump diode wavelength spread. Laser numerical simulations, are in fact a useful tool to
predict general device behaviour since the era of telecom Erbium-doped fiber amplifiers
and have now reached a high degree of sophistication (D’Orazio et al. 2005; Giles and
Desurvire 1991; He et al. 2006; Magne et al. 1994). The configuration that will be analyzed is one of the more common in laser industry, in
term of mass use, being used, for example, for marking processes, and is based on the
Master-Oscillator-Power-Amplifier (MOPA) scheme where the YDFA is seeded by a few
hundred of mW input and provide an output power of about 20 W (O’Neill 2008; Zhang
et al. 2012a; Pask et al. 1995; Snitzer 1966). We will evaluate the extra pump power
required to achieve a desired gain in case of non-optimal pump diode wavelength. We will
consider amplification of both 1030 and 1064 nm signals. Table 1 Active fiber parameters 2 Simulation We investigated a 20 W, 17 dB gain (i.e. with a 0.4 W seed) Yb-doped germane-silicate
continuous wave amplifier (Paschotta et al. 1997; Zervas 2014; Zhang et al. 2012a; Pask
et al. 1995; O’Neill 2008), being a quite common design for the large market of lasers for
marking. The doping level was set to be compatible with a fiber length ranging from 3 to
13 m. We simulated the device under continuous wave condition since results may be
extended in term of efficiency to a configuration were a ns pulsed seed is used (O’Neill
2008). Of course, in case of long fiber lengths, the amplifier pulsed amplification will suffer
from several nonlinear effects, here not considered. This was also the reason to use
maximum length of 13 m above which nonlinear effects may be detrimental. The speci-
fications of the active fiber are summarized in Table 1. The active fiber is a Yb-doped
germanium silicate fiber, doping concentration, Nyb, is 3 9 1025 m-3, and input signal Table 1 Active fiber parameters
Parameters
Yb-germanosilicate
Core diameter
5 lm
Doping concentration
3 9 1025 ions/m-3
Signal power
400 mW
Gain
17 dB
Pump cladding radius
50 lm
Radius of propagating Gaussian seed
4.8 lm
123 Table 1 Active fiber parameters
Parameters
Yb-germanosilicate
Core diameter
5 lm
Doping concentration
3 9 1025 ions/m-3
Signal power
400 mW
Gain
17 dB
Pump cladding radius
50 lm
Radius of propagating Gaussian seed
4.8 lm Table 1 Active fiber parameters
Parameters
Yb-germanosilicate
Core diameter
5 lm
Doping concentration
3 9 1025 ions/m-3
Signal power
400 mW
Gain
17 dB
Pump cladding radius
50 lm
Radius of propagating Gaussian seed
4.8 lm
123 Parameters
Yb-germanosilicate
Core diameter
5 lm
Doping concentration
3 9 1025 ions/m-3
Signal power
400 mW
Gain
17 dB
Pump cladding radius
50 lm
Radius of propagating Gaussian seed
4.8 lm Table 1 Active fiber parameters 123 3 12 Numerical modeling of the impact of pump wavelength on… Page 3 of 7
500 500 power Ps is 400 mW with a targeted 17 dB gain. The absorption and emission cross-
sections for Yb-doped germanium silicate glass fiber by Paschotta et al. (1997) are shown
in Fig. 1 (Paschotta et al. 1997; O’Connor and Shiner 2011; Weber et al. 1983), and
commercial software (RP Fiber Power) was used for the simulation (Paschotta 2011). Fig. 1 Absorption and emission
cross-section of ytterbium-doped
germanosilicate glass (Paschotta
et al. 1997) 2 Simulation In
the simulation, we used the standard ytterbium two level scheme and we included the
impact of amplified spontaneous emission (ASE) using emission and absorption cross
section data of Fig. 1 (Paschotta 2011). No phenomena, as photodarkening, depending on
the local inversion level, have been considered, yet, for the given configuration they should
not impact the comparison we investigate (Taccheo et al. 2011; Gebavi et al. 2015). The
fiber radius was set at r = 5 lm, while the length of the fiber has been varied from 3 to
13 m. A Gaussian field distribution of 4.8 um radius was used for the propagation of signal
radiation, while the pump radius is set at 50 lm, with a top-hat profile to simulate a double
cladding fiber with a 100 lm diameter outer cladding. The used software (Paschotta 2011) utilizes the Finite Difference Time Domain with a
fixed number of mesh points, N, which the software employs to solve the differential
equations. In order to avoid gain calculation errors we performed a preliminary set of
simulation, showing gain calculations reaches a stable value for N [ 50, a safeguard value
of 1000 was chosen. 3.1 Pump power for seed wavelength of 1030 and 1064 nm Figure 2a shows the required pump power versus fiber length and pump wavelength with a
1030 nm seed. We notice that for a 3 m fiber length, the minimum pump power is 47,2 W
at 914 nm and then increases as the pump diode wavelength diverges from optimum value
to reach 60.5 W at 930 nm, and 64.7 W at 890 nm as shown in Fig. 2b. As the fiber length
increases the optimum wavelength red-shift and the minimum pump power decreases as
well as the difference between minimum pump power and the power at the edges: 26.5 W
at 890 nm wavelength, 23.8 W at 918 nm wavelength (minimum) and 25.3 W at 930 nm
wavelength. However we notice that pulsed amplification in case of long fibre will be
limited by non-linear effects (Paschotta et al. 1997; Zervas 2014; Pask et al. 1995), so long
fibre case may not be practical for pulsed amplification. Ytterbium cross-section (10^-25m^-2)
Wavelength (nm)
Fig. 1 Absorption and emission
cross-section of ytterbium-doped
germanosilicate glass (Paschotta
et al. 1997)
123 Ytterbium cross-section (10^-25m^-2)
Wavelength (nm) Wavelength (nm) 500
Page 4 of 7 A. Albalawi et al. (a) 890
895
900
905
910
915
920
925
930
Pump wavelength (nm)
20
25
30
35
40
45
50
55
60
65
70
pump power(W)
L3
L6
L10
L13
(a)
(b)
Fig. 2 a Pump power versus fiber length and pump wavelength with seed wavelength of 1030 nm,
b section of a showing the pump power variation versus wavelength for fiber lengths of 3, 10 and 13 m 890
895
900
905
910
915
920
925
930
Pump wavelength (nm)
20
25
30
35
40
45
50
55
60
65
70
pump power(W)
L3
L6
L10
L13
(b) (a) Pump wavelength (nm) Fig. 2 a Pump power versus fiber length and pump wavelength with seed wavelength of 1030 nm,
b section of a showing the pump power variation versus wavelength for fiber lengths of 3, 10 and 13 m Figure 3 illustrates the same simulations for a 1064 nm seed. While the absolute power
is a slightly different from the case of the 1030 nm wavelength seed of Fig. 2a, the
difference between optimum case and edge pump wavelength excess power is very similar. 3.1 Pump power for seed wavelength of 1030 and 1064 nm For example in case of the fiber length of 3 m, minimum pump power is 50.4 W at
wavelength of 914 nm, and reaches 69.3 W at 890 nm wavelength. 3.2 Normalized pump power penalty 3 a Pump power versus fiber length and pump wavelength with seed wavelength of 1064 nm,
b section of a showing the pump power variation versus wavelength for fiber lengths of 3, 10 and 13 m 123 123 Numerical modeling of the impact of pump wavelength on… Page 5 of 7
500 3
4
5
6
7
Fiber Length (m)
8
0
9
5
890 894
10
10
898
15
902
Pump wavelength (nm)
11
906
20
910
percentage of pump
914
12
25
918 922
30
13
926
35
930
40
0
5
10
15
20
25
30
35
40
percentage of pump power change(%)
power change(%)
(a) 3
4
5
6
7
Fiber Length (m)
8
0
9
5
890 894
10
10
898
15
902
Pump wavelength (nm)
11
906
20
910
percentage of pump
914
12
25
918 922
30
13
926
35
930
40
0
5
10
15
20
25
30
35
40
percentage of pump power change(%)
5
5
5
5
5
5
10
10
10
10
10
15
15
15
20
20
20
25
30
890
894
898
902
906
910
914
918
922
926
930
Pump wavelength (nm)
3
4
5
6
7
8
9
10
11
12
13
Fiber Length (m)
power change(%)
(a)
(b)
Fig. 4 a Normalized pump power penalty versus fiber length and pump wavelength with a 1030 nm
wavelength seed, b bidimensional representation 5
5
5
5
5
5
10
10
10
10
10
15
15
15
20
20
20
25
30
890
894
898
902
906
910
914
918
922
926
930
Pump wavelength (nm)
3
4
5
6
7
8
9
10
11
12
13
Fiber Length (m)
(b) (a) percentage of pump power chang Pump wavelength (nm)
914 918 922 Fig. 3.2 Normalized pump power penalty Since each specific amplifier may require a different pump power level, we decided to
define a normalized power penalty as excess pump power percentage required to reach the
desired gain, 17 dB in our case. The normalized pump power penalty (Pn) is defined as: Pn ¼ Ppw k; L
ð
Þ Popt kmin; L
ð
Þ
Popt kmin; L
ð
Þ
100
ð1Þ ð1Þ where Ppw (k, L) is the pump power at a given fiber length, L, and for a specific pump
wavelength k, and Popt (kmin, L) is the minimum pump power required at the optimum
wavelength kmin and for the same fiber length L. where Ppw (k, L) is the pump power at a given fiber length, L, and for a specific pump
wavelength k, and Popt (kmin, L) is the minimum pump power required at the optimum
wavelength kmin and for the same fiber length L. (a) 890
895
900
905
910
915
920
925
930
Pump wavelength (nm)
20
25
30
35
40
45
50
55
60
65
70
pump power(W)
L3
L6
L10
L13
(a)
(b)
Fig. 3 a Pump power versus fiber length and pump wavelength with seed wavelength of 1064 nm,
b section of a showing the pump power variation versus wavelength for fiber lengths of 3, 10 and 13 m 890
895
900
905
910
915
920
925
930
Pump wavelength (nm)
20
25
30
35
40
45
50
55
60
65
70
pump power(W)
L3
L6
L10
L13
(b) (a) Fig. 3.2 Normalized pump power penalty 4 a Normalized pump power penalty versus fiber length and pump wavelength with a 1030 nm
wavelength seed, b bidimensional representation 3
4
5
6
7
Fiber Length (m)
8
0
9
5
890 894
10
10
898
15
902
Pump wavelength (nm)
11
906
20
910
percentage of pump power change(%)
914
12
25
918 922
30
13
926
35
930
40
0
5
10
15
20
25
30
35
40
t
f
h
(%)
(a) 3
4
5
6
7
Fiber Length (m)
8
0
9
5
890 894
10
10
898
15
902
Pump wavelength (nm)
11
906
20
910
percentage of pump power change(%)
914
12
25
918 922
30
13
926
35
930
40
0
5
10
15
20
25
30
35
40
percentage of pump power change(%)
5
5
5
5
5
5
10
10
10
10
10
15
15
15
20
20
20
25
30
890
894
898
902
906
910
914
918
922
926
930
Pump wavelength (nm)
3
4
5
6
7
8
9
10
11
12
13
Fiber Length (m)
(a)
(b)
Fig. 5 a Normalized pump power penalty versus fiber length and pump wavelength with a 1064 nm
wavelength seed, b bidimensional representation 5
5
5
5
5
5
10
10
10
10
10
15
15
15
20
20
20
25
30
890
894
898
902
906
910
914
918
922
926
930
Pump wavelength (nm)
3
4
5
6
7
8
9
10
11
12
13
Fiber Length (m)
(b) Pump wavelength (nm) Fig. 5 a Normalized pump power penalty versus fiber length and pump wavelength with a 1064 nm
wavelength seed, b bidimensional representation Figure 4a illustrates that normalized excess pump power using data from Fig. 2. Note
for sake of clarity we shows on the right side (Fig. 4b) the same data in a bidimensional
plot. Here we notice more clearly that short fibre lengths are definitively far more sensitive
to diode pump wavelength spread: up to 37.2 % normalized pump power penalty is
reached in case of using a 890 nm pump laser diodes in a 3 m long amplifier, while
normalized pump power penalty is 12.5 % at 890 nm for a 13 m fibre length. Similar results and behaviours are obtained when the signal wavelength is set at
1064 nm as shown in Fig. 5. Considering Figs. 3.2 Normalized pump power penalty 4 and 5 we notice that power penalty is very similar and therefore
some guidelines can be drawn. As example if we set a 10 % normalized pump power
penalty the allowed batch wavelength spread is between 901 and 924 nm for short fibre. We also note that penalty grows non linearly with the wavelength offset and varies from
10 % to over 25 % when moving from 898 to 890 nm pump wavelength. Results also
indicates the laser performance will be more sensitive with respect pump wavelength offset
if short fibre lengths are planned to be used, as in the case of short amplifiers for short pulse
amplification. 123 500
Page 6 of 7 A. Albalawi et al. 4 Conclusion This paper shows pump diode wavelength spread may cause an increase in the required
pump power, thus increasing the diode driver current and likely accelerating pump diode
ageing. The paper proposes a simple model to quantify the pump power penalty associate
to the use of non optimum wavelength pump laser diode. This allows to define the tolerable
pump wavelength spread. As general guideline pump power penalty is higher for shorter
fibers, preferred to reduce nonlinear phenomena, and the penalty is more severe for pump
wavelength red shifted with respect the optimum pump wavelength. As example a max-
imum of 10 % penalty is achieved within a -13/?10 nm interval with respect to the
optimum pump wavelength of 914 nm for a 3 m long fiber. In case of highly doped
amplifiers schemes using very short fibre the system will be even more sensitive. Acknowledgments Ali Albalawi acknowledges support of Cultural Bureau in London (UKSACB). This
article is based upon work from COST Action MP1401 supported by COST ‘‘(European Cooperation in
Science and Technology)’’. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter-
national License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the
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https://malariajournal.biomedcentral.com/counter/pdf/10.1186/s12936-024-04872-8
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English
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Time series analysis of malaria cases to assess the impact of various interventions over the last three decades and forecasting malaria in India towards the 2030 elimination goals
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Open Access Open Access © The Author(s) 2024. 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. Time series analysis of malaria cases
to assess the impact of various interventions
over the last three decades and forecasting
malaria in India towards the 2030 elimination
goals endra P. Singh1, Harsh Rajvanshi1,2, Praveen K. Bharti3, Anup R. Anvikar3 and Altaf A. Lal1,4,5* Abstract Background Despite the progress made in this decade towards malaria elimination, it remains a significant public
health concern in India and many other countries in South Asia and Asia Pacific region. Understanding the histori-
cal trends of malaria incidence in relation to various commodity and policy interventions and identifying the factors
associated with its occurrence can inform future intervention strategies for malaria elimination goals. Methods This study analysed historical malaria cases in India from 1990 to 2022 to assess the annual trends
and the impact of key anti-malarial interventions on malaria incidence. Factors associated with malaria incidence were
identified using univariate and multivariate linear regression analyses. Generalized linear, smoothing, autoregressive
integrated moving averages (ARIMA) and Holt’s models were used to forecast malaria cases from 2023 to 2030. Results The reported annual malaria cases in India during 1990–2000 were 2.38 million, which dropped to 0.73 mil-
lion cases annually during 2011–2022. The overall reduction from 1990 (2,018,783) to 2022 (176,522) was 91%. The key
interventions of the Enhanced Malaria Control Project (EMCP), Intensified Malaria Control Project (IMCP), use of biva-
lent rapid diagnostic tests (RDT-Pf/Pv), artemisinin-based combination therapy (ACT), and involvement of the Accred-
ited Social Health Activists (ASHAs) as front-line workers were found to result in the decline of malaria significantly. The
ARIMA and Holt’s models projected a continued decline in cases with the potential for reaching zero indigenous cases
by 2027–2028. Important factors influencing malaria incidence included tribal population density, literacy rate, health
infrastructure, and forested and hard-to-reach areas. Conclusions Studies aimed at assessing the impact of major commodity and policy interventions on the incidence
of disease and studies of disease forecasting will inform programmes and policymakers of steps needed dur-
ing the last mile phase to achieve malaria elimination. It is proposed that these time series and disease forecasting
studies should be performed periodically using granular (monthly) and meteorological data to validate predictions
of prior studies and suggest any changes needed for elimination efforts at national and sub-national levels. Keywords Time series forecasting malaria, Interrupted time series, Malaria elimination *Correspondence:
Altaf A. Lal
altaf.lal@gmail.com
Full list of author information is available at the end of the article *Correspondence:
Altaf A. Lal
altaf.lal@gmail.com
Full list of author information is available at the end of the article Correspondence:
Altaf A. Lal
altaf.lal@gmail.com
Full list of author information is available at the end of the article Malaria Journal Malaria Journal Singh et al. Malaria Journal (2024) 23:50
https://doi.org/10.1186/s12936-024-04872-8 Background to use curated intervention efforts in a context-specific
manner for the high-burden areas that are posing prob-
lems for disease elimination. Several malaria forecasting
studies have been conducted in India, China, Burundi,
Mali, Afghanistan, Bhutan and Ethiopia [5–11]. However,
the studies conducted in India either used hospital based
passive malaria cases [12] or data from selected Indian
states [13–15], which did not allow for the time series
analysis and predictive malaria forecasting of the entire
country.h to use curated intervention efforts in a context-specific
manner for the high-burden areas that are posing prob-
lems for disease elimination. Several malaria forecasting
studies have been conducted in India, China, Burundi,
Mali, Afghanistan, Bhutan and Ethiopia [5–11]. However,
the studies conducted in India either used hospital based
passive malaria cases [12] or data from selected Indian
states [13–15], which did not allow for the time series
analysis and predictive malaria forecasting of the entire
country.h Malaria, a global public health issue, has been success-
fully eliminated from 41 countries through time-tested
case management and vector control strategies. Despite
these promising strides, in the year 2021, the World
Health Organization (WHO) estimated an alarming 247
million malaria cases and 619,000 deaths worldwide. Of these cases, the WHO South East Asia (SEA) region
accounted for about 2% of the global malaria burden. Amongst the SEA countries, India contributed approxi-
mately 79% of the malaria cases and 83% of the malaria-
attributed deaths in the region [1]. The three inter-related objectives of this study were:
(1) identification of the trends of malaria cases in India
over the period 1990 to 2022 using time series analysis
in order to forecast future malaria case burden for 2023
and 2030; (2) perform segmented regression on inter-
rupted time series data to assess the impact of major
interventions adapted in the national malaria programme
over the period 1990 to 2022; and (3) conduct analysis
to determine the association of extraneous independent
factors with malaria incidence for the period of 2011 to
2021. Several countries in the WHO SEA region have
eliminated malaria or are close to eliminating malaria. Maldives and Sri Lanka are malaria-free. Thailand,
Timor-Leste, Bhutan and Nepal are referred to as “E2025”
countries as they aim to achieve elimination by 2025. Background Countries of the Asia Pacific region have also committed
to the malaria elimination goal of 2030, with the major-
ity of cases concentrated in the island nations of Papua
New Guinea and the Solomon Islands, with an Annual
Parasitic Incidence of 65 and 119, respectively [1]. There
is a common concern of emergence and spread of drug
resistance from the Greater Mekong Sub region, which
combined with the insecticide resistance poses a threat
to realizing the goals of malaria elimination in the region.h For forecasting analysis and assessment of the impact
of commodity and policy interventions, country-wide
malaria data over 3 decades was used. For analysis of pre-
dictors of malaria, the state-wise annual malaria data was
used. The WHO Global Technical Strategy has set the objec-
tive to achieve at least 90% case reduction in the global
malaria burden and zero indigenous malaria cases by
2030 [2]. Despite the commitment, not all countries
are on track to meet the 2030 global target of reducing
malaria case incidence and death rates by at least 90%. Certain countries in the region grapple with a complex
interplay of socio-economic, ecological, and health sys-
tem-related factors, which have proven formidable obsta-
cles in their malaria elimination endeavours. Methods
Study design
Th
l This ecologic study employed an explanatory time-trend
design to study the annual trend of malaria cases during
1990–2022 and forecast future malaria cases, assessing
the impact of major anti-malarial interventions during
the study period and assessing the extraneous factors
associated with malaria incidence in India. India’s malaria epidemiology is complex across diverse
demography, topography, and socio-cultural landscapes,
which presents a risk to sustain the reduction in malaria
cases and achievements of the elimination goals in a
timely manner [3]. The country has implemented the
National Strategic Plan for Malaria Elimination 2017–22
[4] and the National Framework for Malaria Elimina-
tion (NFME) in 2016–2030 [3], describing the strategies
of early diagnosis and prompt treatment, vector control,
community engagement, and inter-sectorial coopera-
tion, to achieve the national malaria elimination goal of
2030. The national programme has emphasized special
focus on 27 high-priority districts with moderate to high
malaria transmission. © The Author(s) 2024. 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. Singh et al. Malaria Journal (2024) 23:50 Singh et al. Malaria Journal (2024) 23:50 Page 2 of 11 Prediction of malaria cases from 2023 to 2030h The training set had
been used to train the forecasting model, while the test-
ing set was reserved to assess the model’s performance
on new and unseen data. The linear trend, quadratic,
cubic, centred Moving Average (MA), LOWESS, Sim-
ple Exponential Smoothing (SES), Double Exponential
Smoothing (ETS), Auto-correlation Integrated Moving
Average (ARIMA), Holt’s additive and Holt’s multiplica-
tive regression models were used for forecasting analysis. g
g
y
Evaluating the accuracy of time series forecasting mod-
els is essential to ensure that the predictions are reli-
able and trustworthy. Measuring performance of time
series prediction model provides capability of the fore-
cast to the real values. The most common performance
statistics used are: Mean Forecast Error (or Forecast
Bias) indicates the tendency of a model to overestimate
or underestimate where positive bias means the model
tends to overestimate, while negative bias means it tends
to underestimate; Mean Absolute Error (MAE) meas-
ures absolute average magnitude of error (difference
between actual value and predicted value) in prediction
without considering the direction; Mean Squared Error
(MSE) measures the average of the squares of the errors,
which gives more weight to the large errors; Root Mean
Squared Error (RMSE) is the square root of the MSE
which is easier to interpret. It provides an estimate of the
standard deviation of the forecast errors; Mean Absolute
Percentage Error (MAPE) measures the relative accu-
racy of a forecast by calculating the percentage difference
between predicted and actual values; Mean Absolute
Standard Error (MASE), Akaike Information Criterion
(AIC) and Bayesian Information Criterion (BIC). The
model accuracy was measured with the lowest Root
Mean Square Error (RMSE), Akaike Information Crite-
rion (AIC) and Bayesian Information Criterion (BIC),
Mean Absolute Percent Error (MAPE), Mean Absolute
Standard Error (MASE) in the time series data for the The major change points have been identified in the
years 1997, 2006, 2013, and 2016, to determine the
impact of respective interventions using the General-
ized Least Square (GLS) model along with autocor-
relation via a corARMA function in R 4.3.2 (The R
Project for Statistical Computing) to an Interrupted
Time Series (ITS) malaria data by dividing into pre-
intervention and post-intervention segment periods
(equation below) [24]. Two dummy variables such as
a binary indication of whether the intervention has
taken place at the time(x) and time elapsed since the
intervention were created. Data sources Retrospectively reported annual malaria cases for the
period of 1990 to 2022 was obtained from the World
Malaria Reports (WMRs) 2012 and 2023. State-wise
annual malaria incidence data for the period of 2011
to 2021 and the timelines of different interventions for
malaria control in the country was obtained from the
public domain of NCVBDC New Delhi [16]. The extra-
neous variables associated with malaria incidence were
collected from Census of India, 2011; India State of For-
est Report 2021, Forest Survey of India; Rural Health Sta-
tistics, National Health Mission, Government of India;
Monthly rainfall data series for districts, states, and sub-
divisions and all India, Additional Director General of
Meteorology, Ministry of Earth and Science, India Mete-
orological Department [17–20]. Information obtained through disease forecasting and
time series analysis of historical data presents an oppor-
tunity for the policymakers and programme managers Singh et al. Malaria Journal (2024) 23:50 Page 3 of 11 Prediction of malaria cases from 2023 to 2030h Prediction of malaria cases from 2023 to 2030
The data from WMR used in this study was from the
years 1990 to 2022, which allowed for a robust non-
seasonal disease forecasting analysis to predict future
cases of malaria. The dataset was divided into two sub-
sets: a training set and a testing set. The training set had
been used to train the forecasting model, while the test-
ing set was reserved to assess the model’s performance
on new and unseen data. The linear trend, quadratic,
cubic, centred Moving Average (MA), LOWESS, Sim-
ple Exponential Smoothing (SES), Double Exponential
Smoothing (ETS), Auto-correlation Integrated Moving
Average (ARIMA), Holt’s additive and Holt’s multiplica-
tive regression models were used for forecasting analysis. Evaluating the accuracy of time series forecasting mod-
els is essential to ensure that the predictions are reli-
able and trustworthy. Measuring performance of time
series prediction model provides capability of the fore-
cast to the real values. The most common performance
statistics used are: Mean Forecast Error (or Forecast
Bias) indicates the tendency of a model to overestimate
or underestimate where positive bias means the model
tends to overestimate, while negative bias means it tends
to underestimate; Mean Absolute Error (MAE) meas-
ures absolute average magnitude of error (difference
between actual value and predicted value) in prediction
without considering the direction; Mean Squared Error
(MSE) measures the average of the squares of the errors,
which gives more weight to the large errors; Root Mean
Squared Error (RMSE) is the square root of the MSE
which is easier to interpret. It provides an estimate of the
standard deviation of the forecast errors; Mean Absolute
Percentage Error (MAPE) measures the relative accu-
racy of a forecast by calculating the percentage difference
between predicted and actual values; Mean Absolute
Standard Error (MASE), Akaike Information Criterion
(AIC) and Bayesian Information Criterion (BIC). The
model accuracy was measured with the lowest Root
Mean Square Error (RMSE), Akaike Information Crite-
rion (AIC) and Bayesian Information Criterion (BIC),
Mean Absolute Percent Error (MAPE), Mean Absolute
Standard Error (MASE) in the time series data for the The data from WMR used in this study was from the
years 1990 to 2022, which allowed for a robust non-
seasonal disease forecasting analysis to predict future
cases of malaria. The dataset was divided into two sub-
sets: a training set and a testing set. Estimation of trend and impact analysis
of interventions year following the introduction of interventions along
with the sustained effect showing a declining rate over
the following years. The annual trends of the time series data of malaria cases
during 1990–2022 were plotted using centred moving
averages, exponential, linear, and locally weighted regres-
sion lines. Over the last 3 decades, at different time-
points, several commodities and policy interventions
have been introduced in India. Some of these interven-
tions were novel, and some helped in strengthening and
scaling up the existing strategies. The major interven-
tions used in the present study were: (1) In 1997, the
Enhanced Malaria Control Programme (EMCP) was
operational in 181 selected tribal dominant districts
of the country with the assistance of the World Bank. Under EMCP, case detection was improved with robust
surveillance particularly in high endemic, hard to reach
tribal dominated areas; (2) Further, in the year 2006, the
World Bank-assisted Intensified Malaria Control Project
(IMCP) was launched in inaccessible, high endemic and
Plasmodium falciparum dominant areas of the country
[21]. Emphasis was given to early diagnosis and prompt
treatment; (3) On-spot diagnosis using monovalent rapid
diagnostic test for P. falciparum (RDT-Pf) and arte-
misinin-based combination therapy (Artesunate + Sulf-
adoxine-Pyrimethamine (ACT-SP) as first-line treatment
of P. falciparum introduced in 2005; (4) Accredited Social
Health Activists (ASHAs) engaged to provide diagnosis
and treatment of malaria cases at village level in 2009; (5)
Bivalent RDTs for diagnosis of P. falciparum and Plas-
modium vivax (RDT-Pf/Pv) infections and long lasting
insecticidal nets (LLINs) for vector control in 2009 [22];
(6) Revised national drug policy with introduction of
artemether-lumefantrine combination (ACT-AL) as first-
line drug to treat confirmed P. falciparum cases in North
Eastern states of the country in 2013; (7) Scaling up of
LLINs coverage in malaria-endemic areas from 2015; and
(8) Launch of NFME in 2016 [3, 4, 23]. outcome = gls(malaria cases ∼time + intervention
+ postintervention time, data = xxx, method
= "ML", correlation = corARMA
p = n, q = n, form =∼time
) Association of extraneous factors with malaria
incidenceh The univariate and multivariate generalized linear
regression model was used to determine the extraneous
independent factors associated with state-wise annual
parasite incidence (API) data from 2011 to 2021 as the
trend of malaria over the study period was roughly lin-
ear. Homoscedasticity test with residual plot, normality
test with Q-Q plot and multi-collinearity test were also
performed. The multi-collinearity effect between the
independent variables included in the model was tested
using a pairwise correlation matrix. The analysis did not
reveal any strongly correlated variables. Hence, all the
independent variables were included in the multivariate
model, such as the proportion of the tribal population
and literacy rate as per the 2011 Census (in the demogra-
phy domain), vacant positions of ASHAs and peripheral
health workers, number of health facilities (in the health
infrastructure domain), and geographical areas under i
During the 3 decades i.e. 1990–2000, 2001–2010 and
2011–2022, the annual mean P. falciparum and P. vivax
cases were 1.00, 0.85, 0.42 and 1.38, 0.90, 0.30 million,
respectively. The annual percent decline during 3 decades
was 4.02, − 2.05, and − 12.51 in P. falciparum and − 1.57,
− 2.11 and − 14.75 in P. vivax, respectively (Table 1). The
mean percent change during 3 decades in P. falciparum
and P. vivax did not differ significantly (p > 0.05). Simi-
larly, the difference in mean percent change between total
malaria cases and two species (P. falciparum and P. vivax)
was also not found significant statistically (p > 0.05). Table 1 Trend of malaria during 3 decades of 1990–2000, 2001–2010, and 2011–2022
CI: confidence interval; Q1: first quartile; Q3: third quartile
Species
Period
Yearly malaria cases (millions)
Yearly percent decline
Mean
95% CI
Median
Q1
Q3
Mean
95% CI
Median
Q1
Q3
Lower
Upper
Lower
Upper
Malaria
1990–2000
2.38
2.14
2.63
2.22
2.12
2.66
0.64
− 7.42
8.69
2.19
− 11.07
4.89
2001–2010
1.75
1.61
1.89
1.80
1.56
1.87
− 2.15
− 6.84
2.54
1.34
− 5.16
2.45
2011–2022
0.73
0.45
1.01
0.86
0.26
1.09
− 14.31
− 27.67
− 0.95
− 17.76
− 21.80
− 0.46
P. falciparum
1990–2000
1.00
0.90
1.09
1.01
0.88
1.14
4.02
− 4.78
12.82
1.38
− 4.60
16.15
2001–2010
0.85
0.80
0.90
0.84
0.80
0.89
− 2.05
− 7.19
3.10
− 2.55
− 9.56
4.38
2011–2022
0.42
0.25
0.59
0.49
0.14
0.68
− 12.51
− 29.80
4.78
− 17.40
− 24.22
− 5.48
P. Annual trends of malaria during 1990–2022 Annual trends of malaria during 1990 2022
During 1990–2000, 2001–2010 and 2011–2022, 2.38
(95% CI 2.14, 2.63), 1.75 (95% CI 1.61, 1.89), and 0.73
(95% CI 0.45, 1.01) million malaria cases were reported
annually, respectively. The annual percent decline was
0.64 (95% CI − 7.42, 8.69), − 2.15 (95% CI − 6.84, 2.54),
and − 14.31 (95% CI − 27.67, − 0.95), respectively, during
these 3 decades (Table 1). The per cent decline in malaria
cases during the 3rd decade (2011–2022) was signifi-
cantly higher than in 1990–2000 (coefficient = − 14.94;
95% CI − 27.95, − 1.94; p = 0.026); and 2001–2010 (coef-
ficient = − 12.15; 95% CI − 25.16, − 0.99; p = 0.05). Prediction of malaria cases from 2023 to 2030h The slopes of the inter-
rupted time trends of malaria cases between these two
segments were compared to the estimated quantum of
change and their statistical significance. The immedi-
ate effect showed a decline in malaria cases during the Singh et al. Malaria Journal (2024) 23:50 Page 4 of 11 forest, hilly terrain and annual average rainfall (in geo-
climatic domain). test period. Accurate forecasting using ARIMA requires
normally distributed stationary data with constant mean
and variance over time. The present study used annual
time series data points due to the non-availability of sea-
sonal malaria data. Therefore, a seasonality test was not
performed. Augmented Dickey-Fuller test (ADF Test)
was used to test whether a given time series is station-
ary or not. The regular differencing method was used to
make stationary time series observations over the time
period. In the differencing method, each observation
was replaced by a difference between the current and
previous observation [25]. The present study follows the
EPIFORGE 2020 checklist for reporting forecasting and
prediction research [26]. The API is the number of malaria-positive cases per
thousand population. Since API is highly dependent on
the annual blood examination rate (ABER), it was stand-
ardized with the 10% constant ABER in the analysis. The
multicollinearity between the independent variables was
resolved before applying the multivariate linear regres-
sion model. All the statistical analyses were performed in
R 4.3.2 (The R Project for Statistical Computing). CI: confidence interval; Q1: first quartile; Q3: third quartile Prediction of malaria from 2023 to 2030h The statistical accuracy was found highest in ARIMA
(1,2,2) model, with the lowest RMSE (64,631), AIC
(774.15), BIC (779.48), MAPE (32.57), and MASE (0.34)
indicator scores. The next lowest values of these param-
eters were in Holt’s additive and followed by Holt’s
multiplicative models (Table 3). The MASE in Holt’s mul-
tiplicative model is more than 50% (0.51) which showed
it to be an inappropriate model of forecasting for the pre-
sent time series data. However, the 95% confidence inter-
val in the predicted malaria cases was wider in ARIMA
(1,2,2), as compared to both Holt’s models. The forecast-
ing of the malaria cases for the next eight years (2023–
2030) showed that the rapid decline from 2017–2022 was
likely to continue, assuming the extraneous factors would
be constant and there are no outbreaks. These obser-
vations imply that the target level of zero indigenous
malaria cases would be achieved by 2027–2028 (Fig. 3). Association of extraneous factors with malaria
incidenceh vivax
1990–2000
1.38
1.19
1.58
1.27
1.19
1.65
− 1.57
− 11.47
8.34
− 0.92
− 10.95
8.41
2001–2010
0.90
0.80
1.00
0.94
0.77
1.01
− 2.11
− 8.47
4.25
− 1.79
− 6.60
5.79
2011–2022
0.30
0.19
0.42
0.34
0.13
0.40
− 14.75
− 27.86
− 1.64
− 15.92
− 20.13
− 6.45 Singh et al. Malaria Journal (2024) 23:50 Singh et al. Malaria Journal (2024) 23:50 Page 5 of 11 due to the fewer follow-up years after the change point
(Table 2; Fig. 2). The temporal trend in malaria over the period of 1990
to 2022 revealed a 91.26% (95% CI − 91.29, − 91.22)
reduction in the reported malaria cases. Overall, the
average annual declining rate in the reported malaria
cases was − 5.84% (95% CI − 11.56, − 0.12). Most of the
annual decline was observed in 2017–2018 by 49%, which
could be attributed to robust implementation of moni-
toring and evaluation frameworks of case management
and vector control strategies that were part of the NFME
2016–30 (Fig. 1). Impact of major anti‑malarial intervention
from 1990–2022 The analysis of ITS has revealed that EMCP, which
focused on tribal-dominated hilly forests and P. falcipa-
rum-prone areas, had the most significant impact and
effects sustained during the post-intervention period
(p < 0.0001). IMCP provided early case detection and
prompt treatment using RDT-Pf and ACT-SP, which
contributed to a significant yearly cases reduction of
malaria (p = 0.002). ASHAs (since 2009) and diagnosis
using RDT-Pf/Pv (since 2013), and treatment with ACT-
AL (since 2013) in the North Eastern region states led to
an additional reduction in malaria cases, which was sig-
nificant (p = 0.048). The NFME and scaling-up of LLINs
distribution in hard-to-reach high malaria endemic areas
had the highest impact in the reduction of malaria cases. However, in the GLS regression model, the coefficient
was not found statistically significant (p = 0.101), possibly Factors associated with malaria incidence Univariate and multivariate linear regression analy-
sis revealed that populations classified under Sched-
uled Tribes (indigenous people in India) were positively
associated, and literacy rate was inversely associated
with the API, both of which were statistically signifi-
cant (p ≤ 0.001). Health infrastructure, which included Fig. 1 Trend of malaria cases in India from 1990 to 2022 showing time series (red line) and smoothing curves: (1) Centred moving average (black
line); (2) Exponential curve (green line); (3) Linear curve (grey line); and (4) Lowess curve (blue line) Fig. 1 Trend of malaria cases in India from 1990 to 2022 showing time series (red line) and smoothing curves: (1) Centred moving average (black
line); (2) Exponential curve (green line); (3) Linear curve (grey line); and (4) Lowess curve (blue line) Fig. 1 Trend of malaria cases in India from 1990 to 2022 showing time series (red line) and smoothing curves: (1) Centred moving average (black
line); (2) Exponential curve (green line); (3) Linear curve (grey line); and (4) Lowess curve (blue line) Page 6 of 11 Singh et al. Factors associated with malaria incidence Malaria Journal (2024) 23:50 Table 2 Interrupted time series segmented regression analysis showing the impact of major anti-malarial interventions from 1990 to
2022
* EMCP: enhance malaria control programme; IMCP: intensified malaria control programme; RDT: rapid diagnostic test; Pf: plasmodium falciparum; Pv: plasmodium
vivax; ACT: artemisinin combination therapy; SP: sulfadoxine pyrimethamine; ASHA: accredited social health activist; AL: artemether lumefantrine; NFME: national
framework of malaria elimination; LLIN: long lasting insecticidal nets
Year of
change point
Interventions*
Factors
Beta coefficient
Standard error
t
P value
1997
EMCP
Intercept
− 366,103,779
63,332,205
− 5.781
< 0.0001
Year
184,914
31,777
5.819
< 0.0001
Immediate effect
− 406,760
133,185
− 3.054
0.005
Sustained effect
− 274,754
32,080
− 8.565
< 0.0001
2006
IMCP + RDT(PF) + ACT (ASP)
Intercept
67,536,474
30,212,557
2.235
0.033
Year
− 32,693
15,125
− 2.161
0.039
Immediate effect
− 70,923
194,286
− 0.365
0.718
Sustained effect
− 70,867
20,480
− 3.460
0.002
2013
ASHA + RDT(PF + PV) + ACT(ASP/AL)
Intercept
112,158,649
18,390,013
6.099
< 0.0001
Year
− 55,050
9190
− 5.990
< 0.0001
Immediate effect
− 77,516
232,003
− 0.334
0.741
Sustained effect
− 69,221
33,475
− 2.068
0.048
2016
NFME + RDT(PF + PV) + ACT(ASP/
AL) + LLIN (scale-up)
Intercept
122,813,847
15,355,727
7.998
< 0.0001
Year
− 60,384
7668
− 7.875
< 0.0001
Immediate effect
− 58,668
271,886
− 0.216
0.831
Sustained effect
− 94,662
55,948
− 1.692
0.101 Table 2 Interrupted time series segmented regression analysis showing the impact of major anti-malarial interventions from 1990 to
2022 * EMCP: enhance malaria control programme; IMCP: intensified malaria control programme; RDT: rapid diagnostic test; Pf: plasmodium falciparum; Pv: plasmodium
vivax; ACT: artemisinin combination therapy; SP: sulfadoxine pyrimethamine; ASHA: accredited social health activist; AL: artemether lumefantrine; NFME: national
framework of malaria elimination; LLIN: long lasting insecticidal nets * EMCP: enhance malaria control programme; IMCP: intensified malaria control programme; RDT: rapid diagnostic test; Pf: plasmodium falciparum; Pv: plasmodium
vivax; ACT: artemisinin combination therapy; SP: sulfadoxine pyrimethamine; ASHA: accredited social health activist; AL: artemether lumefantrine; NFME: national
framework of malaria elimination; LLIN: long lasting insecticidal nets Fig. 2 Segmented regression analysis in interrupted time series to assess the impact of major anti-malarial Interventions change point from 1990
to 2022. The four major change points are depicted in the graph. Factors associated with malaria incidence EMCP: enhance malaria control programme; IMCP: intensified malaria control
programme; RDT: rapid diagnostic test; Pf: Plasmodium falciparum; Pv: Plasmodium vivax; ACT: artemisinin combination therapy; SP: sulfadoxine
pyrimethamine; ASHA: accredited social health activist; AL: artemether lumefantrine; NFME: national framework of malaria elimination; LLIN: long
lasting insecticidal nets Fig. 2 Segmented regression analysis in interrupted time series to assess the impact of major anti-malarial Interventions change point from 1990
to 2022. The four major change points are depicted in the graph. EMCP: enhance malaria control programme; IMCP: intensified malaria control
programme; RDT: rapid diagnostic test; Pf: Plasmodium falciparum; Pv: Plasmodium vivax; ACT: artemisinin combination therapy; SP: sulfadoxine
i
h
i
ASHA
di d
i l h
l h
i i
AL
h
l
f
i
NFME
i
l f
k f
l i
li
i
i
LLIN l Fig. 2 Segmented regression analysis in interrupted time series to assess the impact of major anti-malarial Interventions change point from 1990
to 2022. The four major change points are depicted in the graph. EMCP: enhance malaria control programme; IMCP: intensified malaria control
programme; RDT: rapid diagnostic test; Pf: Plasmodium falciparum; Pv: Plasmodium vivax; ACT: artemisinin combination therapy; SP: sulfadoxine
pyrimethamine; ASHA: accredited social health activist; AL: artemether lumefantrine; NFME: national framework of malaria elimination; LLIN: long
lasting insecticidal nets the vacant position of ASHA workers (community-level
health provider) per 1000 population and the vacant
position of health workers at peripheral health facilities such as sub-centres, primary health centres, community
health centres, and district hospitals (facility-level health
provider) per 1000 population was positively associated Singh et al. Factors associated with malaria incidence Malaria Journal (2024) 23:50 Page 7 of 11 Page 7 of 11 Table 3 Measuring the accuracy of ten prediction models using nine summary statistics criteria
ME: mean error; RMSE: root mean square error; MAE: mean absolute error; MPE: mean percent error; MAPE: mean absolute percent error; MASE: mean absolute scaled
error; ACF: auto correlation function; AIC: akaike information criterion; BIC: bayesian information criterion
Models (n = 10)
Summary statistics (n = 9)
ME
RMSE
MAE
MPE
MAPE
MASE
ACF1
AIC
BIC
Linear
Training
− 2.71e−11
305,647.6
224,512.4
− 6.74
17.31
1.34
0.65
Test
− 4.33e + 05
436,429.1
433,277.9
− 248.19
248.19
2.59
Quadratic
Training
2.72e− 11
250,728.0
189,867.5
− 1.92
11.90
1.13
0.66
Test
8.41e + 04
148,494.2
122,609.9
49.43
70.07
0.73
Cubic
Training
− 2.52e− 11
262,603.7
191,917.9
− 2.08
11.82
1.15
0.67
Test
1.38e + 05
197,841.6
155,947.6
80.50
90.17
0.93
Moving average
Training
− 70,280.17
183,319.75
145,459.7
− 10.08
13.57
1.05
0.51
Test
5797.66
11,713.42
10,721.0
2.98
6.02
0.08
Loess
Training
− 7111.25
47,878.78
21,823.28
− 0.65
1.34
0.32
− 0.12
Test
342,636.37
355,731.07
342,636.37
197.43
197.43
5.06
Simple Exponential
Training
− 67,907
216,932
164,871
− 10.29
15.6
1.05
0.25
Test
− 2,009,565
2,015,929
209,565
− 1147.84
1147.84
932.35
936.84
Double Exponential
Training
− 68,756
216,914
164,812
− 10.56
15.36
0.97
0.23
Test
− 2,113,922
2,122,632
2,113,922
− 1213.69
1213.69
932.34
936.83
ARIMA(1,2,2)
Training
− 47,088
197,391
157,307
− 3.96
11.62
0.94
− 0.06
Test
− 43,021
64,631
57,451
− 24.39
32.57
0.34
774.15
779.48
Holt’s additive
Training
− 54,609
213,525
162,359
− 5.72
11.74
0.97
0.24
Test
24,779
77,470
63,311
14.82
35.48
0.38
852.06
857.67
Holt’s multiplicative
Training
− 42,161
225,610
172,941
− 6.12
13.15
1.03
0.35
Test
− 86,303
90,667
86,303
− 49.43
49.43
0.51
855.31
860.92 Table 3 Measuring the accuracy of ten prediction models using nine summary statistics criteria ME: mean error; RMSE: root mean square error; MAE: mean absolute error; MPE: mean percent error; MAPE: mean
error; ACF: auto correlation function; AIC: akaike information criterion; BIC: bayesian information criterion with API (p ≤ 0.001). Whereas the number of health facil-
ities per square km of geographical area was inversely
associated with API (p = 0.04). Further analysis revealed
that geographical areas under forest cover, hilly terrain,
and annual average rainfall (mm) were positively associ-
ated with API (p < 0.0001). Factors associated with malaria incidence In the multivariate model, 37%
of the variance in the API was attributed to the above
independent factors included in the analysis (Table 4). This study has used reported malaria cases data from
the WMRs instead of the controversial estimated cases
data. In India, efforts are being made to bridge the gap
between reported and estimated cases with the increased
community level awareness about the significant value of
accessing public health systems. The mandatory report-
ing of malaria cases data by the private sector in almost
all states of the country in the last 18 months and the
introduction of newer data reporting tools such as the
Integrated Health Information Platform are expected to
shrink this gap. Discussionh The use of longitudinal historical data for assessing the
impact of intervention and forecasting studies is crucial
for identifying interventions that have the most impact
on achieving malaria elimination goals at national and
sub-national levels. For conducting malaria forecasting
analysis, the data quantity, quality, timelines, and con-
sistency in reporting are key requirements. Generalized
linear, ARIMA, and Holt–Winter’s models are the most
commonly used statistical models in malaria forecast-
ing studies. The selection of the appropriate forecast-
ing model depends on the predictive accuracy, which
is largely determined by RMSE, MAPE, MASE, MAE,
MAD, 95% confidence intervals, and visual observation. In addition, AIC and BIC are also used as model-fitting
criteria [12, 27, 28]. The experiences from the Malaria Elimination Dem-
onstration Project (MEDP) conducted for four years in
Mandla, Madhya Pradesh have confirmed that treat-
ments that are home-based or use alternate systems
of medicine do not add significantly to the case data of
state national programs. Through active surveillance
and RDT-based diagnostics, it was found that malaria-
attributable fever never went beyond 1%, so the individu-
als who receive treatment at home or receive treatment
through alternative systems will not add significantly to
the malaria case burden [29]. The present study has revealed a linear declining
trend of malaria cases in India, with about a 91% reduc-
tion from 1990 to 2022. Between 1990 and 2000, there Page 8 of 11
Singh et al. Malaria Journal (2024) 23:50
Fig. 3 Forecast analysis using time series data from 1990 to 2022 and prediction of malaria cases from 2023 to 2030 using (A) ARIMA and B Holt’s
additive models. The various interventions from 1990 to 2022 are depicted in both models Singh et al. Malaria Journal (2024) 23:50 Page 8 of 11 Fig. 3 Forecast analysis using time series data from 1990 to 2022 and prediction of malaria cases from 2023 to 2030 using (A) ARIMA and B Holt’s Fig. 3 Forecast analysis using time series data from 1990 to 2022 and prediction of malaria cases from 2023 to 2030 using (A) ARIMA and B Holt’s
additive models. The various interventions from 1990 to 2022 are depicted in both models Fig. 3 Forecast analysis using time series data from 1990 to 2022 and prediction of malaria ca
additive models. The various interventions from 1990 to 2022 are depicted in both models P. Discussionh The impact of EMCP was due to a number of interven-
tions introduced through EMCP, particularly in tribal-
dominated high-burden areas of the country. RDTs for P. falciparum in the national programme. While the terms
EMCP or IMCP are not mentioned now, but the compo-
nents of the intervention have been continued with the
addition of various new tools and methodologies. increased travel time and distance to healthcare facili-
ties significantly affect the likelihood of seeking care. For
instance, a study in Uganda indicated that as travel time
to a health facility increased, the probability of seeking
care for malaria symptoms decreased [31]. Circular asso-
ciations may arise if, for example, high malaria incidence
deters healthcare professionals from working in these
areas, further exacerbating staff shortages and weaken-
ing the healthcare infrastructure. Consequently, this can
lead to a vicious cycle where increasing malaria incidence
and decreasing healthcare provision could reinforce each
other.h ARIMA and Holt’s time series regression models are
the most common tools for disease forecasting [30]. In
the present study, the ARIMA (1,2,2) was the most fitted
model to predict malaria cases compared to nine other
models. The ARIMA (1,2,2) model predicted that zero
malaria cases might be achieved in the year 2028. How-
ever, there was a wide range of 95% Confidence Inter-
val (CI). Further, Holt’s additive model predicted the
achievement of zero malaria cases by the year 2026 to
2028. In a state-wide study done in Odisha, the authors
found that Holt’s Winter was the most fitted model across
varying endemicities, which predicted a slowing down of
the decline in 2014–2016, hence, missing the state elimi-
nation goal [13].h The present study has also found that tribal-dominated,
hilly and forested areas have a greater risk of malaria
infection. This finding is complemented by a study done
in the state of Madhya Pradesh, where it was found that
communities with high literacy had a lower burden
of malaria, possibly because of better health-seeking
behaviour [32]. Discussionh falciparum infections, enhanced surveillance under
the IMCP, introduction of LLINs, and ASHAs. Com-
plimenting the interventions done during the above
period, in 2011–2022, the addition of NFME, bivalent
RDTs, AL, and scaling-up of LLINs further contributed
to about 14% yearly decline in malaria cases. was an approximately 0.64% yearly increase in malaria
cases. From 2001 to 2010, there was an annual malaria
case reduction of about 2%, which could be attributed
to the combination of several policy and commodities
interventions such as the introduction of monovalent
RDTs, replacement of chloroquine with ACT-SP for P. falciparum infections, enhanced surveillance under
the IMCP, introduction of LLINs, and ASHAs. Com-
plimenting the interventions done during the above
period, in 2011–2022, the addition of NFME, bivalent
RDTs, AL, and scaling-up of LLINs further contributed
to about 14% yearly decline in malaria cases. Singh et al. Malaria Journal (2024) 23:50 Page 9 of 11 Table 4 Regression analysis of factors associated with the Annual Parasite Incidence (standardized with annual blood examination
rate)
FACTORS
Univariate
Multivariate (R2 = 0.37)
Beta coefficient (95% CI)
P value
Beta coefficient (95% CI)
P value
Demographics
Scheduled Tribe (%)
0.04 (0.03, 0.05)
< 0.0001
0.02 (0.01, 0.03)
0.001
Literacy (%)
− 0.10 (− 0.13, − 0.08)
< 0.0001
− 0.09 (− 0.012, − 0.07)
< 0.0001
Health infrastructure
Proportion of vacant position of ASHA worker/000 pop
10.98 (8.65, 13.30)
< 0.0001
5.75 (2.27, 9.24)
0.001
Proportion of vacant position of health worker*/000 pop
7.73 (6.84, 8.61)
< 0.0001
8.95 (7.26, 10.63)
< 0.0001
Number of Health Facilities (SC, PHC, CHC, SDH, DH)/sq km
− 3.87 (− 5.22, − 2.51)
< 0.0001
− 1.46 (− 2.85, − 0.06)
0.04
Geography
Forest covered (%)
0.04 (0.03, 0.05)
< 0.0001
0.02 (0.01, 0.03)
< 0.0001
% of Hilly Districts
0.01 (0.05, 0.01)
< 0.0001
0.02 (0.02, 0.03)
< 0.0001
Climate
Annual Rainfall mm
0.0004 (0.0001, 0.0007)
0.002
0.0005 (0.0003, 0.0008)
< 0.0001
Constant
5.54 (3.88, 7.20)
< 0.0001 Table 4 Regression analysis of factors associated with the Annual Parasite Incidence (standardized with annual blood examination
rate) Table 4 Regression analysis of factors associated with the Annual Parasite Incidence (standardized with annual blood examination
rate) EMCP had the highest impact, possibly because in
the year 1996, the number of malaria cases was higher. Discussionh The phenomenon of high literacy and
low malaria burden is supported by the national trends
where most malaria burden is found in inaccessible hilly
forested terrains and tribal-dominated areas with poor
healthcare infrastructure and low levels of literacy [33].h The Goalkeepers Report 2021 by the Bill and Melinda
Gates Foundation (BMGF) has predicted that the global
malaria cases would be 32 new cases per 1000 people in
2030, which is almost the same as the reported malaria
burden of 31 new cases per 1000 people in 2020. The
report gives a range of 21–42 cases per 1000 popula-
tion as the best and the worst situations in 2030, respec-
tively. A similar trend has been predicted for South East
Asia + East Asia + Oceania, predicting zero to one case The present study has found that the vacant positions
of health providers at the community and facility levels
were associated with an increased risk of malaria inci-
dence. It has also revealed that if the health care facilities
per square kilometre were to be increased, particularly in
hard-to-reach malaria high-endemic areas, the incidence
of malaria could decrease. However, while assessing the
relationship between healthcare facilities and malaria
incidence in remote areas, studies have found that Singh et al. Malaria Journal (2024) 23:50 Singh et al. Malaria Journal (2024) 23:50 Page 10 of 11 reduction of 91%. Factors contributing to this substantial
decrease include focused interventions such as EMCP,
IMCP, ASHAs’ contribution, RDT-Pf/Pv and ACT
deployment, the NFME, and scaling-up of LLINs
distribution. per 1000 population as the best and the worst situations
in 2030, respectively. In comparison, in sub-Saharan
Africa, 68 to 195 malaria cases per 1000 population are
predicted as the best and worst-case scenarios in 2030. For India, the prediction showed one to four cases per
1000 population in 2030, indicating a potential miss of
the national malaria elimination goal [34]. However, the
present study has suggested that India might be able to
achieve the national malaria elimination goal, subject to
the absence of disease outbreaks, climatic changes, emer-
gence of anti-malarial drug resistance, and other inde-
pendent factors. It is also critical to use context specific
and curated elimination protocols for each of the 27 high
burden districts in India to eliminate indigenous trans-
mission. Conclusionhi The significant decrease in malaria incidence in India
from 1990 to 2022 highlights the successful implemen-
tation of various anti-malarial strategies and interven-
tions. This study reveals a significant negative trend in
malaria cases over the past 3 decades, with a remarkable Availability of data and materials Data analysed in the present study was obtained from public domain and the
references have been cited. The analysed data is available with corresponding
author. Author contributions
MPS HR
d AAL MPS, HR, and AAL conceptualized the study. MPS and AAL performed data
analysis and interpretation of the data. MPS, HR and AAL wrote the manu-
script. PKB and ARA participated in review of data and writing of the manu-
script. All authors reviewed the final draft of the manuscript for submission. Funding g
This study was conducted with the financial support from the Foundation for
Disease Elimination and Control of India (FDEC-India), which is a non-profit
organization under Corporate Social Responsibility (CSR) of Sun Pharmaceuti-
cal Industries, Mumbai, India. The funding agency did not have any role in the
study design, data collection and analysis. Author details
1 1 Foundation for Disease Elimination and Control of India, Mumbai, India. 2 Asia Pacific Leaders’ Malaria Alliance, Singapore, Singapore. 3 Indian Council
of Medical Research–National Institute of Malaria Research, New Delhi, India. 4 Sun Pharmaceutical Industries Ltd, Mumbai, India. 5 Global Health and Phar-
maceuticals, Inc, Atlanta, GA, USA. Received: 21 December 2023 Accepted: 6 February 2024 Received: 21 December 2023 Accepted: 6 February 2024 Declarations This study used reported annual malaria cases in the
absence of granular and monthly malaria data of the
country in public domain. Therefore, seasonality and
geographical variability in malaria cases could not be
analysed in the time series modelling. Further, in the pre-
sent forecasting model, the effect of climatic variables
such as rainfall, humidity, and temperature have not been
quantitated, although these are significant covariates of
malaria as reported by other studies [38]. The data in the
context of roads and telecommunications infrastructure
were also not analysed in the present study. Competing interests The authors declare no competing interests. Discussionh If this is accomplished in three to four years, as
was demonstrated in Mandla through MEDP, and there
are no disease outbreaks and issue related to climate
change and drug resistance a malaria free India in 2030
is possible. Out of these predictive models, the ARIMA and Holt’s
additive have shown reliable predictive capabilities, indi-
cating that the decreasing malaria cases are likely to
continue, forecasting zero indigenous malaria cases by
2027–2028. The study also identified the most impactful
combination of intervention packages. Disease modelling
studies will have the most impact during the last mile of
disease elimination, provided the information is used in
real-time by the programmes and policymakers. It is pro-
posed that such time series and disease modelling efforts
should be repeated periodically to validate prior predic-
tions of recent years and suggest any changes needed in
the interventions required for a malaria-free India. A study conducted in Odisha state reported about
three times higher annual declining rates in malaria inci-
dence during the intensified post-intervention period
(2009–2013) compared to the pre-intervention period
(2003–2008). This study attributed the drop to integrated
vector control measures, rapid diagnosis and prompt
treatment, service decentralisation, inter-sectoral con-
vergence, and behaviour change communication (13). LLINs, RDTs with ACT and biological vector control
interventions helped in a significant reduction in malaria
cases during 2011–2021 in the Karnataka state [35]. The
present study has also found that optimal coverage of
ITNs/LLINs effectively decreased the malaria caseload. Similarly, ITNs/LLINs were found to be a cost-effective
intervention tool in Bangladesh [36] and Sri Lanka,
which have been malaria-free since 2016 [37].h Ethics approval and consent to participate No primary data or samples were collected for the present study. Therefore,
ethical approval was not required. 1.
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High-level listening comprehension in advanced English as a second language: Effects of the first language and inhibitory control
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Bilingualism
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cc-by
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Bilingualism: Language and
Cognition
cambridge.org/bil Bilingualism: Language and
Cognition Bilingualism: Language and
Cognition Introduction The ability to draw inferences from auditory and written input is crucial for comprehension
and successful educational outcomes and is especially relevant in linguistically diverse contexts
where learners have heterogeneous language backgrounds but are educated in the predomin-
ant, privileged language of the country. Such a case is South Africa, where tertiary education is
almost exclusively received through the medium of English (Madadzhe, 2019), though it is not
the first language (L1) for the majority of the population but for only 9.6% of citizens
(Statistics South Africa, 2012). This makes South Africa a unique context to investigate lan-
guage effects in listening comprehension in a sample that scarcely (if at all) comprises
‘pure’ monolinguals but is home to individuals that widely differ in their L1 and degree of
multilingualism. Accordingly, the main aim of this study is to investigate how advanced
English proficient South African adults differing in their L1 (English or Zulu) perform on lis-
tening text comprehension in the language of instruction (English). We first provide an over-
view of the language and educational context in South Africa, followed by an account of the
cognitive and linguistic processes involved in text comprehension in both reading and listening
modalities. Mandy Wigdorowitza,b
, Ana I. Pérezc
and Ianthi M. Tsimplia Mandy Wigdorowitza,b
, Ana I. Pérezc
and Ianthi M. Tsimplia Keywords: listening comprehension; L1 background;
comprehension monitoring; revision;
inhibitory control; South Africa Address for correspondence:
Mandy Wigdorowitz,
St John’s College,
Theoretical and Applied Linguistics Section,
9 West Rd, Cambridge CB3 9DP
E-mail: mandy.wigdorowitz@gmail.com Address for correspondence:
Mandy Wigdorowitz,
St John’s College,
Theoretical and Applied Linguistics Section,
9 West Rd, Cambridge CB3 9DP
E-mail: mandy.wigdorowitz@gmail.com Research Article aTheoretical and Applied Linguistics, University of Cambridge, Cambridge, United Kingdom; bDepartment of
Psychology, University of Johannesburg, Johannesburg, South Africa and cDepartment of Experimental
Psychology, University of Granada, Granada, Spain Cite this article: Wigdorowitz M, Pérez AI,
Tsimpli IM (2023). High-level listening
comprehension in advanced English as a
second language: Effects of the first language
and inhibitory control. Bilingualism: Language
and Cognition 1–15. https://doi.org/10.1017/
S1366728923000135 Abstract English is imposed as the language of instruction in multiple linguistically diverse societies
where there is more than one official language. This might have negative educational conse-
quences for people whose first language (L1) is not English. To investigate this, 47 South
Africans with advanced English proficiency but different L1s (L1-English vs. L1-Zulu) were
evaluated in their listening comprehension ability. Specifically, participants listened to narra-
tive texts in English which prompted an initial inference followed by a sentence containing an
expected inference or an unexpected but plausible concept, assessing comprehension monitor-
ing. A final question containing congruent or incongruent information in relation to the text
information followed, assessing the revision process. L1-English participants were more
efficient at monitoring and revising their listening comprehension. Furthermore, individual
differences in inhibitory control were associated with differences in revision. Results show
that participants’ L1 appears to supersede their advanced English proficiency on highly
complex listening comprehension. Received: 25 March 2022
Revised: 23 January 2023
Accepted: 26 January 2023 Received: 25 March 2022
Revised: 23 January 2023
Accepted: 26 January 2023 © The Author(s), 2023. Published by
Cambridge University Press. This is an Open
Access article, distributed under the terms of
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted re-use, distribution
and reproduction, provided the original article
is properly cited. High-level listening comprehension in
advanced English as a second language: Effects
of the first language and inhibitory control cambridge.org/bil High-level text comprehension processes Comprehension is the process of extracting meaning from visual
and/or auditory stimuli as opposed to a mere verbatim record of
the presented content. It involves the interplay between world-
knowledge (top-down processing) in conjunction with the accu-
mulated content-based information of the text (bottom-up pro-
cessing) so that the comprehenders can construct an integrated
and coherent mental representation of the content that clarifies
its meaning (for reviews, see Aryadoust, 2019; McNamara &
Magliano, 2009). This mental representation is referred to as a
SITUATION MODEL (Kintsch, 1998). As each word or phrase is pro-
cessed, it is actively integrated into the situation model of compre-
henders such that text coherence can be achieved. At the lexical or
sentence level, word meanings are retrieved and systematically
grouped into meaningful grammatical units, while at the dis-
course level, other higher-level cognitive skills are engaged to be
able to construct a unified, coherent, and accurate representation
of the content in real time (Rapp, van den Broek, McMaster,
Kendeou & Espin, 2007; van Dijk & Kintsch, 1983). Relevant
higher-level comprehension processes that play a role in text
coherence include INFERENCING, MONITORING, and REVISION. (3) In the lake by my house there are many animals that make
quacking noises. Yesterday the sounds were really loud, so I
went to see which animals were making them. g
(4a) On a big rock in the middle of the lake, I could see the ducks (4a) On a big rock in the middle of the lake, I could see the ducks
(4b) On a big rock in the middle of the lake, I could see the g
(4b) On a big rock in the middle of the lake, I could see the
turtles Moreover, in situations where upcoming information does not
match with a previously generated inference as the narrative
unfolds and further information is presented, comprehenders
need to review their existing interpretation and update their situ-
ation model so that the new and unexpected content becomes
meaningfully integrated into the discourse context, in a process
known as REVISION (Rapp & Kendeou, 2007). Participants need
to update their situation model in order to identify whether the
content is congruent (4a →5a or 4b →5b) or incongruent (4a
→5b or 4b →5a) in relation to preceding information. South African linguistic and educational context material in English, and the conception that English is a global
lingua franca that can lead to more long-term social and eco-
nomic opportunities and mobility (Madadzhe, 2019; Mesthrie,
2002). Similarly, English is ubiquitous across government,
media, and industry, so it is indeed privileged in this regard
and considered the country’s linguistic bridge. y
g
g
It can therefore be assumed that South Africans entering uni-
versity have prolonged and sustained exposure to English both in
and out of the school context as well as high levels of proficiency
to meet the language requirements necessary to be accepted to an
institution of higher education.1 Few studies have investigated dif-
ferences between highly English proficient adult learners, but who
enter educational systems with different L1s. We are particularly
interested in L1 as opposed to LINGUALISM STATUS (monolingual,
bilingual) because L1 is a highly informative indicator of founda-
tional language background in the South African context, given
the (mis)alignment that it has in comparison to the medium of
tertiary education. Importantly, the question of whether indivi-
duals’ L1 supersedes their proficiency in listening text compre-
hension has not previously been explored. There is, however,
concrete evidence to suggest that comprehension ability is pre-
dicted by proficiency (Filippi, Leech, Thomas, Green & Dick,
2012),
particularly
in
school-aged
children
(Babayiğit
&
Shapiro, 2020; Melby-Lervåg & Lervåg, 2014; Pretorius &
Spaull, 2016), but it is unclear whether young adults situated in
a multilingual context who have advanced proficiency in
English (i.e., the language of learning and teaching) but differ
in their L1 (in this case, L1-Zulu) are able to perform equally
well
on
a
demanding
comprehension
task
compared
to
L1-English participants. (1) The mother has two daughters and one son (2) The mother has two daughters and one dog (adapted from
Dijkstra, Van Hell & Brenders, 2015) COMPREHENSION MONITORING is the active, metacognitive strategy
that facilitates awareness of what is being comprehended, whereby
efficient comprehenders are able to rapidly notice information
that does not align with previously presented content so as to dis-
ambiguate their predictions (Baker, 1989; McNamara, 2007;
Wagoner, 1983; Zhang, 2017). A consistent outcome is a time
cost on unexpected compared to expected sentences because of
noticeable disruptions to text coherence (O’Brien, Rizzella,
Albrecht and Halleran, 1998; Rinck & Weber, 2003). 1See, for example https://www.wits.ac.za/internationalstudents/requirements/english-
language-proficiency/ (last accessed 19/01/2023). South African linguistic and educational context Young adults completing tertiary education in South Africa are exposed to regular comprehen-
sion of complex and novel content. Learners entering a university are required to, at min-
imum, understand, speak, read, and write in standard, university-level English if they are to
be adept and competitive (South African Department of Education, 2008). While this
seems like an unreasonable expectation given how few South Africans have English as an
L1, the post-apartheid government has attempted to put educational safeguards in place
that aim to ensure adequate English competency by tertiary education, while still promoting
multilingualism (Constitution of the Republic of South Africa, 1996; Department of
Education, 1997). While, in principle, the language-in-education policy is linguistically inclu-
sive, in reality, multilingual education still remains imbalanced, with English (and to a lesser
extent, Afrikaans) being prioritised and disproportionally supported (Drummond, 2016; van
Wyk, 2014). English medium schools are by far the most prevalent across South Africa
(Webb, Lafon & Pare, 2010), so over the past two decades the majority of South Africans
have received education in English as the language of learning and teaching throughout
their schooling career. This disparity is multidimensional but in a large part due to the ele-
vated status that English occupies, as well as access to educational resources and teaching © The Author(s), 2023. Published by
Cambridge University Press. This is an Open
Access article, distributed under the terms of
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted re-use, distribution
and reproduction, provided the original article
is properly cited. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 2 information arises that deviates from one’s expectations (Bubic,
von Cramon & Schubotz, 2010; Pickering & Gambi, 2018). For
example, in (1) the target word “son” is highly semantically con-
straining and expected, while in (2) the target word “dog” is less
semantically constraining – that is, unexpected, but still plausible. When presented with examples such as (2) where the inference is
not expected, prediction errors (e.g., processing delays, decline in
accuracy) are likely to arise, especially where the strength of the
generated prediction is robust, or the content is presented in a
second language (Kaan & Grüter, 2021). Importantly, prediction
errors are not confined to single sentences like (1) and (2) but
may emerge in longer narratives where knowledge-based inferences
have been generated based on the establishing content, as in (3). South African linguistic and educational context For instance,
following passage (3), participants have to adjust their expecta-
tions with reference to additional incoming content that is either
expected and aligns (4a) or unexpected and misaligns (4b) with
the prior information. Efficient comprehenders are able to rapidly
notice information that does not align with previously presented
content in order to disambiguate their predictions (McNamara,
2007; Zhang, 2017). https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press High-level text comprehension processes longer-true, irrelevant, and outdated information that disrupts the
text’s coherence (Kendeou et al., 2013). Outdated information is
expected to decay in line with the presentation of new, harmoni-
ous information and should be deactivated from the current men-
tal model, losing its accessibility (Zwaan & Radvansky, 1998). Much of the time this is borne out such that comprehenders
can successfully update their situation model (de Vega, 1995;
Rapp & Taylor, 2004). However, it has also been demonstrated
that while revision can be achieved, processing delays may be
incurred as a direct consequence of maintaining the outdated
information in the situation model (de Vega, Urrutia & Riffo,
2007; Guéraud, Harmon & Peracchi, 2005; O’Brien, Cook &
Guéraud, 2010); Rapp & Kendeou, 2007). In such cases, compre-
hension can be disrupted. longer-true, irrelevant, and outdated information that disrupts the
text’s coherence (Kendeou et al., 2013). Outdated information is
expected to decay in line with the presentation of new, harmoni-
ous information and should be deactivated from the current men-
tal model, losing its accessibility (Zwaan & Radvansky, 1998). Much of the time this is borne out such that comprehenders
can successfully update their situation model (de Vega, 1995;
Rapp & Taylor, 2004). However, it has also been demonstrated
that while revision can be achieved, processing delays may be
incurred as a direct consequence of maintaining the outdated
information in the situation model (de Vega, Urrutia & Riffo,
2007; Guéraud, Harmon & Peracchi, 2005; O’Brien, Cook &
Guéraud, 2010); Rapp & Kendeou, 2007). In such cases, compre-
hension can be disrupted. (5a) There were animals with brown and green feathers making
noise (5b) There were animals with brown and green shells making
noise (adapted from Pérez, Joseph, Bajo & Nation, 2016) The majority of research conducted on high-level comprehension
processes has focussed on reading rather than listening compre-
hension (for a review, see McNamara & Magliano, 2009) and
even fewer studies have evaluated comprehension from a multi-
modal perspective (Hu & Jiang, 2011; Manfredi, Cohn, Andreoli
& Boggio, 2018; Pérez, Schmidt, Kourtzi & Tsimpli, 2020; Wu
& Ma., 2016). Despite this asymmetry, the processes involved in
reading and listening comprehension appear to overlap in critical
ways and rely on similar bottom-up and top-down mechanisms
(Rost, 2005, 2016). 2While L2 does not always strictly refer to participants’ second language, we use the
term to denote a language acquired after the L1. High-level text comprehension processes They also act simultaneously and in parallel
by drawing on various types of knowledge sources including lin-
guistic, world, and context-based knowledge (Vandergrift, 2007)
as well as depend on the proficiency/language background of
the listener, their working memory capacity, and the amount of
inhibitory control needed to process the content (Kim, 2015;
Pérez, Paolieri, Macizo & Bajo, 2014; Rapp et al., 2007). In fact,
although for proficient listeners the process can be largely auto-
matic, listeners have some control over their degree of engage-
ment and attentional allocation of the input being attended to
(Bodie, Worthington, Imhof & Cooper, 2008). That is, listening
can be effortful with more challenging contexts (e.g., L2 compre-
hension) compared to less demanding contexts (e.g., L1 compre-
hension). Crucially, there is a gap in the literature of listening
comprehension when the language tested is not the participant’s
L1 and when multilingualism is characteristic of the individual
and societal context. y
g
g
p
Finally, a study investigating high-level cognitive processes
during reading comprehension in the L1 and L2 also found that
executive control (in addition to L2 proficiency) explained indi-
vidual differences in the revision process (Pérez, Hansen &
Bajo, 2019). Specifically, efficient revision in the L1 was related
to a balance between proactive control (the ability to sustain
information related to a meaning goal in anticipation of a cue)
and reactive control (the ability to reactivate meaning goals as a
response to a cue), whereas better revision in the L2 was asso-
ciated with strong proactive control. Thus, a relevant matter
here is to understand whether in developing and multilingual
countries such as South Africa, where English is imposed as the
official language in tertiary education, L1-English speakers have
better performance on high-level listening comprehension ability,
or whether people with a different L1 background (L1-Zulu) but
high L2-English2 proficiency perform equally well. The present study The main aim of this study was to investigate how advanced
English proficient South African adults differing in their L1
(English or Zulu) perform on listening text comprehension in
the language of instruction (English). The present study expands
on research that has assessed inferential monitoring and revision
during reading and multimodal comprehension (Pérez et al.,
2016, 2020) by presenting narrative texts only in the auditory
modality. By comparing L1-English vs. L1-Zulu populations in
their ability to perform inferencing, monitoring, and revision,
we can understand the importance of L1 experience over and
above task proficiency in individuals from multilingual contexts. An additional aim was to understand if the effects derived from
the main goal were explained by individual differences in IC,
especially regarding the revision process. High-level text comprehension processes Revision of the situation model is achieved only if comprehension
of the text is adequately regulated by inhibiting the original inter-
pretation that was encoded into the situation model in favour of
the new, updated interpretation – an updating process that is
both linguistically and cognitively demanding (Kendeou, Smith
& O’Brien, 2013; Rapp & Kendeou, 2007). Central to the revision
process is a distinction between the processes of updating and
outdating, whereby the former involves the encoding and integra-
tion of new information into the mental representation of the text,
while the latter entails a disregarding and replacement of the no INFERENCING is the ability to deduce information from content
without it being explicitly referred to (Cook & O’Brien, 2017;
Graesser, Singer & Trabasso, 1994; Zwaan & Radvansky, 1998). An important inference affecting language processing is predic-
tion – the capacity to precedingly anticipate incoming informa-
tion or adapt to situations where conflicting or ambiguous https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press 3 Bilingualism: Language and Cognition addition, following the establishing text, participants were then
presented with one of two comprehension sentences that brought
either congruent or incongruent information in reference to the
content of the prior text, as in (5a) and (5b). This time, readers
with lower verbal (but not visuospatial) working memory had
longer reading times and made more regressions on congruent
sentences only after being exposed to the unexpected word (“tur-
tles” →“shells”). The authors suggested that the revision process is
more cognitively demanding than the monitoring process and it
depends
on
(verbal)
working
memory
capacity
(also
see
Carretti, Borella, Cornoldi & De Beni, 2009). Importantly, a simi-
lar relationship has been found with IC and its influence on the
quality of the mental representation that is constructed during
reading
and
oral
single
word
comprehension
(Arrington,
Kulesz, Francis, Fletcher & Barnes, 2014; Blumenfeld & Marian,
2011). Interestingly, the effect of working memory in revision dis-
appears when IC is taken into account to explain individual dif-
ferences in multimodal (auditory-verbal and visual-pictorial)
comprehension in L1-English speakers (Pérez et al., 2020) since
IC is thought to encompass aspects of working memory (Tiego,
Testa, Bellgrove, Pantelis & Whittle, 2018). IC is therefore an
important executive function to consider when measuring the
process of revision in text comprehension, but it has not been
solely assessed during listening comprehension. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press High-level text comprehension and inhibitory control In
this sentence, the unexpected condition requires participants to
re-evaluate their mental representation in relation to their initial
inference, while in the expected condition no re-evaluation is
necessary since the content confirms the prediction biased in
the establishing context. Only the last word of the critical sentence
is manipulated for each narrative with the rest of the wording
being identical, thus allowing for participants to experience either
expected or unexpected information at the same point in the crit-
ical sentence in all cases. RL after listening to the critical sentences
gives the “target word RL”. Importantly, to extract a more pure
processing time, the target word RL is divided by the baseline
RL (i.e., “pure target word RL”), which is used as an index of com-
prehension monitoring. g
p
g
According to the second aim, IC is not expected to explain
individual differences for young adults in the monitoring pro-
cess, because as compared to the revision process, text moni-
toring is easier and less cognitively demanding (Pérez et al.,
2016; Hypothesis 7). However, for the revision process, we pre-
dict IC to modulate the effect of listening comprehension per-
formance given that comprehenders with higher IC could be
better at managing this type of conflict by suppressing prior
conflicting information (Pérez et al., 2020), and in particular,
we expect IC to be associated with conditions that tap into
the revision process specifically (unexpected-congruent and
unexpected-incongruent conditions), such that those with bet-
ter IC will answer the comprehension sentences more effi-
ciently
and/or
with
lower
RLs
(Hypothesis
8). Finally,
L1-Zulu participants with higher IC could also show a more
comparable pattern in the revision process, similar to the one
manifested by L1-English participants (Hypothesis 9; Pérez
et al., 2019). Lastly, a final comprehension sentence is again auditorily pre-
sented also in one of two possible conditions: a congruent sen-
tence in which the content relates to the information from the
target concept in the critical sentence (e.g., “feathers” for
“ducks” or “shells” for “turtles”); or an incongruent sentence
where the content does not relate to the target concept in the crit-
ical sentence (e.g., “shells” for “ducks” or “feathers” for “turtles”). Here, participants have to revise the content presented to them
and indicate whether the comprehension sentence matches the
information presented in the critical sentence. High-level text comprehension and inhibitory control Inhibitory control (IC) and working memory have routinely been
associated with comprehension ability (Pérez et al., 2014; Tarchi,
Ruffini & Pecini, 2021). In a significant study for the present
research, Pérez et al. (2016) investigated how adults monitor
and revise their situation model after encountering unexpected
information. Using the mismatch detection paradigm, partici-
pants were presented with a series of short narrative texts where
the first sentences primed a knowledge-based inference as in
(3), followed by a sentence that either brought an expected
(“ducks”) or unexpected but plausible (“turtles”) word, as in
(4a) and (4b). Results revealed that readers were able to detect a
mismatch on unexpected compared to expected words, indicating
a good ability to monitor inferential information in real time. In To test our aims, we used an auditory version of the mismatch
detection task (Pérez et al., 2016) – henceforth, AMDT. In this Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 4 be
higher
(Hypothesis
3)
and
accuracy
to
be
lower
(Hypothesis 4) for comprehenders in conditions where there is
prediction
interference
(expected-incongruent,
unexpected-
congruent, unexpected-incongruent), specifically where revision
is needed, i.e., for critical sentences containing unexpected target
words followed by congruent or incongruent comprehension
sentences. Similarly, we predict RLs to be higher (Hypothesis
5) and accuracy to be lower (Hypothesis 6) in L1-Zulu partici-
pants compared to L1-English participants on all but the
expected-congruent (easiest) condition, given the mismatch
between Zulu participants’ L1 and the language of the task,
while for L1-English participants, no such interference should
emerge. These findings would indicate difficulties for L2
English speakers to revise their situation model during high-
level listening comprehension in English. task, narrative texts comprising three parts (establishing, critical,
and comprehension) are auditorily presented to participants in
succession (see Table 1). The first two sentences establish the con-
text of the story by describing a scenario that biases a knowledge-
based inference generated at the level of the situation model
(“ducks”). Response latency (RL) after listening to the establishing
sentences provides a “baseline RL”. A third sentence is then pre-
sented in one of two possible conditions that either aligns and is
consistent with the prior primed narrative content (expected con-
dition: “ducks”) or misaligns and is unlikely but still provides valid
and plausible information (unexpected condition: “turtles”). Aims and hypotheses The first aim of this study is to investigate the effect of L1 on
high-level comprehension processes in text listening comprehen-
sion in English proficient young South African adults completing
a tertiary education, and secondly, to evaluate whether individual
differences in IC modulate prior effects. Accordingly, our hypoth-
eses are as follows. For the COMPREHENSION MONITORING PROCESS, we
expect greater prediction interference (increased pure target word
RLs) for all comprehenders when they are presented with unex-
pected compared to expected critical words (Hypothesis 1). Furthermore, L1-Zulu participants are predicted to respond com-
paratively slower to the unexpected condition than L1-English
participants because of the misalignment between their L1 and
that of the task, while no differences between groups are antici-
pated for the expected condition where no conflict arises
(Hypothesis 2). Participants Participants were 47 South Africans between 18-24 years old
(Mage = 19.53, SD = 1.37) who were completing an undergradu-
ate degree at an English-medium university in Johannesburg
(years of tertiary education: M = 1.11, SD = .98). Based on
their reported language background, participants were divided
into two groups: L1-Zulu (n = 22; Mage = 19.59, SD = 1.37) and
L1-English (n = 25; Mage = 19.48, SD = 1.39) speakers, where
L1 is operationalised as the first language participants learned
as a child. Participants also listed their home, spoken, and writ-
ten languages, the language(s) they were formally taught in dur-
ing primary and secondary schooling, as well as their use of
English among various interlocutors. Descriptive details about
this
linguistic
information
can
be
found
in
Appendix
A. Importantly, all participants were highly proficient in
English, but, as expected, L1-Zulu participants used English
for fewer years on average than L1-English participants: 14.71
compared to 18.80 years, t(44) = −5.51, p < .001. High-level text comprehension and inhibitory control RL from the
onset of the question mark to the onset of the “Yes/No” response
to the comprehension sentence gives the “sentence RL”. Similar to
the monitoring process, this measure is divided by the baseline RL
to obtain a more pure processing time (i.e., “pure sentence RL”),
and this measure works as an index of revision. Finally, accuracy
scores are also recorded in the comprehension sentence, as a
second measure of revision. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press 3Items can be accessed in the supplementary material of Pérez et al. (2020). They
include four practice items (one per condition) and items 1, 2, 3, 5, 7, 11, 14, 15, 16,
17, 18, 19, 20, 21, 23, 24, 26, 27, 28, 29, 31, 32, 34, 35, 36, 37, 40, 42, 43, 44, 45, 47,
48, 49, 51, 53, 54, 57, 58, 60. Since the original (Pérez et al., 2016) and multimodal
(Pérez et al., 2020) studies were both carried out in the United Kingdom, the content
of each narrative text was evaluated so that only the 40 texts most culturally appropriate
and applicable to the South Africa context were retained. For instance, a text was excluded
if it referred to the monarchy or snowfall – concepts South Africans may be less
familiar with. Inhibitory control y
The flanker task from the Attention Network Task was used to
assess the executive function of IC (Fan, McCandliss, Sommer,
Raz & Posner, 2002). In this task, five horizontal arrows are pre-
sented, and participants need to indicate the direction of the cen-
tral arrow (left or right). Surrounding arrows can either point in
the same (congruent condition) or opposite (incongruent condi-
tion) direction compared to the central arrow. Twenty-four prac-
tice trials followed by 288 main trials were presented over three
blocks (96 trials per block, presented in a counterbalanced
order with four cue conditions by three target conditions). Accuracy rate across the task was high and approaching ceiling
across all conditions (M = 96.31%; SD = 3.69). Incorrect trials
(3.69%) and extreme values under 200ms and over 1200ms
(2.50%) were removed. Reaction times (in milliseconds) were
recorded for each trial and averaged across each condition to cal-
culate the conflict effect (mean RT incongruent - mean RT con-
gruent flanker conditions; Ms = 699.40 (SD = 71.26) and 579.77
(SD = 70.18) for incongruent and congruent conditions, respect-
ively). Higher conflict effect scores mean worse IC (i.e., inhibitory
cost), whereas the opposite is true for lower conflict effect scores. The IC index was included in the final model to understand
whether individual differences in this measure explained differ-
ences in the monitoring and revision processes. As it was mentioned, RLs were recorded at three different
points: (1) from the onset of the fixation cross after listening to
the establishing sentences to the onset of the spacebar key press
(baseline RL); (2) from the onset of the fixation cross after listen-
ing to the critical sentence to the onset of the spacebar key press
(target word RL); and (3) from the onset of the question mark to
the onset of the ‘“Yes/No” response to the comprehension sen-
tence (sentence RL) (see Table 1). Accordingly, to assess compre-
hension monitoring, we calculated a “pure target word RL” index
by dividing the target word RL by the baseline RL, whereas revi-
sion was evaluated by a “pure sentence RL” index, which was
extracted by dividing the sentence RL by the baseline RL. These
indexes avoided any influence from RL speed. Finally, accuracy Control measures To ensure we had a valid presentation of participants’ ENGLISH
PROFICIENCY, both self-assessed and objective measures were
used. The self-assessed measure was evaluated by means of the
Contextual Linguistic Profile Questionnaire (CLiP-Q), whereas
objective proficiency was evaluated with an expressive vocabulary
task, a verbal fluency task, and a reading comprehension task (see
descriptive statistics in Table 2). https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Establishing sentences Note. RL = Response latency. aCritical sentence RL / baseline RL; bComprehension sentence RL / baseline RL. scores were also recorded in the comprehension sentence, as a
second measure of revision. four-sentence narrative texts sourced from Pérez et al. (2020)3, in
one of the two possible critical sentence conditions (expected or
unexpected)
and one of the two possible comprehension
sentence conditions (congruent or incongruent). As such, four
versions were created and counterbalanced so that each of the
40 narratives were presented to participants only once in one of
the four cross-conditions, with 10 trials per cross-condition
(expected-congruent, expected-incongruent, unexpected-congruent,
unexpected-incongruent). The trials were presented in rando-
mised order across two blocks. To begin each trial, the establish-
ing sentence was played when participants pressed the spacebar. Immediately afterwards, a fixation cross appeared in the centre
of the screen. Participants pressed the spacebar again to cue the
soundtrack of the critical sentence, and when it was finished, a
second fixation cross appeared. Participants pressed the spacebar
to hear the final comprehension sentence. This time, after the
comprehension sentence was played, a question mark appeared
and participants were instructed to press “Yes” (‘M’ key) if this
sentence matched with the information presented in the critical
sentence, or “No” (‘Z’ key) if it did not match, as quickly and
as accurately as possible. Materials High-level listening comprehension processes High-level listening comprehension processes
Text listening comprehension was evaluated by means of the
AMDT (see Table 1). Participants were presented with 44 (4 prac-
tice, 40 experimental) culturally appropriate audio-recorded yp
For the REVISION PROCESS, given that RLs have been found to
increase and accuracy to decrease when sentence coherence is
disrupted in general and as a consequence of proficiency (e.g.,
Pérez et al., 2016, 2019, 2020), we expect pure sentence RLs to Bilingualism: Language and Cognition 5 Table 1. Example of a narrative story from the auditory mismatch detection task showing sentence type and narrative information, factors and conditions, cognitive
processes, response recording, and dependent variables assessed in each process. Table 1. Example of a narrative story from the auditory mismatch detection task showing sentence type and narrative information, factors and conditions, cognitive
processes, response recording, and dependent variables assessed in each process. Sentence type and narrative information
Factor and
conditions
Cognitive
process
Response recording
Dependent
variable
Establishing sentences
In the lake by my house there are many animals
that make quacking noises. Yesterday the sounds
were really loud, so I went to see which animals
were making them. Context
establishment
Inferencing
(“ducks”)
From fixation cross (+) at end of
establishing sentence to spacebar
press to cue onset of critical sentence
Baseline
RL
Critical sentence
On a big rock in the middle of the lake, I could
see the “ducks/turtles”. Expectancy
Expected = ducks
Unexpected =
turtles
Monitoring
From fixation cross (+) at end of
critical sentence to spacebar press
to cue onset of comprehension
sentence
Pure target
word RLa
Comprehension sentence
There were animals with brown and green
“feathers/shells” making noise. Congruency
Congruent or
Incongruent
Revision
From question mark (?) at end of
comprehension sentence to answer
of Yes/No question
Pure sentence
RLb and
Accuracy
Note. RL = Response latency. aCritical sentence RL / baseline RL; bComprehension sentence RL / baseline RL. 4A Principal Component Analysis (PCA) was run on all English proficiency measures.
Two components with eigenvalues > 1 emerged, with self-assessed proficiency loading as
one component and objective proficiency as a second component. No differences were
found between groups on either self-assessed, F(1, 45) = 2.06, p = .16, η2 = .04, or objective
proficiency, F(1, 45) = 2.73, p = .11, η2 = .06, but English proficiency scores were positively
correlated, r = .46, p < .001. Contextual linguistic diversity The CLiP-Q (Wigdorowitz, Pérez & Tsimpli, 2020) was used to
assess contextual and individual linguistic diversity. It is a https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 6 Table 2. Mean differences for participants’ background measures by language group. Range
L1-English
L1-Zulu
F
p
η2
Self-assessed English proficiencya
Speaking
6–10
9.00 (1.00)
8.55 (1.01)
2.39
.13
.05
Understanding
6–10
9.12 (1.09)
8.64 (1.00)
2.48
.12
.05
Reading
6–10
9.12 (1.01)
8.73 (1.20)
1.48
.23
.03
Writing
6–10
8.96 (1.17)
8.59 (1.10)
1.23
.27
.03
Objective English proficiency
Expressive vocabularyb
10–17
14.84 (1.18)
13.91 (1.93)
4.11
.05
.08
Verbal fluency
15–42
25.08 (4.48)
23.32 (5.13)
1.58
.22
.03
Reading comprehensionc
11–29
20.60 (.07)
20.09 (3.07)
.23
.64
.01
Working memory
.71–.96
.86 (.06)
.84 (.07)
1.23
.27
.03
Socio-economic status
Socio-economic status index
1.5–6.0
3.81 (1.30)
3.09 (1.05)
4.26
* .045
.09
School typed
2–6
4.04 (.94)
3.23 (.97)
9.11
** .004
.17
Note. arange 1 (very low) to 10 ( perfect); bnorm scores from the WAIS-IVSA; crange 0 to 30, drange 2 to 6. * = p < .05, ** = p < .01. Table 2. Mean differences for participants’ background measures by language group. comprehensive language profiling measure that captures partici-
pants’ demographic information (e.g., age, gender), language
background (e.g., L1, L2), use (e.g., home, school), proficiency
(e.g., speaking, writing), and socio-economic status (SES), empha-
sising the importance of sociolinguistic context. It has been vali-
dated and used in the South African context (Wigdorowitz, Pérez
& Tsimpli, 2022). As part of the CLiP-Q, participants evaluated
their English proficiency across the modalities of speaking, under-
standing, reading, and writing on a Likert scale from 1 (very low)
to 10 ( perfect). These scores were used as indicators of self-
assessed English proficiency. Reading comprehension
Reading comprehension was assessed using the Adult Reading
Test (ART, second edition; Brooks, Everatt & Fidler, 2016). Participants were presented with one practice and three main pas-
sages that increased in length and difficulty based on the Flesh
Kincaid grade levels – a readability formula assessing the approxi-
mate reading grade level of a text (Flesch, 1949; Kincaid,
Fishburne, Rogers & Chissom, 1975). After reading each main
passage, participants were asked 10 pre-specified comprehension
questions based on fact, memory, or inference to assess differing
levels of understanding and retention. Contextual linguistic diversity Correct answers received a
score of 1 with a total comprehension score being the sum of all
questions (range = 0–30).4 Working memory
k g
y
Working memory was tested by a 2-back task, which measures the
storage and updating of information (Kirchner, 1958; Mackworth,
1959). In this task, participants are required to monitor a
sequence of digits to determine whether the present digit is the
same as the digit that was presented n-digits back; in this case,
two digits back, or 2-back. Of a total of 60 digits presented in
the main trial, 20 met the 2-back criteria and required a ‘J’ key
press, while 40 did not, requiring no button press, and were con-
sidered ‘false alarms’. Before the start of the main trials, partici-
pants were presented with 10 practice trials comprising three
‘hits’ and seven ‘false alarms’. A composite A’ (A-prime) score
was calculated based on participants’ hit rates and false alarm
rates, following the A scores outlined by Zhang and Mueller
(2005). A higher A’ score is indicative of better working memory
performance, where participants have fewer misses and incorrect
key presses. Expressive vocabulary To test expressive vocabulary, we used the South African version
of the WAIS-IVSA Vocabulary subtest (Wechsler, 2008, 2014). Participants were verbally and visually presented with a series of
30 words that increased in difficulty for which they were
instructed to provide oral definitions of each. Each definition
is awarded a score of 0, 1, or 2 (except for the first three
words, whose maximum scores are 1; range = 0–57). This scoring
system allows for easy discrimination between high-level and
superficial-level
responses
to
the
same
item. Scores
were
summed and converted to age-normed standard scores (M = 10,
SD = 3; range = 0–19), with higher scores indicating better
performance. Procedure Students who volunteered to participate completed the CLiP-Q
online
prior
to
attending
the
experimental
session. Experimental sessions were carried out at a university with a
researcher and one participant at a time. Each session lasted
between one-to-two hours and took place in one sitting with
regular breaks. The verbal fluency task was audio recorded and
subsequently transcribed, the 2-back, ANT, and AMDT tasks
were
completed
on
PsychoPy
(Peirce,
2007),
and
the
WAIS-IVSA vocabulary subtest
and ART-2 were
manually
recorded using scoring templates from the assessment manuals. The order of assessments was counterbalanced across one of
four conditions where each participant completed the next con-
secutive order. To ensure uniformity, however, all participants
began the session with the verbal fluency task and the fourth
task was always the vocabulary subtest. Participants gave their
informed consent prior to completing the CLiP-Q online and
again before testing, and received monetary compensation. To determine the optimal structure for the random and fixed
components, we followed the procedure outlined by Zuur, Ieno,
Walker, Saveliev and Smith (2009). We first looked for the best
random structure using maximum likelihood, while the full
fixed structure (i.e., three-way interaction) was retained (Barr,
Levy, Scheepers & Tily, 2013). More specifically, the random
structure was tested by running an ANOVA between all possible
models containing the various combinations of intercepts and/
or slopes, using group and condition as random slopes, while
keeping the full fixed structure. The converging model that con-
tained the lowest Akaike information criterion (AIC) and
Bayesian information criterion (BIC) values was selected. Once
the best random structure was identified, we then tried to obtain
the best fixed structure. To do this, we ran stepwise model com-
parisons from the most complex model (i.e., three-way inter-
action) to the simplest model (i.e., main effects), by selecting
the significant χ2 test for the log-likelihood, using maximum like-
lihood. Third, F and p values were provided by the ANOVA func-
tion of the lmerTest package using Satterthwaite’s method and χ2
and p values were provided by the ANOVA function of the car
package using Type II Wald chi-square tests (Fox et al., 2021). To qualify the two-way interactions, the testInteractions function
of the phia package (De Rosario-Martínez, 2015) and emmeans
function of the emmeans package (Lenth, Buerkner, Herve,
Love, Riebl and Singmann, 2021) were used for post-hoc analyses
with Bonferroni correction. 5A PCA was also applied to the SES measures, where it was found that all indicators
loaded as one component (eigenvalue > 1). L1-English participants had a significantly
higher SES index score than L1-Zulu participants, F(1, 45) = 9.11, p < .01, η2 = .17.
6When SES was added to the statistical models the overall results remained the same Socio-economic status effects models (LME) with the lmer function of the lme4 R pack-
age (Bates et al., 2021), whereas accuracy, as it is a binomial meas-
ure (0 or 1), was analysed through mixed-effects logistic
regression (MELR) models with the glmer function of the same
package. All models included Participants and Items as random
factors, and Condition (expected vs. unexpected for monitoring;
expected-congruent vs. expected-incongruent vs. unexpected-
congruent vs. unexpected-incongruent for revision), Group
(L1-English vs. L1-Zulu), and the centered values of IC, as the
fixed factors. Thus, the full fixed structure of each model was
always a three-way interaction (condition*group*IC) as well as
the combination of all its lower-level interactions and main
effects. Using the simr package (Green & MacLeod, 2016), we
ran power analyses with 1,000 simulations and alpha set at .05
to assess whether the sample size was sufficient to detect effects
in our models. A composite SES score was captured from the CLiP-Q and calcu-
lated from an index of 12 household assets (0 = no, 0.5 = yes,
summed), annual household income (value range according to
tax brackets; 1 = less than R195,850 to 6 = R708,311 and above),
and maternal and paternal level of education (1 = primary school
to 6 = Master’s or higher). Participants’ type of primary and sec-
ondary school was used as an additional indicator of SES (1 = gov-
ernment township/rural school, 2 = government suburban school,
3 = private school, summed across primary and secondary school-
ing; range 2–6).5 Data analyses Initial analyses to assess normality and control for confounds
were carried out. Using ANOVAs, we compared the continuous
scores of each background measure across language group (see
Table 2). No differences were found in any of the self-assessed
English proficiency measures, the objective English proficiency
measures, and working memory (all ps > .05), suggesting that
the groups were comparable in these abilities. However, both
SES measures presented differences between groups, where the
L1-English group had a higher SES score than the L1-Zulu
group. Unfortunately, these SES differences are reflective of the
inherent inequality between the two sample groups that were
selected
from
South
Africa
(Cornwell
&
Inder,
2008;
Leibbrandt, Finn & Woolard, 2012), and therefore, SES cannot
be teased apart from the statistical analyses.6 Verbal Fluency The verbal fluency task assesses lexical retrieval efficiency, tapping
into both proficiency and varying levels of executive control
(Friesen, Luo, Luk & Bialystok, 2015). In our version, participants
were required to generate exemplars from two semantic categor-
ies: living (animals) and non-living (fruits), under a 30-second
time restriction per condition. One point was awarded for each
new and correct word and no points were given for repeated
words or if words did not belong to the specified category (e.g.,
carrot reported as a fruit). Summed scores for the combined cat-
egories were calculated. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Bilingualism: Language and Cognition Bilingualism: Language and Cognition 7 Socio-economic status https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press 6When SES was added to the statistical models, the overall results remained the same. Target word in the text: Monitoring process Target word in the text: Monitoring process SE = .06), t(1410) = –3.66, p < .01. No other differences were
found between these conditions ( ps > .31). A main effect of
group also emerged, F(1, 46.11) = 13.85, p < .001, ηp
2 = .23, where
L1-English participants (M = 1.22, SE = .06) were significantly
faster to respond to the comprehension sentences than L1-Zulu
participants (M = 1.43, SE = .06). A third main effect of IC
emerged, F(1, 45.67) = 9.48, p < .01, ηp
2 = .17, where participants
with higher conflict costs (i.e., lower IC) were faster to respond
to the comprehension sentences in general. In addition, the two-
way interaction of group × condition was marginally significant,
F(3, 1395.88) = 2.46, p = .06, ηp
2 = .01, and the interaction of con-
dition × IC was significant, F(3, 1390.83) = 2.84, p < .05, ηp
2 = .01. No other interactions were significant (all ps > .17). An LME model with Participants and Items as the random fac-
tors,
and
Condition
(expected
vs. unexpected),
Group
(L1-English vs. L1-Zulu), and IC as the fixed factors, was run
on the pure target word RL index. A significant main effect of
condition emerged, F(1, 1437.76) = 83.26, p < .001, ηp
2 = .05,
which showed that participants were faster to respond to expected
words (M = 1.17, SE = .03) compared to unexpected words (M =
1.43, SE = .03). A marginal effect of group was also found, F(1,
46.87) = 3.54, p = .07, ηp
2 = .07, where L1-Zulu participants (M =
1.35, SE = .04) were marginally slower to respond to the critical
sentences than L1-English participants (M = 1.25, SE = .04). More importantly, a two-way interaction of group × condition
emerged, F(1, 1437.76) = 9.32, p < .01, ηp
2 = .01 (see Figure 1),
where post-hoc comparisons revealed a significant difference
between groups for the unexpected, χ2(1) = 9.73, p < .01, but not
the expected condition, χ2(1) = 0.01, p ≈1.00. Specifically, while
both groups were equally efficient at responding to words in the
expected condition, L1-Zulu participants were slower to respond
to the critical sentence than L1-English participants when this
sentence contained an unexpected word. No other effects were sig-
nificant (all ps > .56). Target word in the text: Monitoring process These findings suggest that while both groups
were equally efficient at comprehending coherent information, the
L1-Zulu group took longer to monitor inconsistent information in
English than the L1-English group, suggesting a less efficient mon-
itoring process. In addition, none of the monitoring process effects
were qualified by individual differences in IC. g
p
Post-hoc comparisons in the interaction of group and con-
dition revealed that compared to the L1-English group, the
L1-Zulu group took longer to respond to the comprehension
sentences
when
presented
with
incongruent
information:
expected-incongruent, t(185) = –3.15, p < .05, and unexpected-
incongruent, t(230) = –3.72, p < .01, conditions; but this did not
happen when information was congruent: expected-congruent,
t(171) = -1.01, p = .97, and unexpected-congruent, t(187) = -2.40,
p = .25, conditions, where L1-Zulu participants responded more
similarly to the L1-English group (see Figure 2). Therefore, as it
occurred in the monitoring process, our results indicate that the
two groups did not differ when the story was completely coherent
(i.e., expected-congruent), and they had to confirm the informa-
tion in the comprehension sentence (i.e., unexpected-congruent). In
contrast,
L1-Zulu
participants
were
less
efficient
than
L1-English
participants
when
the
comprehension
sentence
required the disconfirmation of the presented information (i.e.,
expected-incongruent and unexpected-incongruent) as well as
when it demanded revision of the initial interpretation followed
by content that did not align with the unexpected information
(i.e., unexpected-incongruent). Pure sentence RL index Pure sentence RL index
A second LME model with Participants and Items as the
random factors, and Condition (expected-congruent vs. expected-
incongruent vs. unexpected-congruent vs. unexpected-incongruent
for revision), Group (L1-English vs. L1-Zulu), and IC as the fixed
factors, was performed on the pure sentence RL index. The main
effect of condition was significant, F(3, 1394.88) = 12.04, p < .001,
ηp
2 = .03, where post-hoc comparisons showed that the pure sen-
tence RL index was higher on the unexpected-incongruent condi-
tion (M = 1.54, SE = .06) in comparison to the other three
conditions: (a) expected-congruent (M = 1.23, SE = .06), t(1405)
= –5.30, p < .001, (b) expected-incongruent (M = 1.22, SE = .06),
t(1410) = –5.27, p < .001, and (c) unexpected-congruent (M = 1.33, Moreover, pairwise comparisons in the interaction of condi-
tion and inhibitory control showed significant differences between
the regression slopes of the four conditions, χ2(3) = 8.52, p < .05,
where participants with higher inhibitory cost (lower IC) took
longer in the unexpected-incongruent condition compared to
the other three conditions. In contrast, participants with lower
inhibitory cost (higher IC) showed no differences between condi-
tions (see Figure 3). Results Our results are divided into two sections, depending on the
AMDT structure. First, we present the RL results obtained after
the presentation of the critical sentence containing the target
word (i.e., pure target word RL index), regarding the comprehen-
sion monitoring process. Subsequently, we present the two mea-
sures
assessed
in
the
comprehension
sentence
(i.e.,
pure
sentence RL index and accuracy), in relation to the revision
process. In this paper we focused on the fixed effects of the
models but summary details regarding model fit and random
effects of each model are provided in Appendix B, as well as
Pearson
correlations
between
each
variable
in
Appendix
C. Results of the power analyses confirmed that the sample size
for each model had sufficient power: pure target word RL index
= 100% (CI = 99.63–100%), pure sentence RL index = 88.90%
(CI = 86.79–90.78%), and accuracy = 100% (CI = 99.63, 100%)
(Brysbaert, 2019). p
y
For the RL analyses, only accurate data was used. Furthermore,
outliers were identified using the boxplot function, where data
points are considered outliers if they lie outside 1.5 times the
interquartile range above the upper quartile and below the
lower quartile (Becker, Chambers & Wilks, 1988). A total of 92
(5.86%) outliers were removed from the pure target word RL
index (monitoring process), and 134 (8.54%) outliers were
removed from the pure sentence RL index (revision process). These two pure RL indexes were analysed through linear mixed https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 8 Accuracy th mean comprehension sentence response latency scores, representing the interaction between condition and inhibitory control. whether individual differences in IC explained any effects asso-
ciated with listening comprehension processes. unexpected-incongruent) and the L1-English group manifested
better comprehension (and therefore, better revision) than the
L1-Zulu group. Accuracy An MELR model with Participants and Items as the random fac-
tors, and Condition (expected-congruent vs. expected-incongru-
ent vs. unexpected-congruent vs. unexpected-incongruent for
revision), Group (L1-English vs. L1-Zulu), and IC as the fixed fac-
tors, was run on accuracy to the comprehension sentence. Similar
to the previous measure, a significant main effect of condition
emerged, F(3, 66.47) = 22.16, p < .001, where post-hoc compari-
sons showed lower accuracy on the unexpected-incongruent con-
dition,
(M = .72,
SE = .11),
compared
to
the
other
three
conditions: (a) expected-congruent, (M = .91, SE = .16), z = 7.10,
p < .001, (b) expected-incongruent, (M = .86, SE = .14), z = 5.22,
p < .001, and (c) unexpected-congruent, (M = .87, SE = .14), z =
5.53, p < .001. There was also a significant main effect of group,
F(1,
5.78) = 5.78,
p < .01,
with
higher
accuracy scores
for
L1-English participants (M = .86, SE = .09) compared to L1-Zulu
participants (M = .81, SE = .09). No other conditions or interac-
tions were significant ( ps > .19). Therefore, all participants per-
formed worst in the most difficult revision condition (i.e., Fig. 1. Mean and standard error values of the pure target word response latency
index obtained after listening to the critical sentence of the AMDT, between the lan-
guage groups across each of the expectancy conditions. Fig. 1. Mean and standard error values of the pure target word response latency
index obtained after listening to the critical sentence of the AMDT, between the lan-
guage groups across each of the expectancy conditions. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Bilingualism: Language and Cognition 9 Fig. 2. Mean and standard error values of the pure sentence response latency index obtained after listening to the comprehension sentence of the AMDT, between
the language groups across each of the four expectancy and congruency conditions. Fig. 2. Mean and standard error values of the pure sentence response latency index obtained after listening to the comprehension sentence of the AMDT, between
the language groups across each of the four expectancy and congruency conditions. Fig. 3. Scatter linear graph with mean comprehension sentence response latency scores, representing the interaction between condition and inhibitory control. Fig. 3. Scatter linear graph with mean comprehension sentence response latency scores, representing the interaction between condition and inhibitory control. Discussion The comprehension monitoring process was evaluated by manipu-
lating the expectancy of the target word presented in the critical
sentence, which either aligned (expected) or misaligned (unex-
pected) but was still plausible with the generated inference made
from the establishing context. Accordingly, the pure target word
RL index for the critical sentence was used to evaluate whether
comprehenders had generated the knowledge-based inference
facilitated by the establishing context, and subsequently detected
a mismatch between this prediction and the unexpected word. This study is the first to evaluate high-level listening comprehen-
sion in young South African adults with advanced proficiency in
English – the language of tertiary education and the country’s lin-
gua franca – but who have differing L1s that either match
(English) or differ from (Zulu) the language of the listening com-
prehension task. Our first goal was to understand how these
groups monitor and revise inferential information while listening
to narrative stories. We were additionally interested in exploring https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 10 Rohde & Shafer, 2014) and recent research suggests that the
mechanisms underlying sentence processing, and prediction in
particular, is comparable in L1 and L2 comprehenders, where
results should be explained in terms of individual differences
between groups (Kaan, 2014). Accordingly, there does appear to
be a processing advantage for individuals with an L1 that matches
the language of the task, even when all participants have advanced
proficiency, which is attributable to a heightened processing sen-
sitivity to variation within the semantic context. Interestingly, 12
(54.5%) L1-Zulu participants reported English as being their most
proficient language. This result may too be a consequence of the
quantity and quality of English input and usage between the
groups, given that L1-English participants reported to use
English with a variety of interlocutors (e.g., family, friends)
more so than L1-Zulu participants (see Appendix A). Further
research exploring questions of typological distance between lan-
guages as well as input and usage quantity and quality in multi-
lingual contexts as it relates to the monitoring process in
listening
comprehension
should
therefore
be
avenues
of
exploration. Revision in high-level listening comprehension During successful comprehension of unfolding narratives, the
process of revision is required such that comprehenders need to
review the existing text interpretation and update their situation
model so that the new and potentially unexpected content can
become meaningfully integrated into the discourse context. This
process is cognitively demanding and largely dependent on the
proficiency
and
language
experience
of
comprehenders
(Kendeou et al., 2013; Pérez et al., 2015, 2016, 2020). Until
now, however, no study has investigated whether L1, as either cor-
responding with (English) or differing from (Zulu) the language
of the task, has an effect on the revision process under the condi-
tion that English proficiency for the whole sample is advanced. Revision was assessed by combining the expectancy of the target
word presented in the critical sentence (expected vs. unexpected)
with the congruency of the critical information presented in the
comprehension sentence (congruent vs. incongruent), by means
of both the pure sentence RL index in and accuracy to the com-
prehension sentence. Crucially, revision was only required after
the presentation of the unexpected target word, to (dis)confirm
the comprehension sentence. p
In line with the general predictions, we found that RLs were
higher (Hypothesis 3) and accuracy lower (Hypothesis 4) on the
unexpected-incongruent condition (“turtles” →“feathers”) in
comparison to the other three conditions, with no differences
between them. These results indicate that participants were least
efficient when they were required to disconfirm information
that was coherent with their initial inference (e.g., “feathers” →
“ducks”). In fact, this was considered the most difficult revision
condition due to the need to be able to discard no longer probable
information. In contrast, participants were able to quickly and
accurately respond to information that was either coherent with
their initial interpretation or incoherent with this interpretation
but confirmed by the comprehension sentence. Comparisons can further be drawn with acoustic studies
assessing monosyllabic word comprehension in the presence of
noise and/or reverberation interference. Monolinguals consist-
ently outperform highly proficient L2-English bilinguals during
English speech perception in noise tasks, suggesting that meaning
extraction is compromised during speech signal detection in L2
listening (Rogers, Lister, Febo, Besing & Abrams, 2006; Shi,
2010; Tabri, Chacra & Pring, 2011). Importantly, these results
are not explained by proficiency but rather by participants’ L1
background
as
it
relates
to
the
language
of
the
task. Discussion Previous studies on comprehension monitoring within both
the reading modality and auditory-verbal and visual-pictorial
modality at the sentence and discourse level have demonstrated
a general expectancy effect (Baker, 1989; Hu & Jiang, 2011;
Pérez et al., 2016, 2019, 2020; Vorstius, Radach, Mayer &
Lonigan, 2013). In line with these findings, increased RLs in the
unexpected compared to the expected condition confirmed that
our participants had generated an initial inference and were
able to monitor their comprehension across the auditory modality
(confirming Hypothesis 1). This suggests that the initial inference
generated from the establishing sentence was more likely to be
activated in the situation model than the alternative interpretation
when participants encountered the target word in the critical sen-
tence. Notably, these results also indicate that a similar compre-
hension monitoring process is evident across various forms of
content presentation, whereby the ability to monitor inferential
information across different modalities is efficient and largely
modality independent. Importantly, we also observed that L1 background played a
role in comprehension monitoring efficiency when there was pre-
diction interference. As predicted (Hypothesis 2), L1-Zulu parti-
cipants
were
slower
to
process
sentences
containing
the
unexpected word compared to L1-English participants, while
there were no group differences in the expected condition. Thus, monitoring was less efficient for comprehenders who,
although highly proficient, had a different L1 to that of the task
when there was misalignment between the target word in relation
to the previously presented content. A similar reading time effect
was observed in an English monolingual sample with a slightly
different paradigm involving three conditions evaluating compre-
hension monitoring (a neutral condition, which contained general
information following the establishing content; a no-revise condi-
tion, which was similar to the expected condition containing a
likely concept; and a revise condition, which contained an unex-
pected but plausible concept) (Pérez, Cain, Castellanos & Bajo,
2015). Similar results have also been found in behavioural and
electrophysiological studies evaluating comprehension monitor-
ing, where highly proficient L2 speakers are less sensitive to
upcoming content and do not anticipate information to the
same extent compared to L1 speakers, likely because more time
is required to deal with unexpected information in a non-
foundational
language
(Chambers
&
Cooke,
2009;
Kaan,
Kirkham & Wijnen, 2016; Martin, Thierry, Kuipers, Boutonnet,
Foucart and Costa, 2013). This effect may be related to typological
differences between languages, though (Foucart, Martin, Moreno
& Costa, 2014). https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Revision in high-level listening comprehension Comprehension monitoring may therefore draw on automatised
processing which may be harder to attain if content is not pre-
sented in the L1. Using the same paradigm but assessing reading comprehen-
sion, Pérez et al. (2016) found that readers spent less time reading
congruent compared to incongruent sentences but only for narra-
tives preceded by an expected (but not unexpected) concept. Furthermore, when text containing an unexpected concept was
followed by a congruent (but not incongruent) sentence, readers Earlier
theories
of
sentence
processing
postulated
that
non-L1 comprehenders have a Reduced Ability to Generate
Expectations (RAGE; Kaan, Dallas & Wijnen, 2010; Grüter, Bilingualism: Language and Cognition 11 or equivalent (Filippi, Ceccolini, Periche-Tomas & Bright 2020)
in bilinguals compared to monolinguals. A preliminary interpret-
ation of these findings proposes that self-evaluation of perform-
ance may be related to the language background of participants,
such that confidence is reduced in relation to the number of par-
ticipants’ spoken languages. Applied to this study, it is likely that
L1-Zulu participants were less confident in their judgement of
auditorily presented information tapping revision purely because
English is not their L1 and/or they are regularly exposed to
numerous languages, making them more sensitive to resolving
conflict
and
accepting
inaccurate
information. While
the
L1-Zulu participants have the linguistic and cognitive resources
to deal with prediction interference, and indeed perform remark-
ably well on other language tasks and reading comprehension (see
Table 2), they may be less assured in their interpretation of audi-
tory content, specifically when it is complex and requires the can-
cellation or questioning of previously generated knowledge and/or
inferences. While metacognition was not directly assessed in this
study, it is worth exploring its influence in future research to shed
light on the effect of self-awareness on non-L1 comprehension
processing, given that our interpretation is speculative. were slower to respond than when it contained an expected con-
cept. Crucially, for listening comprehension, the only difference
found relates to the condition where incongruent content follows
an unexpected concept. While for reading comprehension, it
appears that prior context facilitates (or impedes) reading when
narrative consistency is maintained (or disrupted), for listening
comprehension, the effect is evinced only in the most difficult
revision condition involving an unexpected target concept fol-
lowed by an incongruent comprehension sentence. Inhibitory control in high-level listening comprehension As a second goal, we explored whether high-level comprehension
processes were modulated by individual differences in IC. Sentence processing as explained by language differences may
be accounted for by processing loads during L2 comprehension,
such
that
when
information
is
processed
in
a
non-L1
language, it requires additional cognitive resources and may be
processed at a lower, usually lexical level, leaving fewer resources
for conceptual/semantic processing (Frey, 2005; Perfetti, Yang &
Schmalhofer, 2008). Comparably, the process of revising outdated
inferential content has been found to involve IC, whereby irrele-
vant information is efficiently disregarded while pertinent infor-
mation is at the forefront of one’s focus (Pérez et al., 2016). This may be borne out during high-level listening comprehension
where great inhibitory capacity is required to efficiently process
ongoing content, especially for non-L1 comprehenders. As such,
IC was evaluated as a factor modulating listening comprehension
performance for L1-English and L1-Zulu groups. In terms of language differences, L1-English comprehenders
were significantly faster than L1-Zulu comprehenders to respond
to the comprehension sentence. Importantly, a two-way inter-
action of group × condition also emerged for RLs, where the
L1-Zulu group took longer to respond to the comprehension sen-
tence than the L1-English group in the expected-incongruent
(“ducks” →“shells”), and unexpected-incongruent (“turtles” →
“feathers”)
conditions,
compared
to
the
easiest
expected-
congruent condition (“ducks” →“feathers”, almost confirming
Hypothesis 5). The same was also predicted for the unexpected-
congruent condition (“turtles” →“shells”), but although means
suggested a similar RL trend, this effect was not significant
( p = .25). Overall, our findings indicate that non-L1 comprehen-
ders experienced more difficulties than L1 speakers in dealing
with interference prediction, especially when the hardest revision
condition was involved during high-level listening comprehen-
sion. These results are consistent with Pérez et al. (2019), who
found that participants were less efficient at revising their situ-
ation model during a reading comprehension task when it was
presented in their L2 compared to their L1, even though these
comprehenders were highly proficient in both languages. p
g
g
p
As expected, individual differences of IC were not associated
with the comprehension monitoring process (Hypothesis 7). This lack of effect is consistent with previous studies showing
that text monitoring of knowledge-based predictive inferences is
a more passive process and not very cognitively demanding, espe-
cially in highly proficient individuals (Pérez et al., 2015, 2016). Revision in high-level listening comprehension This supports
the claim that revising content that is disconfirmed by one’s initial
inference followed by a further iteration of incongruency is a cog-
nitively taxing and difficult comprehension process. y
g
p
p
Importantly, some research has found a prolonged influence of
initially encoded information from early parts of a narrative text,
such that the initial content exerts an effect on comprehension
even when newer content is qualified as the narrative unfolds
(Guéraud et al., 2005; Raap & Kendeou 2007), while other
research suggests that comprehenders can successfully amend
their knowledge to reflect new states of affairs that emerge over
the course of a narrative text (de Vega, 1995; Diakidoy,
Kendeou & Ioannides, 2003). Depending on the type and diffi-
culty
of
conditions
presented,
both
arguments
hold. Comprehenders are sufficiently able to modify earlier representa-
tions of a text with updated content, specifically when there is
only one disruption to coherence, but experience processing dif-
ficulties when listening to narratives that include prediction inter-
ference across two instances of coherence. https://doi.org/10.1017/S1366728923000135 Published online by Cambridge University Press Inhibitory control in high-level listening comprehension In
contrast, IC was related to the revision process, suggesting that it
is more cognitively demanding than the monitoring process
(Pérez et al., 2016, 2020). Accordingly, participants with lower
IC were faster at responding to comprehension sentences in gen-
eral, but no differences in accuracy emerged. While not an
expected finding, it may be the case that participants with greater
IC were more deliberate and considered when responding to com-
prehension sentences, thereby being slower while still manifesting
an accurate interpretation. However, further research would be
necessary to clarify this general effect. For the accuracy analysis, general scores were relatively high,
with L1-English and L1-Zulu participants achieving accuracy
scores of 85.8% and 81.6%, respectively. However, L1-English
comprehenders were more accurate than L1-Zulu comprehen-
ders, suggesting a more accurate inferential comprehension
once more in favour of L1-comprehenders (partially confirming
Hypothesis 6). Nonetheless, no interaction with condition
emerged, so these differences did not seem to be due to difficul-
ties in the revision process. A possible way to address these differences may be accounted
for in terms of metacognition – an awareness and evaluation
of one’s own cognitive processes and thoughts (Flavell, 1979;
Fleming & Lau, 2014). Indeed, metacognition has been found
to be lower (Folke, Ouzia, Bright, De Martino & Filippi, 2016) More
importantly,
IC
also
interacted
with
condition,
where participants with lower IC (i.e., high inhibitory cost) took
longer to respond to the most difficult condition (unexpected-
incongruent) when they were required to revise their situation
model and replace their initial interpretation for a new one, Mandy Wigdorowitz, Ana I. Pérez and Ianthi M. Tsimpli 12 substantial and proficiency is advanced. Furthermore, while
there are processing costs associated with comprehension in the
auditory modality of linguistically and cognitively demanding
information, these do not appear to correspond to costs in
other linguistic tasks or reading comprehension, indicating the
remarkable ability of learners with advanced English proficiency
but who have a different L1. Together, these results reinforce
the idea that comprehenders with an L1 similar or dissimilar to
that of the task language, but who have high proficiency in the
language of the task were faster at revising their situation model
when their initial interpretation had changed, because they were
better able to suppress the interference generated by their initial
prediction equally well. Inhibitory control in high-level listening comprehension Since this study was conducted on
South African university students, the results may only be gener-
alisable to similar linguistically diverse and highly educated
populations. Notwithstanding, given that proficiency is consid-
ered a primary predictor of comprehension and educational
outcomes more generally, this study further illuminates the pic-
ture by illustrating that L1 is also important to consider in
comprehension performance, especially in linguistically diverse
contexts. compared to the rest of the conditions. In contrast, this was not
the case for participants with higher IC, who took a similar
amount
of
time
in
all
conditions
(partially
confirming
Hypothesis 8). These findings suggest that participants with
lower IC experience difficulties revising their situation model,
whereas higher IC participants perform comparably across condi-
tions. Similarly, Pérez et al. (2020) found that L1-English compre-
henders
with
higher
IC
performed
more
accurately
on
multimodal comprehension tapping the revision process, thereby
suggesting that participants with higher IC are better able to revise
their situation model when confronted with conflicting informa-
tion across pictorial and verbal modalities. Finally, we did not observe an effect of IC as a consequence of
L1-groups (disconfirming Hypothesis 9), suggesting that L1-Zulu
participants across the IC spectrum were comparable in process-
ing efficiency and accuracy to L1-English participants when
answering comprehension sentences. There does not appear to
be a relationship between IC as it relates specifically to L1 back-
ground in highly proficient young adults. Rather, we found a gen-
eral effect of IC across all participants, irrespective of their L1
background. A possible explanation may be linked to participants’
extensive quantity and high quality of English exposure and use
spanning a number of communicative contexts, coupled with
the fact that all participants could be classified as multilingual
to some degree and might therefore have enhanced executive con-
trol from regular language competition (Bialystok, Craik, Green &
Gollan, 2009). Notably, none of the participants were charac-
terised as ‘typically’ monolingual in the sense that they spoke
and had experience with only one language (see Appendix A). For instance, of the 10 participants who self-reported as monolin-
gual, they indicated that they spoke between two to five languages
(M = 3.40, SD = .84) and could write in one to three of these (M =
2.30, SD = .67), suggesting that this group actively uses more than
one language. Inhibitory control in high-level listening comprehension It has also been well established that an individual’s
languages are concurrently active irrespective of whether only one
is being used at any given time (Kroll, Bobb, Misra & Guo, 2008;
Wu & Thierry, 2010). Accordingly, suppression of non-target
language(s) is a regular cognitive activity for bi/multilinguals,
which is thought to enhance executive function beyond the domain
of language (Abutalebi & Green, 2007; Stocco, Yamasaki, Natalenko
& Prat, 2014). Such cognitive control may be heightened in indivi-
duals living in highly linguistically diverse contexts such as South
Africa, explaining why we did not observe group differences in
IC. In future studies, it would be beneficial to evaluate the role of
other (continuous) language factors, such as dominance, English
age-of-acquisition, degree of exposure to languages within the ambi-
ent context and so forth, since some of these factors have been
found to be determinants of executive control and/or comprehen-
sion ability in multilingual South Africans (Espi-Sanchis &
Cockcroft, 2021; Cockcroft, Wigdorowitz & Liversagem, 2019;
Palane & Howie, 2020). Acknowledgements. This work was supported by the Commonwealth
Scholarship Commission and Cambridge Commonwealth, European &
International Trust to Dr. Mandy Wigdorowitz, and MSCA-COFUND
Athenea 3i-2018 grant (754446) to Dr. Ana I. Pérez: [Grant Number
754446]. We would like to thank Margreet Vogelzang for assistance with cod-
ing the auditory mismatch detection task on PsychoPy and all the participants
who took part in this study. Competing interests. The authors declare none Data availability. The data that support the findings of this study are openly
available in OSF at https://doi.org/10.1017/S1366728923000135 Data availability. The data that support the findings of this study are openly
available in OSF at https://doi.org/10.1017/S1366728923000135 Supplementary Material. For supplementary material accompanying this
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https://discovery.ucl.ac.uk/id/eprint/1532734/1/Walters_Age-related%20references%20in%20national%20public%20health%2C%20technology%20appraisal%20and%20clinical%20guidelines%20and%20guidance.%20Documentary%20analysis_VoR.pdf
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Age-related references in national public health, technology appraisal and clinical guidelines and guidance: documentary analysis
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Age and ageing
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Abstract Background: older people may be less likely to receive interventions than younger people. Age bias in national guidance
may influence entire public health and health care systems. We examined how English National Institute for Health & Care
Excellence (NICE) guidance and guidelines consider age. (
) g
g
g
Methods: we undertook a documentary analysis of NICE public health (n = 33) and clinical (n = 114) guidelines and tech-
nology appraisals (n = 212). We systematically searched for age-related terms, and conducted thematic analysis of the para-
graphs in which these occurred (‘age-extracts’). Quantitative analysis explored frequency of age-extracts between and within
document types. Illustrative quotes were used to elaborate and explain quantitative findings. Results: 2,314 age-extracts were identified within three themes: age documented as an a-priori consideration at scope-setting
(518 age-extracts, 22.4%); documentation of differential effectiveness, cost-effectiveness or other outcomes by age (937 age-
extracts, 40.5%); and documentation of age-specific recommendations (859 age-extracts, 37.1%). Public health guidelines
considered age most comprehensively. There were clear examples of older-age being considered in both evidence searching
and in making recommendations, suggesting that this can be achieved within current processes. g
gg
g
p
Conclusions: we found inconsistencies in how age is considered in NICE guidance and guidelines. More effort may be
required to ensure age is consistently considered. Future NICE committees should search for and document evidence of
age-related differences in receipt of interventions. Where evidence relating to effectiveness and cost-effectiveness in older
populations is available, more explicit age-related recommendations should be made. Where there is a lack of evidence, it
should be stated what new research is needed. Keywords: older-age, NICE guidance, clinical, public health, technology appraisal Age-related references in national public health,
technology appraisal and clinical guidelines
and guidance: documentary analysis
LYNNE F. FORREST1,2, JEAN ADAMS1,3, YOAV BEN-SHLOMO4, STEFANIE BUCKNER5, NICK PAYNE6,
MELANIE RIMMER6, SARAH SALWAY6, SARAH SOWDEN1, KATE WALTERS7, MARTIN WHITE1,3 1Institute of Health & Society, Newcastle University, Newcastle upon Tyne, Newcastle upon Tyne, UK
2School of GeoSciences, University of Edinburgh, Edinburgh, UK
3MRC Epidemiology Unit, University of Cambridge, Cambridge, Cambridgeshire, UK
4School of Social and Community Medicine, University of Bristol, Bristol, UK
5Institute of Public Health, University of Cambridge, Cambridge, Cambridgeshire, UK
6School of Health and Related Research, University of Sheffield, Sheffield, UK
7Centre for Ageing and Population Studies, University College London, UK Address correspondence to: Jean Adams. Tel: (+44) 1223 769 142; Fax: (+44) 1223 330 316. Email: jma79@medschl.cam.ac.uk Age and Ageing 2017; 46: 500–50
doi: 10.1093/ageing/afw235 Age and Ageing 2017; 46: 500–50
doi: 10.1093/ageing/afw235 Age-related references in national public health, technology appraisal and clinical guidelines and receipt of effective interventions occur and identify
strategies to overcome these. health guidelines and technology appraisals (see Box 1). NICE guidance and guidelines are produced in multiple
formats and each final guideline or guidance is accompan-
ied by a variety of supporting documents. We focused on
final documents that professionals searching for these
would be likely to find and use. g
In the UK, the National Service Framework for Older
People (2001) highlighted the need for ‘rooting out age-
discrimination’ (p12) in Health and Social Care [10]. The
UK Equality Act (2010) came into force on 1st October
2012 making age discrimination in the provision of services
and public functions, including health care and public health,
unlawful. Where age is considered a relevant factor in clin-
ical and public health decision-making, the Equality Act
(2010) requires ‘objective justification’ (p10) in accordance
with evidence-based guidelines [11]. Elsewhere, the World
Health Organization is consulting on a Global Strategy and
Action Plan on Ageing and Health with a focus on equitable
access to interventions [12]. Documents available from the list at http://www.nice. org.uk/guidance in July 2014 were considered for inclusion. This list included full versions of public health guidelines
and technology appraisals, and shortened versions of clin-
ical guidelines. We did not include supporting documents or
‘fuller’ versions of clinical guidelines. Guidelines and guid-
ance specific to young people, children, or pregnant women
were excluded. Documents for exclusion were identified by
L.F.F. and independently checked by N.P. Disagreements
were resolved by discussion. One important source of information for those planning
and delivering public health and health care interventions is
national guidance and guidelines. In England, the National
Institute for Health & Care Excellence (NICE) produces
evidence-based guidance (mandatory) and guidelines (advis-
ory) for health, public health and social care (see Box 1). NICE is internationally recognised as a role model in this
area [13]. If age, or other, biases are present in guidance
and guidelines, this may influence entire systems. Analysis and presentation of data We systematically searched three types of NICE documents to
identify all references to age and categorised these into themes. We then explored the frequency of these references to age
overall and within themes, within and across document types. Age-extracts were coded using a framework of themes and
subthemes, which was inductively and iteratively developed
by L.F.F. and checked by S.B. We identified the total num-
ber of age-extracts falling within each theme and subtheme,
as well as the number specifically referring to older-age. A
number of themes were excluded from further consider-
ation. These were use of ‘age’ unrelated to chronological
human age (e.g. the age of studies); age included in job titles Document searching and data extraction A systematic electronic search of included documents was
used to locate all references to ‘age’, ‘old’, ‘elder’ and related
terms, e.g. ‘aged’, ‘older’ and ‘elderly’. When such age-
related terms were identified, the full paragraph around
each (referred to as ‘age-extracts’) was extracted verbatim
for thematic analysis. This ensured that context and mean-
ing were retained during analysis, but meant that more than
one age-related term could be included per age-extract. Age-extracts were the primary unit of analysis. We sought to understand whether and how age is con-
sidered in NICE guidelines and guidance. Document inclusion criteria National Institute for Health & Clinical Excellence (NICE) guidance and guidelines
NICE guidelines make recommendations that are determined by independent committees on a wide range of topics, based on the best available evidence of
what works, and what it costs. NICE also produces social value judgements relating to equity that committees must take into account when making
recommendations. Guidance are developed using similar processes, but are mandatory. We included two types of guidelines and one type of guidance in this
study:
Public health guidelines
These are advisory and make recommendations for populations and individuals in relation to activities, policies and strategies that can help prevent disease or
improve health. NICE clinical guidelines
These are recommendations on how health care professionals and others should care for people with specific conditions. Healthcare professionals are advised
to take the guidelines into account when exercising clinical judgement, while making decisions appropriate to the individual circumstances and wishes of
patients. Technology appraisals
These provide statutory guidance on clinical needs and practice when prescribing drugs or technologies to improve health outcomes or prevent disease, and
thus are mandatory. Box 1. National Institute for Health & Clinical Excellence (NICE) guidance and gu Box 1. National Institute for Health & Clinical Excellence (NICE) guidance and guidelines
NICE guidelines make recommendations that are determined by independent committees on a wide range of topics, based on the best available evidence of
what works, and what it costs. NICE also produces social value judgements relating to equity that committees must take into account when making
recommendations. Guidance are developed using similar processes, but are mandatory. We included two types of guidelines and one type of guidance in this
study:
Public health guidelines
These are advisory and make recommendations for populations and individuals in relation to activities, policies and strategies that can help prevent disease or
improve health. NICE clinical guidelines
These are recommendations on how health care professionals and others should care for people with specific conditions. Healthcare professionals are advised
to take the guidelines into account when exercising clinical judgement, while making decisions appropriate to the individual circumstances and wishes of
patients. Introduction individual, family, community and system level [1, 4–7],
leading to worse outcomes [8]. Given current trends
towards an ageing population [9], it is important to under-
stand where and why age-related differences in access to Substantial research has examined inequity of access to
and receipt of health care and public health interventions
by age [1–3]. Older people may be less likely to receive
potentially beneficial interventions, due to factors at 500 Document inclusion criteria We included three types of documents of most relevance to
health and care practitioners: clinical guidelines, public Box 1. National Institute for Health & Clinical Excellence (NICE) guidance and guidelines
NICE guidelines make recommendations that are determined by independent committees on a wide range of topics, based on the best available evidence of
what works, and what it costs. NICE also produces social value judgements relating to equity that committees must take into account when making
recommendations. Guidance are developed using similar processes, but are mandatory. We included two types of guidelines and one type of guidance in this
study:
Public health guidelines
These are advisory and make recommendations for populations and individuals in relation to activities, policies and strategies that can help prevent disease or
improve health. NICE clinical guidelines
These are recommendations on how health care professionals and others should care for people with specific conditions. Healthcare professionals are advised
to take the guidelines into account when exercising clinical judgement, while making decisions appropriate to the individual circumstances and wishes of
patients. Technology appraisals
These provide statutory guidance on clinical needs and practice when prescribing drugs or technologies to improve health outcomes or prevent disease, and
thus are mandatory. Box 1. National Institute for Health & Clinical Excellence (NICE) guidance and guidelines
NICE guidelines make recommendations that are determined by independent committees on a wide range of topics, based on the best available evidence of
what works, and what it costs. NICE also produces social value judgements relating to equity that committees must take into account when making
recommendations. Guidance are developed using similar processes, but are mandatory. We included two types of guidelines and one type of guidance in th
study:
Public health guidelines
These are advisory and make recommendations for populations and individuals in relation to activities, policies and strategies that can help prevent disease or
improve health. NICE clinical guidelines
These are recommendations on how health care professionals and others should care for people with specific conditions. Healthcare professionals are advised
to take the guidelines into account when exercising clinical judgement, while making decisions appropriate to the individual circumstances and wishes of
patients. Technology appraisals
These provide statutory guidance on clinical needs and practice when prescribing drugs or technologies to improve health outcomes or prevent disease, and
thus are mandatory. Box 1. Results A total of 359 documents containing 2,314 age-extracts
(a mean of 6.4 age-extracts per document) were included in the
analyses. Age-extracts fell into three themes: age documented
as an a-priori consideration at scope-setting; documentation of
differential effectiveness, cost-effectiveness or other outcomes
by age; and documentation of age-specific recommendations. Box 2 provides illustrative quotations from age-extracts
coded within theme 1. Theme 2: documentation of differential effectiveness,
cost-effectiveness or other outcomes by age Table 1 shows the distribution of age-extracts overall
and within themes, across document types. Public health
(relative rate 2.68, 95% CI 2.41 to 2.99, P < 0.001) and
clinical guidelines (relative rate 1.14, 95% CI 1.04 to 1.25,
P = 0.006) contained a greater number of age-extracts than
technology appraisals (comparator), with strong evidence of
statistical heterogeneity (P < 0.001). Detailed considerations of evidence of effectiveness and cost-
effectiveness were not included in the clinical guideline docu-
ments included. While no clinical guidelines described age-
related evidence limitations on cost-effectiveness, 17 (15%)
described age-related limitations in evidence of effectiveness. These primarily related specifically to older-age (n = 12, 11%). Overall, public health guidelines were at least twice as
likely as technology appraisals to report evidence of differ-
ential effectiveness and cost-effectiveness by age, as well as
age-related evidence gaps in evidence of effectiveness and
cost-effectiveness. While the majority of evidence statements
of differential effectiveness by age specifically related to
older-age, this was not the case for cost-effectiveness evi-
dence statements and age-related evidence gaps. g
Table 2 details the framework of included themes and
subthemes, with the number of age-extracts in these by
document type. Below, we discuss key findings within each
theme. Document inclusion criteria Technology appraisals
These provide statutory guidance on clinical needs and practice when prescribing drugs or technologies to improve health outcomes or prevent disease and NICE guidelines make recommendations that are determined by independent committees on a wide range of topics, based on the best available evidence of
what works, and what it costs. NICE also produces social value judgements relating to equity that committees must take into account when making
recommendations. Guidance are developed using similar processes, but are mandatory. We included two types of guidelines and one type of guidance in this
study: dance on clinical needs and practice when prescribing drugs or technologies to improve health outcomes or prevent disease, and 501 L. F. Forrest et al. (e.g. Professor of Old Age Psychiatry); references to other
NICE guidance; and references to children only. appraisals (n = 81, 23%) were aimed at age-specific groups. Very few of any type of document were aimed at older-age
groups specifically (n = 15, 4%). Data extraction and thematic coding were conducted by
L.F.F. Five random age-extracts per theme were checked by
S.B. Disagreements were resolved by discussion. Only public health guidelines documented considering
age at scope-setting, although this was done in less than
half of cases (n = 14, 42%). On 11 occasions, the same
question was listed: ‘Does the effectiveness of the interven-
tion vary with different characteristics within the target
population, such as age?’. This question was not documen-
ted in any technology appraisals or clinical guidelines. We tabulated the total number of age-extracts overall,
and within each theme and subtheme, across document
types. We used Poisson regression (after checking that
assumptions were met) to compare the total number of
age-extracts across document type, using the log number of
documents as the offset. We derived the relative rate of
age-extracts by exponentiating the coefficients and tested
for heterogeneity by using the Wald test for document type. Public health guidelines (n = 25, 76%) were more likely
than clinical guidelines (n = 22, 19%) or technology apprai-
sals (n = 44, 21%) to report statistics describing the prob-
lem addressed by age. Similarly, public health guidelines
were more likely to describe why age might be an important
factor to consider than other documents. However, this
was only done in one-third (n = 11, 33%) of public health
guidelines. Around one-third of all documents identified
age as a risk factor for the problem addressed (n = 105,
29%), particularly older-age (n = 87, 24%). The results section presents quantitative information
with example quotes from age-extracts used to illustrate
and help explain this. Theme 1: age documented as an a-priori consideration
in guidance scope Almost half of clinical guidelines (n = 49, 43%) and a quar-
ter of public health guidelines (n = 8, 24%) and technology A number of evidence statements in public health guide-
lines were based on qualitative work relating to understanding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Distribution of age-extracts overall and within themes across document type
Public health guidelines (n = 33)
Clinical guidelines (n = 114)
Technology appraisals (n = 212)
Total (n = 359)
All themes
Age-extracts, n
476
699
1,139
2,314
Mean age-extracts/document, n
14.4
6.1
5.4
6.4
Theme 1: Age documented as an a-priori consideration in guidance scope
Age-extracts, n
127
213
178
518
Mean age-extracts/document, n
3.8
1.9
0.8
1.4
Theme 2: Documentation of differential effectiveness, cost-effectiveness or other outcomes by age
Age-extracts, n
193
47
697
937
Mean age-extracts/document, n
5.8
0.4
3.3
2.6
Theme 3: Documentation of age-specific recommendations
Age-extracts, n
156
439
264
859
Mean age-extracts/document, n
4.7
3.9
1.2
2.4 Table 1. Distribution of age-extracts overall and within themes across document type Table 1. Distribution of age-extracts overall and within themes across document type 502 . . s, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theme 1: age documented as an a-priori consideration
in guidance scope In those aged 75 and older, the annual incidence is over 300 cases per
100,000 men and over 200 cases per 100,000 women.’ TA105 Laparoscopic surgery for colorectal cancer
Statements of why age is an important factor to consider g
‘More than 250,000 older people (aged 66 and older) living in England in private households reported experiencing maltreatment from a family member.’
PH50 Domestic violence and abuse – how services can respond effectively 21%). Less than half of these recommendations were spe-
cific to older-age (n = 3, 9%). In contrast, only clinical
guidelines prioritised practice recommendations according
to age, but these were only present in one-quarter (n = 31,
27%). Around half were older-age specific (n = 17, 15%). of risk, and of the relevance, appropriateness or acceptability
of an intervention for older populations. Some public health
guidelines also referred to factors that might account for low-
er uptake or effectiveness of interventions in older popula-
tions, such as frailty, poorer health, age-related differences in
perception of relative risk, use of tools designed for younger
people and scarcity of resources. Age-specific research recommendations were present
across guideline types—but were least often found in tech-
nology appraisals. Two-thirds (n = 22, 67%) of public
health guidelines included recommendations for further
age-specific effectiveness evidence and more than one-third
(n = 13, 39%) for further age-specific cost-effectiveness evi-
dence. Comparable figures for clinical guidelines were 31
(27%) and 6 (5%); and for technology appraisals were 4
(2%) and 0. In most cases, less than half of these were
older-age specific. A number of technology appraisals (n = 33, 16%) and
public health guidelines (n = 1, 3%) noted that the age pro-
file of those included in relevant trials was generally younger
to the age profile of those at risk of the problem studied. Illustrative quotes from age-extracts in theme 2 are
shown in Box 3. Theme 3: documentation of age-specific
recommendations Box 4 shows illustrative quotations from age-extracts in
theme 3. Two-thirds of public health guidelines (n = 22, 67%), no
clinical guidelines and two-fifths of technology appraisals
(n = 82, 39%) documented that they took age into account
when making recommendations. Many of these recommen-
dations focused on ensuring people were not excluded
from interventions on the basis of age alone. Theme 1: age documented as an a-priori consideration
in guidance scope Public health (n = 33)
Clinical (n = 114)
Technology appraisal (n = 212)
Total (n = 359)
Documents,
n (%)
Age-extracts,
n
Documents,
n (%)
Age-extracts,
n
Documents,
n (%)
Age-extracts,
n
Documents,
n (%)
Age-extracts,
n
scope
8 (24)
11
49 (43)
98
24 (11)
34
81 (23)
143
1 (3)
3
6 (5)
42
8 (4)
9
15 (4)
54
14 (42)
17
0
0
0
0
14 (4)
17
2 (6)
3
0
0
0
0
2 (1)
3
24 (73)
61
22 (19)
31
44 (21)
54
90 (26)
146
6 (18)
10
14 (12)
15
25 (12)
27
45 (13)
52
10 (30)
18
39 (34)
64
56 (26)
81
105 (29)
163
8 (24)
11
29 (25)
36
50 (24)
64
87 (24)
111
11 (33)
20
18 (16)
20
8 (4)
9
37 (10)
49
ge specific)
6 (18)
9
8 (7)
9
6 (3)
7
20 (6)
25
eness or other outcomes by age
13 (39)
42
0
0
74 (35)
151
87 (24)
193
16 (49)
66
4 (4)
12
33 (16)
61
53 (15)
139
e specific)
15 (46)
47
4 (4)
12
14 (7)
27
33 (9)
86
13 (39)
24
17 (15)
21
24 (11)
35
54 (15)
80
specific)
4 (12)
12
12 (11)
15
9 (4)
14
25 (7)
41
0
0
0
0
80 (38)
167
80 (22)
167
15 (46)
32
2 (2)
2
44 (21)
144
61 (17)
178
er-age
6 (18)
11
2 (2)
2
24 (11)
83
32 (9)
96
7 (21)
9
0
0
26 (12)
40
33 (9)
49
r-age
2 (6)
3
0
0
6 (3)
8
8 (2)
11
er people
7 (21)
19
5 (4)
5
21 (10)
34
33 (9)
58
1 (3)
1
0
0
33 (16)
49
34 (10)
50
0
0
6 (5)
7
13 (6)
16
19 (5)
23
22 (67)
39
0
0
82 (39)
220
104 (29)
259
tions
1 (3)
1
0
0
10 (5)
16
11 (3)
17
7 (21)
15
0
0
0
0
7 (2)
15
3 (9)
9
0
0
0
0
3 (1)
9
0
0
31 (27)
47
0
0
31 (9)
47
fic)
0
0
17 (15)
22
0
0
17 (5)
22
22 (67)
34
31 (27)
60
4 (2)
4
57 (16)
98
pecific)
4 (12)
11
19 (17)
45
2 (1)
2
25 (7)
58
13 (39)
21
6 (5)
7
0
0
19 (5)
28
ge specific)
4 (12)
10
3 (3)
4
0
0
7 (2)
14
18 (55)
47
73 (64)
325
14 (7)
40
105 (29)
412
8 (24)
17
48 (42)
149
8 (4)
28
64 (18)
194 Box 2. Theme 1: age documented as an a-priori consideration
in guidance scope Illustrative quotations from age-extracts within theme 1
Age in guideline scope
‘Are interventions tailored to sub-sets of the smoking population (for example, pregnant women, older smokers) more effective with them than generic
interventions?’ PH1 Brief interventions and referral for smoking cessation
‘How does the effectiveness vary with age, gender, class, ethnicity, etc.?’ PH2 Four commonly used methods to increase physical activity
‘What are the most effective and cost-effective ways for primary and residential care services to promote the mental wellbeing of older people?’ PH16
Occupational therapy and physical activity interventions to promote the mental wellbeing of older people in primary care and residential care
Statistics describing problem considered by age
‘53% of men aged 16–24 achieved the recommended activity levels, compared with 8% of men aged 75 and over. Among women, 29–31% aged 16–54
reached the recommended level. However, the same was only true of 3% of women aged 75 and over.’ PH2 Four commonly used methods to increase physical
activity
Age stated as risk factor for problem considered
‘In people between the ages of 45 and 49 years, the incidence is about 20 per 100,000. In those aged 75 and older, the annual incidence is over 300 cases per
100,000 men and over 200 cases per 100,000 women.’ TA105 Laparoscopic surgery for colorectal cancer
Statements of why age is an important factor to consider
‘More than 250,000 older people (aged 66 and older) living in England in private households reported experiencing maltreatment from a family member.’
PH50 Domestic violence and abuse – how services can respond effectively
L. F. Forrest et al. L. F. Forrest et al. L. F. Forrest et al. Box 2. Illustrative quotations from age-extracts within theme 1 Downloaded from https://academic.oup.com/ageing/article-abstract/46/3/500/2712561 by UCL (Unive Downloaded from https://academic.oup.com/ageing/article-abstract/46/3/500/2712561 by UCL (University College London) user on 04 December 2018 ge s
ed s
s
c o
o p ob e
co s de ed
‘In people between the ages of 45 and 49 years, the incidence is about 20 per 100,000. Summary of findings The Committee agreed that this was not an issue of age discrimination because other factors can also affect whether people are fit enough to
receive chemotherapy, such as comorbidities.’ TA257 Breast cancer (metastatic hormone receptor) – lapatinib and trastuzumab (with aromatase inhibitor)
‘The Committee considered whether NICE’s duties under the equalities legislation required it to alter or to add to its recommendations…the Committee
concluded that…there was no need to alter or add to its recommendations.’ TA282 Idiopathic pulmonary fibrosis – pirfenidone
Target population for recommendations age specific
‘All COPD patients still smoking, regardless of age, should be encouraged to stop, and offered help to do so, at every opportunity.’ CG101 Chronic obstructive
pulmonary disease
‘Offer people aged 80 years and over the same antihypertensive drug treatment as people aged 55–80 years, taking into account any comorbidities.’ CG127
Hypertension
‘Patient-specific factors (including age…) should not be barriers to referral for joint surgery.’ CG177 Osteoarthritis
Further effectiveness/cost-effectiveness research recommended by age
‘How does effectiveness and cost effectiveness vary for…people aged 75 and over?’ PH32 Type 2 diabetes
‘Future studies should be sufficiently powered to detect changes in mental wellbeing…In addition, the outcome measures used should be appropriate to
detect change across different groups of older people.’ PH16 Mental wellbeing and older people Age taken into consideration when making recommendations Age taken into consideration when making recommendations g
g
‘a small population of older patients who are not fit enough to receive chemotherapy may not have access to an alternative treatment and so may be
disadvantaged. The Committee agreed that this was not an issue of age discrimination because other factors can also affect whether people are fit enough to
receive chemotherapy, such as comorbidities.’ TA257 Breast cancer (metastatic hormone receptor) – lapatinib and trastuzumab (with aromatase inhibitor)
‘The Committee considered whether NICE’s duties under the equalities legislation required it to alter or to add to its recommendations…the Committee
concluded that…there was no need to alter or add to its recommendations.’ TA282 Idiopathic pulmonary fibrosis – pirfenidone
Target population for recommendations age specific
‘All COPD patients still smoking, regardless of age, should be encouraged to stop, and offered help to do so, at every opportunity.’ CG101 Chronic obstructive
pulmonary disease
‘Offer people aged 80 years and over the same antihypertensive drug treatment as people aged 55–80 years, taking into account any comorbidities.’ CG127
Hypertension
‘Patient-specific factors (including age…) should not be barriers to referral for joint surgery.’ CG177 Osteoarthritis
Further effectiveness/cost-effectiveness research recommended by age
‘How does effectiveness and cost effectiveness vary for…people aged 75 and over?’ PH32 Type 2 diabetes
‘Future studies should be sufficiently powered to detect changes in mental wellbeing…In addition, the outcome measures used should be appropriate to
detect change across different groups of older people.’ PH16 Mental wellbeing and older people practitioners would be most likely to find. This maximised
the relevance of our findings. However, our findings are not
necessarily generalisable to other types of NICE document,
or to other countries. practitioners and commissioners. In the absence of evi-
dence, guideline developers might choose to highlight the
need for further research. While many public health guide-
lines did this, few technology appraisals and clinical guide-
lines did so. Documentary analysis is labour intensive and time-
consuming [14]. We searched and extracted data from hun-
dreds of large documents. It was not feasible to read all of
these in full. Instead we automated searching for extracts,
but coded these by hand. Duplicate thematic coding helped
increase the validity of this. Summary of findings Two…studies report no
differences in effectiveness according to age.’ PH25 Prevention of cardiovascular disease
‘A meta-analysis of exercise capacity with dual-chamber pacing compared with single-chamber ventricular pacing demonstrated no difference…for patients
older than 75 years…but there was…for patients younger than 75.’ TA88 Dual-chamber pacemakers for symptomatic bradycardia due to sick sinus syndrome and or
atrioventricular block
Limitations or gaps in evidence of effectiveness/cost-effectiveness by age
‘there are…no studies specifically addressing people aged 75 and over.’ CG180 The management of atrial fibrillation
‘No evidence was found of effective or cost-effective interventions to promote mental wellbeing in older people living in residential care.’ PH16 Mental wellbeing
and older people
Age as a reason why interventions not offered/ineffective in older people
‘One qualitative study…found that age was widely perceived to influence access to services…Focus groups revealed that staff appeared to have knowledge of
the benefits for older people but that scarcity of resources prevented them offering more accessible and appropriate services.’ PH15 Identifying and supporting
people most at risk of dying prematurely
‘The PDG [programme development group] considered that people over age 74…might benefit from type 2 diabetes risk assessment and prevention…
However, it recognised that many of the risk-assessment tools are not validated for this age group.’ PH38 Preventing type 2 diabetes – risk identification and
interventions for individuals at high risk
Age of those included in trials different to that those at risk
‘The Committee…noted that the mean age of patients in the trial was 56 years but…the average age of men with gout in UK practice was around 10 years
older.’ TA291 Gout (tophaceous, severe debilitating, chronic) – pegloticase
Age-related references in national public health, technology appraisal and clinical guidelines Box 3. Illustrative quotations from age-extracts within theme 2
Evidence statements of differential effectiveness/cost-effectiveness by age
‘One…study reports a reduction in effectiveness in promoting CVD [cardio-vascular disease] awareness in older participants. Summary of findings This is the first attempt we are aware of to analyse how age
in general, and older-age in particular, is considered in
national health and health care guidelines and guidance. Using NICE as a case study, we found 2,314 age-extracts
across 359 public health and clinical guidelines and technol-
ogy appraisals. These fell into three broad themes: age
documented as an a-priori consideration at scope-setting;
documentation of differential effectiveness, cost-effectiveness
or other outcomes by age; and documentation of age-specific
recommendations. Throughout, there were many documented discussions
of whether age should be taken into account in recom-
mendations and, in documents published since 2012,
whether recommendations should be revised in light of
the Equalities Act (2010). No such revisions were recom-
mended and documents often concluded that intervention
decisions should be made on an individual basis. Evidence
of
effectiveness
and
cost-effectiveness
of
new technologies are often supplied by manufacturers to
NICE technology appraisal panels. Panels appear to spend
substantial time testing and discussing this evidence. Age is
regularly examined during this process (n = 82, 39%), but
mostly in terms of how it is used in cost-effectiveness models. Age was not considered consistently across the three
document types. In general, public health guidelines con-
sidered age more comprehensively, but this was still not
consistent across all public health guidelines. A lack of explicit age-related recommendations within
guidelines and guidance could result in uncertainties for Only public health guidelines made age-specific practice
recommendations, but only in one-fifth of cases (n = 7, 504 Age-related references in national public health, technology appraisal and clinical guidelines Box 3. Illustrative quotations from age-extracts within theme 2
Evidence statements of differential effectiveness/cost-effectiveness by age
‘One…study reports a reduction in effectiveness in promoting CVD [cardio-vascular disease] awareness in older participants. Summary of findings Two…studies report no
differences in effectiveness according to age.’ PH25 Prevention of cardiovascular disease
‘A meta-analysis of exercise capacity with dual-chamber pacing compared with single-chamber ventricular pacing demonstrated no difference…for patients
older than 75 years…but there was…for patients younger than 75.’ TA88 Dual-chamber pacemakers for symptomatic bradycardia due to sick sinus syndrome and or
atrioventricular block
Limitations or gaps in evidence of effectiveness/cost-effectiveness by age
‘there are…no studies specifically addressing people aged 75 and over.’ CG180 The management of atrial fibrillation
‘No evidence was found of effective or cost-effective interventions to promote mental wellbeing in older people living in residential care.’ PH16 Mental wellbeing
and older people
Age as a reason why interventions not offered/ineffective in older people
‘One qualitative study…found that age was widely perceived to influence access to services…Focus groups revealed that staff appeared to have knowledge of
the benefits for older people but that scarcity of resources prevented them offering more accessible and appropriate services.’ PH15 Identifying and supporting
people most at risk of dying prematurely
‘The PDG [programme development group] considered that people over age 74…might benefit from type 2 diabetes risk assessment and prevention…
However, it recognised that many of the risk-assessment tools are not validated for this age group.’ PH38 Preventing type 2 diabetes – risk identification and
interventions for individuals at high risk
Age of those included in trials different to that those at risk
‘The Committee…noted that the mean age of patients in the trial was 56 years but…the average age of men with gout in UK practice was around 10 years
older.’ TA291 Gout (tophaceous, severe debilitating, chronic) – pegloticase
Age-related references in national public health, technology appraisal and clinical guidelines Box 3. Illustrative quotations from age-extracts within theme 2 Evidence statements of differential effectiveness/cost-effectiveness by age Box 4. Illustrative quotations from age-extracts within theme 3
Age taken into consideration when making recommendations
‘a small population of older patients who are not fit enough to receive chemotherapy may not have access to an alternative treatment and so may be
disadvantaged. Study limitations NICE produces a range of different guidelines and guidance. We focused on clinical and public health guidelines and tech-
nology appraisals as we considered these to be most relevant
for practitioners and commissioners. Although multiple docu-
ments related to each guideline and piece of guidance are
available, we focused on the most front-facing of these that It would have been interesting to formally test our quali-
tative assessment that the Equalities Act influenced how
age was considered. However, small numbers in many cells
in Table 2 meant this was not considered appropriate. 505 Comparison with previous findings Others have found, in different contexts, a lack of clear age-
related clinical recommendations [22] and suggested that
NICE make explicit reference to age where there is evidence
of age-related inequities in receipt of interventions [22]. However, little is known about whether, and where, age-
related inequities in receipt of health care and public health
interventions occur. More concerted and systematic action is
required to identify age-related differences in receipt inter-
ventions to guide development of guidelines and guidance. It is unclear how much impact NICE guidance has on
either front-line practice or what services are prioritised for
funding at a local or national level and thus how much the
differences identified here might influence practice [15]. However, it has been suggested that increasing the specifi-
city of guidelines is likely to improve implementation [16]. Implications for research, policy and practice A lack of evidence on effectiveness and cost-effectiveness
in older populations was often noted in included docu-
ments. Some might argue that it is unwise to provide inter-
ventions
without
evidence
of
effectiveness
or
cost-
effectiveness. This is likely to result in age-related differ-
ences in receipt of interventions. Others may feel that older
people should have access to all interventions until such
time as evidence emerges of a lack of effectiveness or cost-
effectiveness. While the Equality Act (2010) would favour
the latter approach, over-provision of ineffective interven-
tions to older people may result in iatrogenesis and waste. Research on the effectiveness and cost-effectiveness of
interventions in older populations is required to overcome
any ambiguity in who should be offered interventions. Until
this is available, clear guidance on how practitioners should
act in the face of absence of evidence is required. L. F. Forrest et al. consider inequalities since at least 2009. While some NICE
processes help ensure embedded equity, others do not. There was inconsistency in the information included in
the different types of documents studied. In particular, the
clinical guideline documents included did not incorporate
the evidence used to make recommendations (although this
is available elsewhere), while public health guidelines and
technology appraisal documents did. This may explain
some of the differences between document types found. Reporting variations may also reflect variations in approach. Nearly half of public health guidelines and one-fifth of
technology appraisals reported evidence of differential
effectiveness and cost-effectiveness by age. Interventions
may be less cost-effective in older people due to their short-
er life expectancy [19]. However, in many instances where
prevalence increases with age, numbers needed to treat will
be smaller in older people and this may increase the cost-
effectiveness of some interventions. Furthermore, the bene-
fits of interventions to older people, in terms of reduced
morbidity, improved quality of life and maintained inde-
pendence, are likely to be different, but no less valuable,
than those to younger people [20]. More consideration of
how age-related differences in benefit can be taken into
account in cost-effectiveness calculations is required as well
as consideration of alternative methodological approaches
that may be more suited for older people [21]. Using documentary analysis meant we only included
information that had been documented. Committees
developing guidance and guidelines may have considered
many issues that were not explicitly recorded in included
documents. We searched specifically for age-related terms. Instances
when age was indirectly referred to, for example in terms of
life expectancy or frailty, may have been excluded, under-
estimating the frequency of age-extracts. However, there is
little reason to believe this would vary systematically across
document types and so constitute bias. We excluded guidance and guidelines with a specific
focus on children, young people and pregnant women. We
did not make judgements about which included docu-
ments should have considered age. However, it could be
argued that all should have done—even if just to state that
guidance and guidelines were relevant across the age
spectrum. Age-related references in national public health, technology appraisal and clinical guidelines ‘fitness’, leaving decisions on assessment of these to practi-
tioners. Variability in how practitioners make such judgements
may be due to unconscious prejudices [24]. Better understand-
ing is required of how the risks and benefits of interventions
are evaluated by practitioners and older people in the absence
of explicit guidance, and what contributions these assessments
may make to inequalities in receipt of interventions. Key points • Age bias in national guidance may influence entire public
health and health care systems. • Age bias in national guidance may influence entire public
health and health care systems. Availability of data and materials All data used in this manuscript are publically available
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17. Culyer AJ, Bombard Y. An equity framework for health tech-
nology assessments. Med Decis Making 2012; 32(3): 428–41. doi:10.1177/0272989×11426484. Received 1 June 2016; editorial decision 8 November 2016 Received 1 June 2016; editorial decision 8 November 2016 508
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https://openalex.org/W2093444067
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https://zenodo.org/records/1786149/files/article.pdf
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German
| null |
Zur Kenntniss der Azophenole
|
Berichte der Deutschen Chemischen Gesellschaft
| 1,906
|
public-domain
| 4,897
|
1 ) Darin sind wir eotgegengesetzter Ansicht als R. C. Farmer und
a) Diese Berichte 28, 2414 [1895].
3, Zur Reduction der AzokBrper mit Phenylhydrazin cfr. R. Walther,
Journ. fiir prakt. Chem. [2] 52, 141 [I8951 und G. Odd0 und E. Paxeddu,
diese Berichte 38, 2752 [1905].
A. Hantzsch, diese Berichte 32, 3089-3100
[lS99].
4) Diese Berichte 23, 11 [1890].
Berichte d. D. chem. Gesellachaft. Jahrg. XXXIX.
222 1 ) Darin sind wir eotgegengesetzter Ansicht als R. C. Farmer und
A. Hantzsch, diese Berichte 32, 3089-3100
[lS99]. 562. Riohard Willstatter und Max Benz:
Zur Kenntniss der ‘Azophenole. [Mittheilung aus dem chem. Laborat. des eidgeu. Polytechuioums in Ziirich.]
(Eingeg. am 2. October 1906; mitgeth. in der Sitzung von Hrn. P. Jacobson.) p-Azophenol wird bei der Einwirkung von Silberoxyd zu Chinon-
azin oxydirt, das in der voranstehenden Mittheilung beschrieben worden
ist. Natiirlich nimmt die clrioofde Verbindung bei der Reduction
wieder zwei Atvme Wasserstoff auf. Es eutsteht also wieder Azo-
phenol. Wenn nun schon das Reductionsprodnct in der Znsammen-
setzung und im Verhalten mit dern Ausgangsmaterial iibereiostimmt,
so zeigt es doch erhebliche Abweichungen in den physikalischen Eigen-
schaften, wie der folgenden Tabelle za entnehmen ist. -
~
____
_ _
-
___
I
Eigenschaften
1
U
B
Aussehen der Krystalle
bernsteingelbeoderhell-
braune Tafelchen
Parbe des Pulvers
dunkelgelb
Parbe deserhitztenpulsers ,
cli rorngriin
Verhalten des erhitzten
Pulvers an feuchter Luft
rea@
nicht
: 10800
Lijslichkeit, in Benzol
1
bei 17O
I
~~
rothlichbraune Tiifelahen
m. starkem bl8ulich. Reflex
blaustichig braunroth
ziegelroth
verbindet sich mit 1 Yol. Ha0
1 : 1500 Der Vergleich ergiebt , dass unser Reductionsproduct aus dem
Azin ni c h t identisch ist mit dem gewohnlichen Azophenol. Wir
schlagen vor, das Letztere als u, die neu aufgefundene Modification
mit ,8 zu bezeichnen. Die beiden Modificationen zeigen aber doch so vie1 Gemeinsames,
dass man nicht daran denken kann, sie als constitutionell verschieden
zu interpretiren. Uebereinstimmend ist bei den zweien das Verbalten
gegen Sauren and gegen Alkalien. Mit gasformigem Ammoniak
bilden sie unter allen Umstanden wasserfreies Diammoniumsalz, das
im Exeiccator oder an der Luft vollig dissociirt. Azobenzol u und $,
so wie sie dabei hinterbleiben, nehmen stets ans der Lnft ein Molekiil
Wasser auf. Auch aus den Losungen krystallisiren beide wasser-
haltig. Von Silberoxyd wird die neue Modification gerade so leicht
angegriffen uod in Chioonazin verwandelt wie gewohnliches Azo-
phenol. Uod gegen Pheoylhydrazio sind die beiden bis zu hohen 3498 4) Diese Berichte 23, 11 [1890].
Berichte d. D. chem. Gesellachaft. Jahrg. XXXIX. 4) Diese Berichte 23, 11 [1890]. Berichte d. D. chem. Gesellachaft. Jahrg. XXXIX. 1 ) Darin sind wir eotgegengesetzter Ansicht als R. C. Farmer und
a) Diese Berichte 28, 2414 [1895].
A. Hantzsch, diese Berichte 32, 3089-3100
[lS99]. 1) H. Klinger und J. Zuurdeeg, Ann. d. Chem. 265, 310 [l883] und
A. Hantzsch und A. Werner, diese Berichte 23, 1243 [lS90].
4 W. R. Orndorff und E. D. Thebaud, Amer. Chem. Journ. 26, 159
[1901], ziehen fiir die 2 Formen des Benzolazoresorcins von W. Will ond
W. Pukall wohl Stereoisomerie in Betracht, aber sie vermuthen nicht geo-
metrisch isomere Azokbrper, sondern Phenylhydrazone.
4 Diese Berichte 86, 4093, 4123, Fussnote 1 [1903] p
y y
4 Diese Berichte 86, 4093, 4123, Fussnote 1 [1903]. 3498 Temperatnren bestandig; sie verbinden sich mit einem Molekiil der
Base eu lrrystallisirenden Salzen. Temperatnren bestandig; sie verbinden sich mit einem Molekiil der
Base eu lrrystallisirenden Salzen. Temperatnren bestandig; sie verbinden sich mit einem Molekiil der
Base eu lrrystallisirenden Salzen. Es ist wohl maglich, daes man die einfachen Azofarbstoffe in
desmotropen Formen im Sinne der Chinon- und Phenol-Formeln
kennen lernen wird. Aber dann miissen solche Desmotrope Unter-
schiede anderer Art zeigen, als a- und $-Azophenol. Diese beiden
Substanzen haben nicht3 von Chinonen an sich'),
ebensowenig wie
p-Oxyazobenzol. Es sind Azokorper. Das zeigt z. B. das er-
wahnte Verhalten gegen Phenylhydrazin. Chinonazin wird in der
Kiilte von Phenylhydrazin energisch reducirt. Bei dem gebildeten
Azophenol bleibt die Reaction stehen; der Wasserstoff tritt an die
Sauerstoffenden des Systems:
) O:/=\:N.N:;=\:O + H~ = HO./-\.N:N. c). OH. \=/
\=/
\L/ Verhielte sich aber Azophenol chinoi'd : Verhielte sich aber Azophenol chinoi'd : /-'. OH,
\L/
o :/=\ :N. NH. \=/ so miisste die Reaction fortschreiten, so wie beispieleweise das Ben-
zoylphenylhydrazon des Chinons nach W. Mc P h e r s o na) von Phenyl-
hydrazin in der Kalte reduzirt wird. Bei hoher Temperatur greift
das Phenylhydrazin natiirlich auch die Azophenole an 3). Nun lie-
fern die beiden Formen Diamrnoniamsalz. Man konnte annehmen, dies
geschehe unter Umlagerung. Allein beim Diseociiren der Ammonium-
salze werden a- und PProduct mit ihren sammtlichen Merkmalen
wiedergewonnen. Ueberhaupt gelingt es recht schwer, CI- und $-Modi-
fication in einander umzuwandeln. Aus
erhielten wir a nur durch
Destillation bei vermindertem Druck unter Zerstorung von vie1 Sub-
stanz. a liefert $, aber nicht ganz einheitlich, beim Kochen mit
feuchtem Aether. Als chinoi'de Verbindung ware Azophenol einbasisch. Von Structnrisomerie kann demnach in unserem Falle nicht die
Rede sein. Eine andere Annahme ware geometrische Isomerie. Diese
MMiiglichkeit haben A. H a n t z s c h und A. W e r n e r 4 ) schon voraus- 222 zuliegen, leider wird ihre Untersuchung erschwert dorch die Unbestan-
digkeit der neuen Form, die beim Auflosen wieder in das alte o-Azo-
phenol iibergeht. Die Azophenole durch ihr Verhalten gegen Ammoniak zu charak-
terisiren, haben uns die grundlegenden Arbeiten von A. Ha n t z s c h 1)
uber Pseudosauren gelehrt. A. H a n t z s c h und F. E. Dollfusa)
haben fiir die Untersuchung der Ammoniakreaction eine sinnreiche
Methode angegeben. Wir zieben es indessen aus verschiedenen Griin-
den vor, auf eine einfacbere Weise, die im experimentellen Theile be-
schrieben wird, das Verhalten der Substanzen i m trocknen Ammoniak-
&om zu priifen, um den Endwerth der Ammoniakaufnahme quanti-
tativ zu bestimmen. I) Diese Berichte 32, 5 i 5 [1599].
Diese Berichte 35, 226, 2724 [1902].
3, P. Weselsky und R. Benedikt, Ann. d. Chem. 196, 343 [1879].
4, Ann. d. Chem. 320, 122, 131 [1902].
5, Ann. d. Chem. 196, 339 [1679]. gesehen in ihrer bahnbrechenden Arbeit : BUeber riiumliche Anordnung
der Atome in stickstoff haltigen Molekii1en.a
Das einzige Beispiel aus
der Klasse der Azokorper, das dafiir zu nennen war, J. V. J a n o w s k y ' s
Trinitroazotoluole, musste bald nachher aus der Literatur gestrichen
werden'). So vie1 uns bekannt, ist in der Folge die Hypothese der
Stemsisomerie fiir die Azoverbindungen nicht weiter discutirt worden 9). Immerhin giebt es einige Fiille in der neueren Literatur, Andeu-
tungen von Isomerieverhaltnissen , die vielleicht als geometrische Iso-
merieen zu rubriciren sein konnten. P. J a c o b s o n und F. Hiinigs-
b e r g e r *) haben eine Anzahl solcher Fiille zusammengestellt, in denen
Modificationen mit kleinen Differenzen beobachtet worden sind. Daran
kniipfen aber P. J a c o b s o n und F. Hiinigsberger die Vermuthung,
dass es sich in den citirten Fallen um Andeutungen der Isomerie
zwischen Oxyazokorpern und Chinonhydrazonen, also um Structur-
isomerie handle. Wir halten es also fur wabrecheinlich, wenn auch nicht fiir be-
wicsen, dass a- und p-Azophenol geometrisch Isomere sind. Urn eine
breitere Ornndlage fiir die Bestimmung der Isomerien bei den Azo-
kiirpern zu gewinnen, ist es erforderlich, die einfachen Verbindnngen
dieser Klasse, soweit sie nur in der alteren Literatur kurz behandelt
worden sind, genauer zu beschreiben. Mit dieser Absicht haben wir
auch o-Azophenol betrachtet. Die o-Verbindung ist der Oxydation mit Silberoxyd nicht zugang-
lich. Auch gegen Ammoniak zeigt sie ein anderes Verhalten, als der
pK6rper. Sie bildet namlich nur ein Monoammoniumsalz. Wenn
man aber o-Azophenol zuvor zum Schmelzen erhitzt oder im Vacuum
vei fliichtigt, so reagirt es absolut nicht mit Ammoniak, selbst bei tage-
langem Aufstellen im Strom des Gases. Dieses Verhalten ist nicht
etwa durch den Zustand der Trockenheit zu erklaren. Vollkommener
Ausschluss von Feuchtigkeit vermag wohl beim o-Azophenol die Re-
action mit Ammoniak zu verzogern, aber die erhitzte Substanz liefert
auch dann kein Ammoniumsalz, wenn man ihr Gelegenheit giebt, Laft-
feuchtigkeit aufzunehnien. Also auch hier scheint eine Isomerie vor- 1) Die Angabe in den Ann. d. Chem. 196, 341 - leicht loslich in
Benzol - beruht auf Irrthum. p - A z o p h e n o l (a). Fiir p - Azophenol sind viele Bildungsweisen angegeben worden. Die Entetehung beim Kuppeln von Phenol mit Diazophenol9 empfieblt
sich nicht ftir die Gewinnung, da die Ausbeute schlecht und das Pro-
duct der Kuppelung sehr unrein ist. Nur zwei Verfahren sind fiir die
Darstellung geeignet. Einmal die Methode von D. Vorliinder'),
Reduction von Diazophenol mit ammoniakalischer L6sung von Kupfer-
oxydulsalz; der Azokorper wird in der Form seines Ammoniumsalzes
in sehr reinem Zustande erhalten, die Ausbeute betragt nach dem Um-
krystallisiren 9 g aus 24 g Aminophenolchlorhydrat (7.2 g nacb der
Literatnrangabe). Zweitens die Kalischmelze von p-Nitrophenol nach
€'. W e s e l s k y und R. Benedikt5), die wir mit einigen Abanderuogen
in folgender Weise ausfiihrten: Gepulvertes p-Nitrophenol (10 g) wurde im Silbertiegel mit 10 g Wasser
angeriihrt, 50 g Aetzkali zugefiigt und gelinde erwgrmt, bis alles gelBst ist. Dann wird erst das iiberschhssige Wasser verdampft und durch weiteres Er-
hitzen auf ca. 200° die Reaction eingeleitet. Sie verlaoft auf diese Art ruhig. ohne dass Verpuffungen vorkommen. Nach Beendigung der Gasentwickdung
nimmt man die dunkelviolettrothe Schmelze in Wasser auf und sguert mit
Salzsgure an. Der ausgeschiedene graubraune Schlamm wird getrocknet und
dann mit Aether ausgekocht. 222* I) Dime Berichte 8, 1499 [1875]. 0.3892 g Sbst.: 0.2495 g AgBr (nach Carius).
ClaHlo02N*.HBr. Ber. Br 27.09. Gef. Br 27.28. Die itherische Losung behandelt man zunichst mit Thierkohle. Dam ant-
hdt sie noch in kleiner Menge als eine hartniickig anhaftende Verunreini-
Bung eine Base, die sich durch wiederholtes Ausrtchiitteln der iitherischen
L6snng mit verdiinnter Schwefelsaure (so lange Rothfiirbung eintritt) beseitigen
lisst. Schliesslich krystallisirt man das Reactionsproduct aus 50-proc. Alkohol
um. Ausbeute 3-3.5
g (36-42 pCt. der Theorie). Azophenol krystallisirt in der von den friiheren Autoren beschrie-
benen wasserhaltigen Form stets aus Alkohol und Aether, wofern man
nicht Wasser bei den Versuchen auf's sorgfaltigste ausgescblossen hat,
und hiiufig auch aus Benzol. Die Krystalle sind hellbraune (aus
Alkohol) oder bernsteingelbe (aus Aether), rhomboGderahnliche, oft
verwachsene Tafelchen mit blaulichem Reflex, im durchfal!enden
Licht grunlicbgelb. Wasserhaltig wird die Substanz auch aus alka-
lischer Losung gefallt und zwar durch Mineralsauren als dunkel citro-
nengelbes Pulver, durch Kohlensaure in Form stark glanzender,
rhomboCderfiirmiger, kleiner Krystalle. Wasserfrei in stark lichtbrechenden, grunlich-braunen immer kreuz-
formig verwachsenen Krystallen kann man das Azophenol aus ent-
wasserter benzoliscber Liisung erhalten. Auch aus der Losung ron
entwasserten Praparatcn in trocknem Aether oder beim Zersetzen des
Sulfats mit atherischem Ammoniak lasst sich die wasserfreie Substanz
isoliren. In Alkohol, Aether, Aceton und Essigester lost sich Azophenol
leicht, in Eisessig ziemlich leicht, in Benzoll) (bei 17O im Mittel von
zwei Versuchen ca. 1 : 10800, bestimmt mit wasserhaltiger Substanz),
Toluol, Petrolather in der Kalte sehr schwer, heiss etwas leichter, in
Wasser sehr schwer. Wasserhaltiges Azophenol ist luftbestandig und verliert auch uber
Schwefelsaure das Wasser niir im Vacuum; natiirlich wird es wasser-
frei bei looo. Bei gewohnlicher Temperatur wird es auch durch die
Reaction mit Ammoniakgas entwassert. Stellt man dann das Ammo-
niumsalz iiber Schwefelsaure auf, so dissociirt es und wasserfreies
Azophenol binterbleibt. s o erhalten , ist Azophenol hygroskopisch
und giebt an feuchter Luft wieder das Eiydrat. Dasselbe gilt fur
Praparate, wie man sie durch Trocknung im Vacuumexsiccator erhalt. Hingegen sind die wasserfreien Krystallisationen und die bei looo ge-
trockneten Praparate der a-Modification an feuchter Luft bestandig ; dies
ist ein charakteristischer Unterschied gegeniiber der ,%Modification, wie
aus der folgenden Tabelle zu ersehen ist. 3497 Reaction von p - A z o p h e n o l (a und p ) m i t Ammoniak. Die Einwirkung von Aminoniakgas auf die Azophenole haben
wir in der Weise untersucht, dass die feingepulverte Substanz in sehr
weiten, niedrigen Wageglasern, die mit gut eingeachliffenen Stopseln
verschliessbar waren, in einem mit Baryumoxyd gefilllten Exsic-
cator aufgestellt wurden. Durch den Tubus desselben liessen wir das
scharf getrocknete Gas einstriimen und mittelst eines Glashahnes aus-
treten, der nach Verdrangung der Luft nur auf gerioge Oeffnung ge-
stellt war. Vor der Wagung wurde natiirlich das Ammoniak in dem
Cliischen durch Luft verdrangt. Siimmtliche Priiparate von p - Azophenol bilden, wie die nach-
stehende Tabelle zeigt, Diammonsalz, und zwar die Krystallisation
mit Wassergehalt unter rascher Abgabe des Wassers, das sich anfangs
in Tropfchen am Wageglas condensirt. Die Salze sind tiefgelb
gefarbt. Eine Beschleunigung der Reaction durch Feuchtigkeit war nicht
augenfiillig. Zwar reagirt die im Vacuum in der Kalte entwasserte
Substanz langsamer mit dem Ammoniak, aber die wasserfreien Kry-
stalle wie die bei looo getrockoeten Praparate addireu rasch. Dabei
scheint das zweite Molekiil wenig langsamer aufgenommen zu werden,
wie das erste. 0.3311 g @-Form addirte in 15 Minuten 24.4 mg, dann in 15 Minuten
16.0 mg, dann in 10 Minuten 3.6 mg, dann in 20 Minuten 5.6 mg, dann in
ca. 100 Miouten 0.S mg NHB. 3497 Tabelle I. Tabelle I. Eotwiisserung und Wasseraufnahme von p-Azophenol (a a n d p). (Berechnet fiir Verlust von 1 Mol. Ha0: 7.76 pCt. von wasserhaltiger Substanz, fin Auf-
nahme vou 1 Mol. HpO: 8.41 pCt. fur wasserfreie Substanz.)
;ewichts-
verlust
iiber
IaSOa im
Vacuum
PrHparat
pCt. a, Krystalle aus Weingeist . . (I, Krystalle aus Weingeist . . a, Krystalle aus Aether . . . c(, Fnllung durch Schwefelsaure
u. Fallung durch Kohlensaure
a, Krpstalle (braune Sternchen)
aus Benzol . . . . . . . . . ,?, Krystalle aus Weingeist . p, Krystalle aus Weingeist . p, Krystalle aus Weingeist . -
-
0.0347
0.0312
0
0.0297
-
-
Substans
0.4086
0.3759
0.9948
0.4542
0.4037
0.4645
0.3962
0.4222
0.3578
-
-
7.64
7.73
0
7.50 -
-
kewichts
verlust
)ei 1000
-
0.0295
0.0814
-
-
0.0009
-
0.0345
0.0299
-
__
pCt. -
-
7.85
8.18 - -
0.19 -
-
8.17
8.35
.___
kewichts. snnahme
an der
Luft
0.0303
0
0.0003
-
-
-
0.0226
0.0329
8.0189 fii~
0.2542
-
3Ct. -
8.02
0
0.03
-
-
- -
6.17
8.49
7.43 wiisserung und Wasseraufnahme von p-Azophenol (a a n d p). Das wasserhaltige Azophenol zeigt dunkelgelbe Pulverfarbe, die
- was noch nicht angegeben worden ist -
beim Entwassern in der Hitze
in ein Chromgriin iibergeht. Stumpfer dunkelgriin wird die Farbe beim
Verwittern im Vacuum; bei der Dissociation des Ammonsalzee wird
das Pulver ofters griinbraun. Die griine Farbe unterliegt keiner Aen-
derung beim Aufbewahren. Verdiinnte Losungen des Farbstoffes sind
gelb gefarbt und tingiren griinlich-gelb. Der Schmelzpunkt ist in der Literatur meist ungenau verzeichnet,
(nur C. J a e g e r l ) beobachtet 214O; P. W e s e l s k y und R. B e n e d i k t
und die meieten andern Autoren 204O); die wasserhaltige und die ent-
wasserte Substanz schmelzen unter Zersetzung bei 215O (uncorr.). Azophenol wird von atzenden und kohlensauren Alkalien, sowie von
Ammoniak mit tief oraugerother Farbe, griingelb tingirend, auf-
genommen, hingegen nicht von Bicarbonat. In concentrirter Schwefelsaure liist es sich leicht , in den Halo-
genwasserstoffvauren nur ein wenig mit carminrother Farbe, wahrend
die Ilauptmenge i n Salz rerwandelt wird. Besonders schon erhllt
man die Additionsproducte mit Halogenwasserstoff in EisessiglBsung
in Form luftbestandiger, stahlblauer Krystalle. I) Dime Berichte 8, 1499 [1875]. l) Diese Berichte 17, 274 [1884]. 3498 Solche halogenwasser8toffsaure Salze scheiden sich auch stet8 bei
der gelinden Einwirknng von Chlor oder Brom auf die Eisessigliisung
von Azophenol aus. Wir fanden die Angaben von R. B o h n und
K. H e u m a n n l ) nicht bestatigt, dam bei der Einwirkung von Chlor
zuerst ein chlorfreier Niederschlag von der Zusammensetzung des
Azophenols ausfalle, der in Alkohol oder Aether fast unliislich sei,
und durch Umfiillen aus alkalischer Liisung wieder gewiihnlicbes Azo-
phenol liefere. Reaction von p - A z o p h e n o l (a und p ) m i t Ammoniak. P h e n y l h y d r a z i n s a l z von A z o p h e n o l (a u n d @) Auch mit organischen Basen, wie Phenylhydrazin und Anilin,
bildet p Azophenol Salze, die von Liisungsmitteln leicht zerlegt werden. Sie entstehen als Brei hellgelber Krystalle beim Verreiben mit den
Basen, und liiaen sich in deren Ueberschuss auf. Wir brachten 1 g
Azophenol (a) mit 3 g Phenylbydrazin in 100 g Benzol bei Siedehitze
in Liisung ; beim Erkalten schieden sich schtine, goldgelbe Prismen l) Diese Berichte 17, 274 [1884]. '1 Die Gewichtszunabmo ist zunschst vie1 grBsser, da Ammoniak rascher aufgenommen wird, als das Wasser entweicht.
2, Der Gewichtsverlust wurde bei diesem Beispiel erst im Vacuum vollstiindig. T a b e l l e 11. Bildung u n d Dissociation des Diammoniumsalzes von p-hzophenoi (a u n d ,8). (Berechnet fiir Aufnahme von 2 Mol. NH3: 15.93 pCt. von wasserfreier Sbst., fiir 2 Mol. NH3-1 Mol. H2O: 6.94 pCt,. von wasserhaltiger Sbst.. fiir Aufnahme von 1 Mol. HPO: 8.41 DCt. von wasserfreier Sbr
Prtiparat
a, Krystalle aus Weingeist . . . . . . a, Ftillung durch Schwefelsiure
. . . . a, Fillung durch Kohlensiiure . . . . . rt, Krystalle aus Weingeist . . . . . . n, Krystalle am Aether . . . . . . . a, Krystalle aus Weingeist . . . . . . a, Krystalle aus Aether . . . . . . . a, Krystalle (braune Sternchen) aus Benzol
a, Chlorhydrat
, . . . . . . . . p, Krystalle aus Weingeist . . . . . . 8, Krystalle aus Weingeist . . . . . . -
Trocknung
lufttrocken 1
lufttrocken 1
lufttrocken
iiber flsS04
im Vacuum 1
iiber &So4
im Vacuum 1
bei 100° 1
bei looo
lufttrocken
(wasserfrei)
uber HgSO,
im Vacuum 1
Substanz
0.6153
0.5409
0.4406
0.5447
0.2878
0.5193
0.3849
0.5846
0.3328
0.4797
0.4187
0.4690
0.4437
~-
I
lufttrocken
0.1907
bei looo 1 i
0.3279
0.3311
Gewichts-
zunahme
fiber NH3
pct. I
0.0418
I
6.79':
0.0340
6.29
0.0295
6.70
0.0367
6.74
0.0197
6.85
0.0757
, 14.58
0.0576
14.96
0.0902
0.0498
0.0758
0.0665
0.07 18
0.0953
0.01 11
0.0501
0.0504
15.43
14.96
15.80
15.88
15.31
2 1.48
(ber. 20.42)
5.82
15.31
15.22 -
Gewichts-
d u s t uber
&SO4
0.0773
0.0673 2,
0.069 1
0.0420
0.0758
0.0592
0.0902
0.0498
0.0265
0.0500
0.0506
Gewichts-
:unahme an
der Luft,
Iezogen auf
wasserfreie
Substanz
0.042 1
0.0426
0.0194
0.0415
0.0488
0 0266
0.041 1
0.0356
0.0372
0.0143
0.0269
0.0270
-
--
pet. 8.46
8.48
7.31
7.99
8.35
7.99
8.57
8.50
7.93 -
8.16
7.57
8.15
'1 Die Gewichtszunabmo ist zunschst vie1 grBsser, da Ammoniak rascher aufgenommen wird, als das Wasser entweicht. 2, Der Gewichtsverlust wurde bei diesem Beispiel erst im Vacuum vollstiindig. T a b e l l e 11. Bildung u n d Dissociation des Diammoniumsalzes von p-hzophenoi (a u n d ,8). (Berechnet fiir Aufnahme von 2 Mol. NH3: 15.93 pCt. von wasserfreier Sbst., fiir 2 Mol. NH3-1 Mol. H2O: 6.94 pCt,. von wasserhaltiger Sbst.. fiir Aufnahme von 1 Mol. '1 Die Gewichtszunabmo ist zunschst vie1 grBsser, da Ammoniak rascher aufgenommen wird, als das Wasser entweicht.
2, Der Gewichtsverlust wurde bei diesem Beispiel erst im Vacuum vollstiindig. HPO: 8.41 DCt. von wasserfreier Sbr
-
-- mit rhombiachem Querschnitt aus, die Krystallbenzol enthielten uud
schnell verwitterten, aeltener benzolfreie Tafeln. Beim Erwarmen auf
ca. 150° tritt die reducirende Wirkung des Phenylhydrazina ein. Daa
Salz der !-Modification
weist keinen Unterschied auf. mit rhombiachem Querschnitt aus, die Krystallbenzol enthielten uud
schnell verwitterten, aeltener benzolfreie Tafeln. Beim Erwarmen auf
ca. 150° tritt die reducirende Wirkung des Phenylhydrazina ein. Daa
Salz der !-Modification
weist keinen Unterschied auf. 1. a. 0.2529 g Sbst. (constant im Vacuum): 40.2 ccm N (17O, 724 mm). 11. ,B. 0.1958g Sbst. (constant im Vacuum): 30.4ccm N (17q 714mm). CisHloOaNa.CsHsN9. Ber. N 17.43. Gef. N I. 17.55, 11. 16.91. 1. a. 0.2529 g Sbst. (constant im Vacuum): 40.2 ccm N (17O, 724 mm). 11. ,B. 0.1958g Sbst. (constant im Vacuum): 30.4ccm N (17q 714mm). CisHloOaNa.CsHsN9. Ber. N 17.43. Gef. N I. 17.55, 11. 16.91. schmelzen mit Aetzkali bei 2@0°; diese Versuche misslangen, hingegen
erhielten wir das griine Pulver der wasserfreien a-Modification bei
Deetillation einer Probe von
im Vacuum, freilich in schlechter Aus-
beute, da der grosste Tbeil der Substanz Zersetzungen anheim fie]. g
g
Wahrend wir bei diesen Behandlungen und dem Umkrystallisiren
der 6-Form nie beim Erhitzen grin werdende Mischpraparate er-
hielten, verwandelt sich a-Azophenol beim Kochen rnit feuchtem Aether
inoffenknndige Mischungen. Der gelb gefarbte Riickstand, den man beim
Verdampfen solcher Aetherliisungen erhalt, nimmt bei raschem Er-
hitzen, namentlich von kleinen, diinn ausgebreiteten Portionen, hoch-
rothe Farbe an, immer bei Versuchen im Reagensglas. Bei lang-
samerem Entwassern griisserer Portionen, in dickerer Schicht , farbt
sich das Pulver theils griin, theils rotb, und damn wird auch nichts
geandert durcb Umkrystallisiren aus Alkohol. I) Ann. d. Chem. 196, 344 [1879]. p - A z o p h e n o l ($). Wenn man Chinonazin (2 g) in Benzol (100 g) lost und unter
allmahlichem Zufijgen von Phenylhydrazin (1 g) gelinde erwarmt, so
fallt ein rotbbrauner Niederschlag (1.8 g) aus. Aus 50-procentigem
Alkohol umkrystaliisirt, zeigte das Reductionsproduct die Zusammen-
setzung des urspriinglichen Azophenols. 0.1648 g Sbst. (krystallffasserhallig): 0.3763 g COa, 0.0786 g HaO. 0.1996 g Sbst. (bei lYOo getrocknet): 0.4944 g COa, 0 0862 g HaO. 0.1648 g Sbst. (krystallffasserhallig): 0.3763 g COa, 0.0786 g HaO. 0.1996 g Sbst. (bei lYOo getrocknet): 0.4944 g COa, 0 0862 g HaO. B
C 62 02 H 5 21 Gef. n 67.55, )) 4.82. Das neue Praparat bildet dunkelrothe und rothlichbraune Blattchen
nnd Tafelchen mit schiinem, blauem Reflex, oft zu sageformigen Ag-
gregaten vereinigt; unter dem Mikroskop erscheinen die Kryatalle in
der Durchsicht griingelb, aber dunkelroth , wo zwei Krystalle sich
kreuzen. Die Farbe des Pulvers ist blausticbig braunroth und wird
bei 1 10-140°
ziegelroth bis hochroth; beim Aufbewahren geht die
Farbe der erhitzen Substanz, auch bei Ausschluss von Feuchtigkeit,
im Laufe einiger Tage in braun iiber, aber bei erneutem Erhitzen
wird das Roth wieder hervorgerufen. Azophenol p! zeigt abnliche Loslichkeitsverhaltnisse wie a; grosser
fanden wir die Loslichkeit in Benzol, namlich bei 17O im Mittel von
3 Versuchen = 1 : 1500. Die Farbe der atherischen Liisung ist mehr
orangestichig gelb als bei a. Der Schmelzpunkt des Rohproductes von
lag bei 208O, der
umkrystallisirten Substanz bei 210O; bei fortgesetztem UmkryetalIisireu
stieg der Schmelzpunkt auf 212O. Dabei wlirde das p-Azophenol auch
im Aussehen der a-Form ahnlicher. Es ist nicht ausgeschloesen, dass es
dabei in eine Mischung der beiden Modificationen ubergegangen ist, allein
beim Erhitzen entstand stets wieder das rein rothe Pulver. Versuche,
die pl-Form in a umzuwandeln, haben wir tlngestellt durch Umfallen
dea Azophenols aue alkalischer LGsung, durch Ueberfiihrung in Chlor-
hydrat und Iaolirung aus dem Salze, endlich durch wiederholtea Ver- 0 - A z o p h e no 1. Das von P. Weselsky und R. Benedikt') beschriebene o-Azo-
phenol haben wir nach der Methode dieser Autoren durch Kalischmelze
von o-Nitrophenol mit der bei der p-Verbindung erwahnten kleinen
Abanderung dargestellt. In
dem Azofarbstoff fanden wir
eine
Beimischung, die zum grossen Theil schon beim Auflosen und Um-
krystallisiren zuriickbleibt , und die sich schliesslich quantitativ be-
seitigen Iasst, wenn man die alkalische Losung dee Azophenols rnit
Benzol wiederholt ausschiittelt. Das indifferente Neben product, rothe
Krystallblatter, tiefblau loslich in Schwefelsaure, griin fluorescirend in
Benzol, war Triphendioxazin. 0.1844 g Sbst.: 0.5098 g Cog, 0.0603 g HaO. 0.1844 g Sbst.: 0.5098 g Cog, 0.0603 g HaO. CieHioOaN2. Ber. C 75.48, H 3.52. o-Azophenol lost sich in 60 Theilen kalten Benzols, leicht in
warmem. Es krystallisirt daraus wie aus Alkohol in goldgelben, stark
gliinzenden Blattchen oder orangefarbigen, langen Nadeln: letztere
zeigen schwachen blaulichen Reflex. Pulver tiefgelb, Schmp. 172O
(nach W e s e l s k y und B e n e d i k t 1710). Es krystallisirt unter allen
Urnstinden wasserfrei. 0-Azophenol reagirt i n iitherischer oder benzol-
lischer Losung weder in der Kalte, noch beim Kochen rnit Silberoxyd. Der o-Azokorper nimmt in trockner Ammoniakatmosphare genau
1 Mol. Ammoniak auf. Er bildet ein kaffeebraunes Ammoniumsalz,
das beim Liegen an der Luft oder uber Schwefelsaure rasch sein
Ammoniak verliert, und zwar obne Hydratbildung. Durch peinlichen I) Ann. d. Chem. 196, 344 [1879]. L, Dies Berichte 38, 3799 [1905]. 3502 Ausschluse von Feuchtigkeit liess sich die Reaction mit Ammoniak
verzcgern, aber nicht verhindern. Oanz anders, wenn das o-Azophenol
znm Schmelzen erhitzt oder im Vacuum sublimirt oder destillirt worden
iet. Ee geht dabei in eine Form iiber, die absolut nicht mit Ammo-
niakgaa reagirt, nicht etwa deshalb, weil sie trockner ist, sondern
auch d a m nicht, wenn man dem Praparat durch laugeres Liegen an
der Luft Gelegenheit bietet , etwas Feuchtigkeit aufzunehmen. Setzt
man aber ein solches Praparat in einem mit wassrigern Ammoniak
beschickten Exeiccator etundenlang einem Ammoniakstrom aus , so
entsteht schliesslich wieder das Ammoniumsalz; auch durch Um-
krystallisiren geht die indifferente Form des Azophenols alsbsld in
die reactionsfiihige zuriick. Tabelle 111. Bildung des Monoammoniumsalzes v o n o-Azophenol. (Ber. fur Aufnahme von 1 Mol. NEB: 7.96 pCt.) PrBp arat
Trocknung
Tabelle 111. Bildung des Monoammoniumsalzes v o n o-Azophenol. (Ber. fur Aufnahme von 1 Mol. NEB: 7.96 pCt.)
Krystalle aus Alkohol . . . KrystalleausAlkohol(B1Bttch.)
Krystalle aus Alkohol(Nade1n)
Krystalle aus Weingeist . . iiber Has04
iiber HI SO4
fiber H, SO4
iiber HsS04
Daaselbe Priiparat, erneut aus
Benzol umkrystallisirt . . Krystalle aus Alkohol . . . Krystalle aus Weingeist . .]I 2 Si?&bei
iiber Has04
bei 120°
Dasselbe Priparat,nach Disso-
ciation des Ammoniumsdzes
iiber HgSOd . . . . . ll
-
ImVacuum destillirt. Substam1
Dasselbe Prgparat , nach er-
ueutem Umkrystallisiren aus
Benzol . . . . . . .I
I
Krystallek Alkohol(B1itttch.) I bei 130O
Krystalle aus Weingeist . .1,36
pso5 Stdn. i.vacuun
iiber
Geschmolzene Substanz . . lufttrocken
lufttrocken
iiber H~SOI
Jersuchs
dauer
Stdn. 21
15
18
14
12
18
18
5und 1E
24 u.38
6
40
7 und 17
32
60
21
Substanz
g
0.2608
0.3111
0.2137
0.344 1
0.3414
0.3492
0.2082
0.2596
0.2005
0.2816
0.2005
0.28 16
0.3042
0.2134
0.2784
0.2676
0.2355
0.285 Z
Gewichtszu-
ahme iib. NB
0.0201
0.0227
0.0154
0.0263
0.0254
0.001 7
0.0144
0.0097 unc
0.0146
0.0002 unC
0.0 I52
0.0005 unc
0.022 1
0.0145
0.02 13
0.0005
0.0003 unc
0.0151
0
0
0
0.0160
Proc. -
7.04
7.26
7.21
7.64
7.44
0.49
6.92
.74 u. i.62
7.58
7.85
7.23
7.56
0.16
7.06
0
0
0
6.14 Tabelle 111. Bildung des Monoammoniumsalzes v o n o-Azophenol. (Ber. fur Aufnahme von 1 Mol. NEB: 7.96 pCt.) 9 Zeitschr. f. Elektrochem. 8, 791 [1902]. a) Journ. f. prakt. Chem. [2] 67, 265 [1903]. 3) Diese Berichte 39, 2346 [190(i]. 4, Ein Theil dieser Untersuchungen bildet den Inhalt einer am 21. M i
dieses Jahree eingereichten Patentanmeldung, in welcher bereits genaue An-
geben und Constanten enthalten sind. 9 Zeitschr. f. Elektrochem. 8, 791 [1902].
a) Journ. f. prakt. Chem. [2] 67, 265 [1903].
3) Diese Berichte 39, 2346 [190(i].
4, Ein Theil dieser Untersuchungen bildet den Inhalt einer am 21. M i
dieses Jahree eingereichten Patentanmeldung, in welcher bereits genaue An-
geben und Constanten enthalten sind.
L, Dies Berichte 38, 3799 [1905]. m -A z o p h en 01. Der m-Azokijrper ist von E. Klappert') (im Laboratorium von Elbs)
durch elektrochemische Reduction des Nitrophenols und dann von K. Elbs
und W. Kirsch? durch Diazotiren und Umkochen aus m-Azoanilin dargestellt
worden. Er lhsst sich aber such (entgegen der Angabe von P. Weselsky und
R. Benedik t) leicht dnrch Kalischmelze von m-Nitrophenol gewinnen. Eine zu
heftige Reaction wird vermieden, wenn man wieder das gepulverte Ausgangs-
material (5 g) rnit gleich vie1 Wasser verriibrt, rnit 20 g Aetzkali langsam
bis zum vcilligen LBsen anwn$rmt und dam erst h6lier erhitzt. Ausbeute an
umkrystallisirtem Azophenol 0.75 g. Hell broncegelbe, metallisch glanzende
Blgttchen; Schmelzpunkt 2050, wie Elbs und Kirsch angeben. Reagirt in
atherischer LBsung nicht mit Silberoxyd. In exsiccatortrocknem Zu-
stand absorbirt es Ammoniak, wobei die Farbe rBthlich braun wird;
die bei erreichter Constanz anfgenommene Menge lag zwischen 1 und
2 Mol. Ammoniak. rn-Azophenol krystallisirt wasserfrei. Das Salz dissociirt ohne Hydratbildung. 0.2392, 0.2997, 0.3292 g Shst. nahmen auf 0.0310, 0.0401, 0.0431 g NHz
entsprechend 12.96, 13.38, 13.09 pet. y
g 0.2392, 0.2997, 0.3292 g Shst. nahmen auf 0.0310, 0.0401, 0.0431 g NHz
entsprechend 12.96, 13.38, 13.09 pet. 663. 3. Hou b e n und Hans Doe s c her: Ueber Hydropinensulfin-
861.~8,
Hydropinencarbithiosiiure, Thioborneol und Thioaempher. [Aus dem T. chemischen Infititut der Universitht Berlin.]
(Eingegangen am 1. October 1906.) Soeben veroffentlichen W. B o r s c h e und W. Lange3) eine Ab-
handlung, deren Inhalt sich zum grossen Theil rnit Untersuchnngen
deckt, die wir vor einiger Zeit gemeinsam unternommen haben, urn
das von dem Einen von uns aus Pinenchlorhydrat gewonnene Hydro-
pinenmagnesinmchlorid noch eingehender mit dem Bornylmagnesinm-
chlorid zu vergleichen und i n seinen Reactionen zu erforschen 4). Wir
theilen daher die bis jetzt gewonnenen Ergebnisse mit. H y d r o p i n e n s u l f i n s a u r e s Natrium. 50 g wasser- und saure-freies , festes Pinenchlorhydrat wurden
nach der von Houben5) augegehenen Methode mittels 50 g Aether und 9 Zeitschr. f. Elektrochem. 8, 791 [1902]. a) Journ. f. prakt. Chem. [2] 67, 265 [1903]. 4, Ein Theil dieser Untersuchungen bildet den Inhalt einer am 21. M i
dieses Jahree eingereichten Patentanmeldung, in welcher bereits genaue An-
geben und Constanten enthalten sind.
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https://openalex.org/W4206183950
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https://accounting.fa.ru/jour/article/download/428/387
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Russian
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Tax aspect of supporting the implementation of the best available technologies
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Učët. Analiz. Audit/Učët, analiz, audit
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cc-by
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ОРИГИНАЛЬНАЯ СТАТЬЯ CC BY 4.0 DOI: 10.26794/2408-9303-2021-8-6-21-30
УДК 336.226.4,336.225.66,336.221.4(045)
JEL H23, H25, E61 Tax Aspect of Supporting the Implementation
of the Best Available Technologies ble ec
olog es
D. A. Smirnovа, A. A. Zavorykinb
Financial University, Moscow, Russia
а https://orcid.org/0000-0003-3112-0585; b https://orcid.org/0000-0002-2426-8488 а https://orcid.org/0000-0003-3112-0585; b https://orcid.org/0000-0002-2426-8488 а https://orcid.org/0000-0003-3112-0585; b https://orcid.org/0000-0002-2426-8488 Налоговый аспект поддержки внедрения наилучших
доступных технологий ологий
Д. А. Смирнова, А. А. Заворыкинb
Финансовый университет, Москва, Россия
a https://orcid.org/0000-0003-3112-0585; b https://orcid.org/0000-0002-2426-8488 о о
Д. А. Смирнова, А. А. Заворыкинb
Финансовый университет, Москва, Россия
a https://orcid.org/0000-0003-3112-0585; b https://orcid.org/0000-0002-2426-8488 АННОТАЦИЯ АННОТАЦИЯ
Статья посвящена исследованию налогового аспекта поддержки внедрения наилучших доступных технологий (НДТ)
в России. Авторами выполнено сравнение отечественного и зарубежного опыта использования экономических инстру-
ментов (в частности, фискальных) для поддержания устойчивого развития и внедрения инновационных технологий
в природоохранную деятельность и приведены аргументы в их пользу. На основе гипотезы «множественного выигрыша»
от применения фискальных инструментов в данной области обосновывается необходимость более широкого подхода
к анализу многофакторности воздействия корректирующих налогов на различные социально-экономические показатели. Доказано, что современная фискальная схема поддержки наилучших доступных технологий должна служить множествен-
ным целям, опираться не только на методы снижения нагрузки для внедряющих инновации предприятий. Предложены
изменения существующего алгоритма поддержки НДТ и системы корректирующих фискальных платежей, которые соот-
ветствуют идеям «множественного выигрыша» и должны способствовать ускорению внедрения необходимых экологи-
чески ориентированных инноваций. Ключевые слова: экологическое налогообложение; наилучшие доступные технологии; экстерналии; налоговое сти-
мулирование; налоговое администрирование; устойчивое развитие Для цитирования: Смирнов Д. А., Заворыкин А. А. Налоговый аспект поддержки внедрения наилучших доступных тех-
нологий. Учет. Анализ. Аудит = Accounting. Analysis. Auditing. 2021;8(6):21-30. DOI: 10.26794/2408-9303-2021-8-6-21-30 21 21 21 21 МЕТОДИКИ И ПРАКТИЧЕСКИЙ ОПЫТ / METHODS AND PRACTICAL EXPERIENCE For citation: Smirnov D. A., Zavorykin A. A. Tax aspect of supporting the implementation of the best available technologies. Uchet.
Analiz. Audit = Accounting. Analysis. Auditing. 2021;8(6):21-30. (In Russ.). DOI: 10.26794/2408-9303-2021-8-6-21-30 ВВЕДЕНИЕ Внедрение НДТ может обеспечиваться раз-
личными средствами экономического механизма
коррекции экстерналий, а прежде всего — фи-
скальными инструментами и субсидированием. Подобный опыт формирует дискуссию по вопросу
эффективности, целесообразности и пропорци-
ональности применения одних инструментов
перед другими [5–9]. На протяжении последних пятидесяти лет ме-
ждународное сообщество в лице Организация
Объединенных Наций (ООН) пыталось выра-
ботать широкий спектр мер по ликвидации со-
циального неравенства, выравниванию темпов
экономического роста стран, поддержке здра-
воохранения, защите населения от последст-
вий изменений состояния окружающей среды,
стимулированию возобновляемых источников
энергии — преодолению трудностей, обуслов-
ленных рыночными экстерналиями [1, 2]. Ука-
занные проблемы легли в основу Целей устой-
чивого развития (ЦУР) ООН и программ ООН
по окружающей среде (ЮНЕП) 1, Таким образом,
государственные органы, научное сообщество
и предприниматели различных стран разраба-
тывают и реализуют элементы экономической
политики, направленные на достижение ЦУР,
но не влияющие отрицательно на уровень на-
ционального благосостояния, конкурентоспо-
собность и состояние окружающей среды. Фор-
мируются различные подходы к экономической,
энергетической и экологической эффективно-
сти производственных процессов. Исходя из вышесказанного, можно обозначить
цель данного исследования как анализ практики
применения различных средств поддержки НДТ
для выработки рекомендаций по формированию
современных экономических мер поддержки
устойчивого развития и коррекции экстерналий. Для ее достижения использовались методы син-
теза, сравнительного и логического анализа. Со-
четание методов позволило всесторонне оценить
эффективность и перспективность применяемых
инструментов. 1 Цели устойчивого развития. URL: https://www.un.org/
sustainabledevelopment/ru/sustainable-development-goals/
(дата обращения: 5.09.2021). ООН. Программа по окружа-
ющей среде.URL: https://www.unep.org/ru (дата обращения:
05.09.2021). МЕТОДИКИ И ПРАКТИЧЕСКИЙ ОПЫТ / METHODS AND PRACTICAL EXPERIENCE МЕТОДИКИ И ПРАКТИЧЕСКИЙ ОПЫТ / METHODS AND PRACTICAL EXPERIENC 2 Transformations to Achieve the Sustainable Development
Goals. URL: https://iiasa.ac.at/web/home/research/twi/ TWI2050_Report_web-small‑071018.pdf (дата обращения:
05.09.2021).
3 Федеральный закон от 21.07.2014 № 219-ФЗ «О внесении
изменений в Федеральный закон «Об охране окружающей
среды» и отдельные законодательные акты Российской
Федерации». URL: http://www.consultant.ru/document/cons_
doc_LAW_165823/ (дата обращения: 25.07.2021). ABSTRACT The article is dedicated to the study of the tax aspect of supporting the implementation of best available technologies
(BAT) in Russia. The authors compare domestic and foreign experience of using environmental protection tools to
support sustainable development and implementation of innovative technologies. The article provides arguments in
favor of using fiscal instruments to support BAT and sustainable development goals and draws some conclusions based
on the results of the recent application of fiscal instruments for environmental protection and sustainable development. The “multiple win” hypothesis is used to justify the application of fiscal instruments for environmental protection, in
particular the need for a broader approach to the analysis of multifactor impact of corrective taxes on various socio-
economic indicators. It is assumed that a modern fiscal scheme for supporting BAT should serve broad purposes, relying
not only on the methods to reduce the burden on enterprises implementing innovations. The authors propose several
changes to the existing scheme of supporting BAT and fiscal payments. These changes are in line with the “multiple win”
idea and should qualitatively contribute to the acceleration of the introduction of environmentally oriented innovations. Keywords: environmental taxation; best available techniques; externalities; tax incentives; tax administration;
sustainable development For citation: Smirnov D. A., Zavorykin A. A. Tax aspect of supporting the implementation of the best available technologies. Uchet. Analiz. Audit = Accounting. Analysis. Auditing. 2021;8(6):21-30. (In Russ.). DOI: 10.26794/2408-9303-2021-8-6-21-30 © Смирнов Д. А., Заворыкин А. А., 2021 www.accounting.fa.ru 22 TWI2050_Report_web-small‑071018.pdf (дата обращения:
05.09.2021). TWI2050_Report_web-small‑071018.pdf (дата обращения:
05.09.2021). МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ В последние годы в России (по аналогии с веду-
щими зарубежными странами) можно наблю-
дать тенденцию, связанную с повышенным вни-
манием к крупным программам экологизации
производства. Такие проекты получают особый
инвестиционный статус и налоговые льготы на
внедрение новых технологий. Важными элементами системы регулирования
природоохранной деятельности и реализации
целей устойчивого развития становятся механиз-
мы коррекции экологических экстерналий. Если
концепция наилучших доступных технологий
впервые нашла применение в Швеции [3], то рос-
сийский опыт ее использования ведет свое начало
только с 2014 г., хотя его корни лежат в проектах
конца ХХ в. НДТ в данном случае определяется
как комплекс решений в области современных
технологий, позволяющих создать такой уровень
экологической, экономической и технической эф-
фективности, который позволит преодолеть сло-
жившиеся «провалы рынка». При применении НДТ
речь идет об опережении темпов роста ВВП над
темпами потребления энергетических ресурсов
при сохранении должного уровня благосостояния
населения за счет передовых производственных
технологий 2 [4]. С одной стороны, эти процессы можно назвать
положительным явлением, но с другой стороны,
есть поводы для беспокойства. С одной стороны, эти процессы можно назвать
положительным явлением, но с другой стороны,
есть поводы для беспокойства. В России данная тенденция берет начало
в 2014 г., когда были приняты изменения в Фе-
деральный закон «Об охране окружающей сре-
ды», которые определили понятие «наилучшие
доступные технологии», методы разработки ин-
формационно-технических справочников, меры
государственной поддержки внедрения НДТ 3. Введенные новации направлены на соответ-
ствие нормам европейского экологического за-
конодательства — Директивам Европарламен-
та и Совета Евросоюза 2008/1/ЕС от 15.01.2008
«О комплексном предупреждении и контроле 3 Федеральный закон от 21.07.2014 № 219-ФЗ «О внесении
изменений в Федеральный закон «Об охране окружающей
среды» и отдельные законодательные акты Российской
Федерации». URL: http://www.consultant.ru/document/cons_
doc_LAW_165823/ (дата обращения: 25.07.2021). Учет. Анализ. Аудит • Т. 8, № 6’2021 23 Д. А. Смирнов, А. А. Заворыкин В России с 2020 г. действуют стимулирующие
коэффициенты (0, 1, 25 и 100) к ставкам платы
за НВОС: нулевой коэффициент применяется
с 01.01.2020 г. при внедрении НДТ на объекте 9. Коэффициент, равный 100, предусматривается
в случае выбросов, превышающих установленные
нормы (с учетом стандартов НДТ) для объектов
I категории, а также превышающих указанные
в декларации 10 для объектов II категории. На-
конец, при внедрении НДТ предусматривается
уменьшение платы за НВОС на фактически про-
изведенные затраты на реализацию мероприятий
по снижению НВОС 11. Согласно Федерального
закона «О промышленной политике в Российской
Федерации» 12 возможно предоставление субсидий
на финансирование создания или модернизации
промышленной инфраструктуры, в том числе при
применении НДТ. 4 Директива Европейского Парламента и Совета Европей-
ского Союза 2008/1/ЕС от 15.01.2008 о комплексном пре-
дотвращении и контроле загрязнений. URL: https://eur-lex.
europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32008L0001
(дата обращения: 25.07.2021). 9 Федеральный закон от 10.01.2002 № 7-ФЗ «Об охра-
не окружающей среды». Ст. 16.3, п. 5. URL: http://www.
consultant.ru/document/cons_doc_LAW_34823/9c0483e7
ceb410b7f80bb26442dd36fc696866e7/ (дата обращения:
25.07.2021). 9 Федеральный закон от 10.01.2002 № 7-ФЗ «Об охра-
не окружающей среды». Ст. 16.3, п. 5. URL: http://www.
consultant.ru/document/cons_doc_LAW_34823/9c0483e7
ceb410b7f80bb26442dd36fc696866e7/ (дата обращения:
25.07.2021).
10 Декларация о плате за негативное воздействие на окру-
жающую среду (Утверждена приказом Минприроды Рос-
сии от 10.12.2020 № 1043). URL: http://www.consultant.ru/
document/cons_doc_LAW_373329/e82b1c2d4d04c3628368049
09bc3eaf97d4bf525/#dst100047 (дата обращения: 25.07.2021).
11 Там же. П. 11. Ст. 16.3 (дата обращения: 25.07.2021).
12 Федеральный закон от 31.12.2014 № 488-ФЗ «О промыш-
ленной политике в Российской Федерации». Ст. 10. URL:
http://www.consultant.ru/document/cons_doc_LAW_173119/.
13 Постановление Правительства РФ от 16.07.2015 № 708
«О специальных инвестиционных контрактах для отдель-
ных отраслей промышленности» (В редакции постановле-
ний Правительства Российской Федерации от 27.12.2016
№ 1501, от 16.12.2017 № 1564, от 01.08.2018 № 895, от
15.01.2020 № 8, от 01.10.2020 № 1585, от 03.12.2020 № 2018).
URL: http://pravo.gov.ru/proxy/ips/?docbody=&nd=102376302
(дата обращения: 25.07.2021). 5 Директива Европейского парламента и Совета Европей-
ского Союза 2010/75/ЕС от 24.11.2010 «о промышленных
выбросах (о комплексном предотвращении загрязнения
и контроле над ним)». URL: http://base.garant.ru/70161770/
(дата обращения: 05.10.2021). 4 Директива Европейского Парламента и Совета Европей-
ского Союза 2008/1/ЕС от 15.01.2008 о комплексном пре-
дотвращении и контроле загрязнений. URL: https://eur-lex.
europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32008L0001
(дата обращения: 25.07.2021).
5 Директива Европейского парламента и Совета Европей-
ского Союза 2010/75/ЕС от 24.11.2010 «о промышленных
выбросах (о комплексном предотвращении загрязнения
и контроле над ним)». URL: http://base.garant.ru/70161770/
(дата обращения: 05.10.2021).
6 Федеральный закон от 10.01.2002 № 7-ФЗ «Об охра-
не окружающей среды. URL: http://www.consultant.ru/
document/cons_doc_LAW_34823/9c0483e7ceb410b7f80bb264
42dd36fc696866e7/ (дата обращения: 25.07.2021).
7 Налоговый кодекс Российской Федерации. (НК РФ).
Ст. 259.3, п. 1, пп. 5. URL: http://www.consultant.ru/
document/cons_doc_LAW_19671/ (дата обращения:
25.07.2021).
8 Т
С
67
1
1 6 Федеральный закон от 10.01.2002 № 7-ФЗ «Об охра-
не окружающей среды. URL: http://www.consultant.ru/
document/cons_doc_LAW_34823/9c0483e7ceb410b7f80bb264
42dd36fc696866e7/ (дата обращения: 25.07.2021). 7 Налоговый кодекс Российской Федерации. (НК РФ).
Ст. 259.3, п. 1, пп. 5. URL: http://www.consultant.ru/
document/cons_doc_LAW_19671/ (дата обращения:
25.07.2021). 10 Декларация о плате за негативное воздействие на окру-
жающую среду (Утверждена приказом Минприроды Рос-
сии от 10.12.2020 № 1043). URL: http://www.consultant.ru/
document/cons_doc_LAW_373329/e82b1c2d4d04c3628368049
09bc3eaf97d4bf525/#dst100047 (дата обращения: 25.07.2021). 8 Там же. Ст. 67, п. 1, пп. 1. МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ возложена роль основного стимулятора. Суб-
сидии же играют роль дополняющего фактора,
который должен содействовать производителю
в самых сложных отраслях, требующих господ-
держки. Чрезмерное поощрение без создания
фискального бремени, ускоряющего принятие
решения субъектами экономики, не помогает
достигнуть нужного эффекта. При этом, как по-
казывает практика стран ЕС, с помощью данных
экономических инструментов можно добиться
перераспределения обязательных (от налогов
на доходы к экологической сфере) и сократить
совокупные общественные издержки. Не менее важной проблемой внедрения НДТ
является недостаточность, а вернее, практическое
отсутствие полноценной системы индикаторов
оценки реализации программ внедрения иннова-
ций 15. Например, градообразующие предприятия
заинтересованы в экологизации инфраструктуры
на территориях присутствия с учетом вноси-
мой платы за НВОС. Однако Бюджетный кодекс
Российской Федерации 16 не предусматривает
создания целевых экологических внебюджетных
фондов, а Министерство финансов РФ не пре-
доставило возможности целевого расходования
средств в счет платы за НВОС, что позволило бы
увидеть движение средств на минимизацию нега-
тивного экологического воздействия в отдельно
взятом регионе и показать прозрачность эколо-
гических мероприятий, и тем самым повысить
доверие бизнеса и населения. Отметим негативный эффект от субсидирова-
ния, который испытывают многие страны — члены
Организации экономического сотрудничества
и развития (ОСЭР) и группы из пяти стран: Бра-
зилии, России, Индии, КНР, ЮАР (БРИКС), когда
данного вида дотациями подменяется сама идея
стимулирования производителей к приходу на
экологичные технологии. Зачастую такие про-
граммы, схожие с отечественной НДТ, помогают
либо поддерживать конкурентоспособность наци-
онального производителя (что и делают страны
ОЭРС, одобряя низкоэкологичные технологии),
либо находятся в руках промышленного лобби,
далекого от целей снижения общественных из-
держек. В долгосрочной перспективе такая по-
литика может негативно сказаться не только на
связанных с экологией общественных издержках,
но и на цене товаров или услуг, производимых по
«старым» технологиям, например на топливных
ценах. Представляется более разумным усилить ак-
цент в части применения фискальных стимулов,
побуждая при этом предприятия к созданию пол-
ноценных схем рециклинга. Очевидно, что абсо-
лютное большинство инструментов коррекции
природоохранной деятельности как в части эко-
номических, так и административно-командных
(командно-контрольных) носят характер обре-
менения для изменения поведения плательщика
в сторону экологизации (исключение, пожалуй,
составляет субсидирование). При этом еще А. Пигу
и П. Сэмуэльсон [1, 2] в прошлом веке отдавали
ведущую роль фискальным инструментам, в част-
ности корректирующим налогам. Складывающаяся с начала 2021 г. МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ загрязнений» 4 и 2010/75/ЕС от 24.11.2010 «О про-
мышленных эмиссиях (комплексное предупре-
ждение и контроль)» 5. В нормативных актах появилось описание
критериев НДТ: минимальный уровень негатив-
ного воздействия на окружающую среду (НВОС)
в расчете на единицу времени или объем произво-
димой продукции (товара), выполняемой работы,
оказываемой услуги либо другие предусмотрен-
ные международными договорами Российской
Федерации показатели; экономическая эффек-
тивность внедрения и эксплуатации НДТ; при-
менение ресурсо- и энергосберегающих методов;
промышленное внедрение этой технологии на
двух и более объектах, оказывающих НВОС. При
этом определено, что господдержка внедрения
НДТ осуществляется при помощи: • предоставления льгот в соответствии с за-
конодательством о налогах и сборах; В качестве мер стимулирования наилучших
доступных технологий также выступают субсидии,
в том числе на компенсацию расходов (уплата
процентов по кредитам в рамках инвестиционных
проектов) и части затрат на НИОКР [10]. Начиная
с 01.01.2019 г. законодательством РФ установлена
возможность заключения специального инвести-
ционного контракта в целях внедрения НДТ 13. • предоставления льгот в отношении платы
за НВОС; • выделения средств федерального бюджета
и бюджетов субъектов Российской Федерации
в соответствии с бюджетным законодательст-
вом 6. В рамках данных мер поддержки при приме-
нении НДТ в Налоговом кодексе Российской Фе-
дерации 7 установлена возможность применения
ускоренной амортизации с учетом повышающего
коэффициента в отношении основных средств,
относящихся к основному технологическому обо-
рудованию. Меры налоговой поддержки НДТ так-
же включают предоставление инвестиционного
налогового кредита, что предусмотрено НК РФ 8. Отметим, однако, что совокупность мер по
стимулированию перехода предприятий на ис-
пользование НДТ в нормативных документах
выражена недостаточно четко. Вместе с тем, не-
смотря на внушительный прирост инвестиций
в экологические программы российских компаний 7 Налоговый кодекс Российской Федерации. (НК РФ). Ст. 259.3, п. 1, пп. 5. URL: http://www.consultant.ru/
document/cons_doc_LAW_19671/ (дата обращения:
25.07.2021). 7 Налоговый кодекс Российской Федерации. (НК РФ). Ст. 259.3, п. 1, пп. 5. URL: http://www.consultant.ru/
document/cons_doc_LAW_19671/ (дата обращения:
25.07.2021). www.accounting.fa.ru 24 МЕТОДИКИ И ПРАКТИЧЕСКИЙ ОПЫТ / METHODS AND PRACTICAL EXPERIENCE (по официальным подсчетам в последние годы
превысивших 150 млрд руб.), вызывает сомнение
значимость для них льготы в части снижения
платы за НВОС 14. возложена роль основного стимулятора. Суб-
сидии же играют роль дополняющего фактора,
который должен содействовать производителю
в самых сложных отраслях, требующих господ-
держки. Чрезмерное поощрение без создания
фискального бремени, ускоряющего принятие
решения субъектами экономики, не помогает
достигнуть нужного эффекта. При этом, как по-
казывает практика стран ЕС, с помощью данных
экономических инструментов можно добиться
перераспределения обязательных (от налогов
на доходы к экологической сфере) и сократить
совокупные общественные издержки. 17 Энергетический бюллетень Аналитического центра
при Правительстве РФ. URL: https://ac.gov.ru/uploads/2-
Publications/energo/2021/%D0%B1%D1%8E%D0%BB%D0%BB
%D0%B5%D1%82%D0%B5%D0%BD%D1%8C_%E2%84%96_99.
pdf (дата обращения: 28.07.2021). 14 Паспорт национального проекта «Экология» [утв. пре-
зидиумом Совета при Президенте РФ по стратегическо-
му развитию и национальным проектам (протокол от
24.12.2018 № 16)]. URL: http://www.consultant.ru/document/
cons_doc_LAW_316096/ (дата обращения: 25.07.2021). 15 Энергетический бюллетень Аналитического центра
при Правительстве РФ. URL: https://ac.gov.ru/uploads/2-
Publications/energo/2021/%D0%B1%D1%8E%D0%BB%D0%BB
%D0%B5%D1%82%D0%B5%D0%BD%D1%8C_%E2%84%96_99.
pdf (дата обращения: 25.07.2021). 14 Паспорт национального проекта «Экология» [утв. пре-
зидиумом Совета при Президенте РФ по стратегическо-
му развитию и национальным проектам (протокол от
24.12.2018 № 16)]. URL: http://www.consultant.ru/document/
cons_doc_LAW_316096/ (дата обращения: 25.07.2021).
15 Энергетический бюллетень Аналитического центра
при Правительстве РФ. URL: https://ac.gov.ru/uploads/2-
Publications/energo/2021/%D0%B1%D1%8E%D0%BB%D0%BB
%D0%B5%D1%82%D0%B5%D0%BD%D1%8C_%E2%84%96_99.
pdf (дата обращения: 25.07.2021).
16 Бюджетный кодекс Российской Федерации (с из-
менениями на 15.07.2021). URL: https://docs.cntd.ru/
document/901714433 (дата обращения: 25.07.2021). МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ позитивная
конъюнктура мирового нефтяного рынка стиму-
лирует российских производителей расширять
поставки сырой нефти на экспорт вместо перера-
ботки на отечественных нефтеперерабатывающих
заводах (НПЗ); при этом применяемые в течение
последних трех лет Правительством Российской
Федерации меры поддержки отрасли не обес-
печивают компаниям достаточной доходности
даже в условиях государственной компенсации
более 60% разницы между экспортной и опто-
вой ценами 17. Указанная проблема актуальна не
только для сложного отечественного топливного Международное сообщество также деклари-
рует использование фискальных инструментов
в сочетании с субсидиями, но именно на первые 15 Энергетический бюллетень Аналитического центра
при Правительстве РФ. URL: https://ac.gov.ru/uploads/2-
Publications/energo/2021/%D0%B1%D1%8E%D0%BB%D0%BB
%D0%B5%D1%82%D0%B5%D0%BD%D1%8C_%E2%84%96_99. pdf (дата обращения: 25.07.2021). 17 Энергетический бюллетень Аналитического центра
при Правительстве РФ. URL: https://ac.gov.ru/uploads/2-
Publications/energo/2021/%D0%B1%D1%8E%D0%BB%D0%BB
%D0%B5%D1%82%D0%B5%D0%BD%D1%8C_%E2%84%96_99. pdf (дата обращения: 28.07.2021). 16 Бюджетный кодекс Российской Федерации (с из-
менениями на 15.07.2021). URL: https://docs.cntd.ru/
document/901714433 (дата обращения: 25.07.2021). Учет. Анализ. Аудит • Т. 8, № 6’2021 25 Д. А. Смирнов, А. А. Заворыкин рынка, но вполне соответствует общемировым
трендам, что свидетельствует о том, что топлив-
ный переход может оказаться слишком резким
явлением для большинства национальных про-
изводителей, и государство должно стремиться
к стимулированию новых технологий, способных
сочетать в себе не только высокую экологичность,
но и большую инновационность, энергоемкость
и энергоэффективность [11]. рынка, но вполне соответствует общемировым
трендам, что свидетельствует о том, что топлив-
ный переход может оказаться слишком резким
явлением для большинства национальных про-
изводителей, и государство должно стремиться
к стимулированию новых технологий, способных
сочетать в себе не только высокую экологичность,
но и большую инновационность, энергоемкость
и энергоэффективность [11]. выигрыша», предполагающую, что в результате
внедрения фискальных инструментов возникают
положительные эффекты, связанные не только
с состоянием окружающей среды и перераспре-
делением налоговой нагрузки, но и затрагива-
ющие здравоохранение, развитие инноваций
и инвестиций, создание новых видов производ-
ства и рабочих мест, повышение уровня жизни
населения, снижение общественных издержек
(расходов на воспроизводство, здравоохране-
ние и т. п.). Элементы гипотезы представлены
на рис. 1, а ее положения были подтверждены
с помощью средств экономико-статистического
анализа [17]. В этой связи во многих публикациях [12–18]
критически оценивается курс стран ОЭСР на
чрезмерную поддержку производителей низко-
экологичной продукции без адекватного стимула
к внедрению НДТ. Эффективные ставки выбросов
углерода (т. е. «цена» выбросов углерода с уче-
том налогов на них, акцизов на использование
энергии и продаваемых разрешений на выбро-
сы) в странах ОЭСР за пределами транспортного
сектора составляет 7,90 евро за тонну CO2
18
. 18 OECD Inventory of Support Measures for Fossil Fuels:
Country Notes. URL: https://www.oecd.org/environment/
oecd-companion-to-the-inventory-of-support-measures-for-
fossil-fuels-country-notes‑5a3efe65-en.htm (дата обраще-
ния: 22.10.2021). 19 Государственный доклад «О состоянии и об охране окру-
жающей среды Российской Федерации в 2018 году». URL:
https://www.mnr.gov.ru/docs/gosudarstvennye_doklady/o_
sostoyanii_i_ob_okhrane_okruzhayushchey_sredy_rossiyskoy_
federatsii/ (дата обращения: 22.11.2021). МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ Это
показывает, что политика этих стран в значитель-
ной степени не учитывает «ценообразование» на
выбросы вредных веществ, но при этом в рамках
лояльности по отношению к производству низ-
коэкологичной энергии они выборочно снижают
налоги на активы, владельцами которых являют-
ся государственные предприятия, субсидируют
бизнес без должной «экологической» мотивации. Поддержка производителей ископаемого топлива
в странах ОЭСР составляет более 60 млрд долл. США в год. В своем реестре они определили около
800 особых налоговых механизмов поддержки
ископаемого топлива. Перспективность фискальных инструментов
при этом является для России не только теорети-
ческой максимой, но и подтверждается практи-
чески. Так, например, в государственном докладе
«О состоянии и об охране окружающей среды
Российской Федерации в 2018 году» 19 подчерки-
вается роль данных инструментов, применяемых
в области охраны окружающей среды, которые
признаются одними из наиболее действенных
факторов, стимулирующих природоохранные
и энергосберегающие инвестиции. Вместе с тем существенным препятствием
для достижения эффективности уже имеющихся
фискальных и административных мер являются
частые нарушения законодательства на местах. Результаты мероприятий, проводимых прокура-
турой, свидетельствуют о том, что повсеместно
распространены факты: загрязнения объектов
окружающей среды хозяйствующими субъек-
тами; незаконного распоряжения природными
ресурсами и их использования; нарушения ор-
ганами государственной власти и местного са-
моуправления порядка выдачи разрешительной
документации; администрирования платежей,
требований при осуществлении контрольных
и надзорных функций. В противовес такой практике, руководящими
принципами экологизации в ЕС являются по-
ложения «налоговой» теории «двойного выиг-
рыша», что означает это улучшение состояния
окружающей среды в результате воздействия
фискальных инструментов (первый «выигрыш»)
и возникновение новых источников доходов от
этих инструментов, позволяющие уменьшить
уровень налоговой нагрузки в ряде значимых
отраслей, например путем снижения ставок НДФЛ
(второй «выигрыш») [19–22]. Остается неудовлетворительным состояние
законности в сфере обращения с отходами про-
изводства и потребления. МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ Органами государст-
венной власти и местного самоуправления не
в полной мере обеспечивается исполнение норма-
тивных актов в данной области, не принимаются Однако, базируясь на опыте реализации те-
ории «двойного выигрыша», России следует
принять за основу гипотезу «множественного www.accounting.fa.ru 26 МЕТОДИКИ И ПРАКТИЧЕСКИЙ ОПЫТ / METHODS AND PRACTICAL EXPERIENCE Множественный выигрыш /
Multiple‐win
Экология / Ecology
Снижение
негативного
воздействия /
Reducing harmful
effects
Сохранение
биоразнообразия /
Conservation of
biodiversity
Повышение качества
жизненного
пространства /
Improving quality of
living space
Ресурсы и
промышленность /
Resources and industry
Бережное отношение
к возобновляемым
ресурсам / Valuing
renewable resources
Экономия
невозобновляемых
ресурсов и
рециклинг / Saving
non‐renewable
resources and
recycling
Социально‐
экономические
показатели / Socio‐
economic indicators
Снижение затрат на
здравоохранение /
Reducing healthcare
costs
Повышение
энергоэффективности
энергопроизводительн
ости / Improving energy
efficiency and energy
productivity
Снижение ущерба от
бытовых отходов /
Reducing damage from
household waste
Повышение качества
и безопасности
товаров / Improving
the quality and safety
of goods
Рост занятости и создание
рабочих мест / Employment
growth
Снижение затрат на
природоохранные
мероприятия / Reducing
the cost of environmental
protection measures
Инновации /
Innovations
Увеличение инвестиций в
новые технологии /
Increased investment in
new technologies
Увеличение числа новых
технологий / An increase
in the number of new
technologies
Экономический рост /
Economic growth
Рис. / Fig. Элементы «множественного выигрыша» от использования фискальных инструментов
в природоохранной деятельности / Elements of the “multiple win” from the use of fiscal instruments
in environmental protection
Источник / Source: разработано А. А. Заворыкиным [18] / developed by A. A. Zavorykin [18]. Экономия
невозобновляемых
ресурсов и
рециклинг / Saving
non‐renewable
resources and
recycling Экономия
невозобновляемых
ресурсов и
рециклинг / Saving
non‐renewable
resources and
recycling Экономический рост /
Economic growth Повышение качества
и безопасности
товаров / Improving
the quality and safety
of goods Рост занятости и создание
рабочих мест / Employment
growth Снижение затрат на
природоохранные
мероприятия / Reducing
the cost of environmental
protection measures Рис. / Fig. Элементы «множественного выигрыша» от использования фискальных инструментов
в природоохранной деятельности / Elements of the “multiple win” from the use of fiscal instruments
in environmental protection
сточник / Source: разработано А. А. Заворыкиным [18] / developed by A. A. Zavorykin [18]. Fig. Элементы «множественного выигрыша» от использования фискальных инструментов
одоохранной деятельности / Elements of the “multiple win” from the use of fiscal instruments
in environmental protection Источник / Source: разработано А. А. Заворыкиным [18] / developed by A. A. Zavorykin [18]. МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ средства региональных бюджетов на их реали-
зацию), не соблюдались сроки внедрения новой
системы в этой области. Зачастую присвоение
статуса «региональных операторов», регулирова-
ние их деятельности и установление предельных
тарифов на услуги, утверждение инвестиционных меры по реализации внесенных в федеральное
законодательство изменений, внедрению меха-
низмов и нового подхода, обусловленных рефор-
мой отрасли. В ряде субъектов Российской Феде-
рации отсутствовали региональные программы
по обращению с отходами (или не были выделены меры по реализации внесенных в федеральное
законодательство изменений, внедрению меха-
низмов и нового подхода, обусловленных рефор-
мой отрасли. В ряде субъектов Российской Феде-
рации отсутствовали региональные программы
по обращению с отходами (или не были выделены Учет. Анализ. Аудит • Т. 8, № 6’2021 27 Д. А. Смирнов, А. А. Заворыкин и производственных программ по части обра-
щения с отходами проводились с нарушением
установленных требований. лированию экологических платежей, их систем-
ность, показатели значимости и целевой харак-
тер. Следовательно, необходимыми элементами
реформы станут: лированию экологических платежей, их систем-
ность, показатели значимости и целевой харак-
тер. Следовательно, необходимыми элементами
реформы станут: Подчеркнем дополнительно, что территори-
альными органами Росприроднадзора должным
образом не обеспечивались ни федеральный госу-
дарственный экологический надзор, ни лицензи-
рование и лицензионный контроль, ни взимание
платы за негативное воздействие на окружающую
среду. Зачастую не принимались исчерпывающие
меры по устранению выявленных нарушений
законодательства. • кодификация в рамках налогового зако-
нодательства значительного спектра платежей
в рамках ограниченного (двумя-тремя пункта-
ми) перечня фискальных инструментов, базовым
из которых должен стать экологический налог; • формирование отдельных элементов пла-
тежей на базе опыта реализации ранее приме-
нявшихся фискальных инструментов, а также
установление целевого характера расходования
получаемых средств; Стоит отметить, что эффективность мер под-
держки НДТ, как и системы фискальных инстру-
ментов природоохранной деятельности в целом,
неразрывно связана с открытостью экономиче-
ских субъектов в части информационного взаи-
модействия с налоговыми и иными контролиру-
ющими структурами. Существенную трудность
в данном вопросе может представлять не столько
нежелание предприятий делиться своими дости-
жениями в области энергоэффективности или
снижения негативного воздействия на окружа-
ющую среду, сколько необходимость раскрытия
нюансов технологий и процессов, внедренных на
производстве. В результате наблюдается стрем-
ление компаний сохранить в тайне информацию,
которая лежит в основе их конкурентоспособности
на рынке. Данный процесс фактически влечет за
собой низкую верифицируемость ряда показа-
телей, важных для стимулирования внедрения
НДТ и экологического налогообложения. В этой
связи при всестороннем рассмотрении вопроса
поддержки НДТ необходимо уделить внимание
цифровым информационным системам контроля
за исполнением экологических нормативов на
предприятиях. МЕТОДОЛОГИЯ И РЕЗУЛЬТАТЫ
ИССЛЕДОВАНИЯ • централизация администрирования значи-
мых фискальных экологических инструментов
в руках одного контрольного органа. При этом отдельное внимание должно быть
уделено использованию современных цифровых
технологий сбора и анализа данных об исполне-
нии экологических нормативов на предприятиях
в целях улучшения качества администрирования
действующих фискальных платежей и мер под-
держки НДТ. При этом отдельное внимание должно быть
уделено использованию современных цифровых
технологий сбора и анализа данных об исполне-
нии экологических нормативов на предприятиях
в целях улучшения качества администрирования
действующих фискальных платежей и мер под-
держки НДТ. Указанные меры предназначены прежде всего
для реализации в рамках деятельности Правитель-
ства Российской Федерации и способны органично
дополнить план мероприятий по повышению
качества состояния окружающей среды согла-
сно Указу Президента Российской Федерации
«О национальных целях и стратегических задачах
развития Российской Федерации на период до
2024 года» 20, в рамках которого был утвержден
национальный проект «Экология», направлен-
ный на улучшение экологической обстановки
в Российской Федерации и объединяющий 11 фе-
деральных проектов. ВЫВОДЫ 20 Указ Президента Российской Федерации от 07.05.2018
№ 204. «О национальных целях и стратегических задачах
развития Российской Федерации на период до 2024 года». URL: http://www.kremlin.ru/acts/bank/43027. Анализ действующей системы поддержки НДТ
в России позволяет сделать вывод о том, что
фактически отсутствуют: единая линия по регу- СПИСОК ИСТОЧНИКОВ 1. Пигу А. С. Экономическая теория благосостояния. М.: Прогресс; 1985. 512 с. 2. Samuelson P. Foundations of Economic Analysis. Harvard University Press; 1947:311–317. 3. Скобелев Д. О., Микаэльссон О., Бхимани Ш. Наилучшие доступные технологии в условиях между-
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welfare. Environmental and Resource Economics. 1998;2(12):137–150. Учет. Анализ. Аудит • Т. 8, № 6’2021 29 Д. А. Смирнов, А. А. Заворыкин REFERENCES Fiscal instruments in the sphere of regulation of environmental protection
and ecological, activity: foreign experience. Economics. Taxes. Law. 2016;6(9):122–128. (In Russ.). 21. Baumol W. J. and Oates W. E. The theory of environmental policy: Second edition. — Cambridge University
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Press; 1988. 299 p. 22. Bovenberg A. L., Van der Ploeg F. Consequences of environmental tax reform for unemployment and Press; 1988. 299 p. 22. Bovenberg A. L., Van der Ploeg F. Consequences of environmental tax reform for unemployment and
welfare. Environmental and Resource Economics. 1998;2(12):137–150. 22. Bovenberg A. L., Van der Ploeg F. Consequences of environmental tax reform for unemployment and
welfare. Environmental and Resource Economics. 1998;2(12):137–150. ИНФОРМАЦИЯ ОБ АВТОРАХ Денис Александрович Смирнов — доктор экономических наук, доцент, профессор Департамента
налогов и налогового администрирования Факультета налогов, аудита и бизнес-анализа, Финансо-
вый университет, Москва, Россия
DASmirnov@fa ru у
р
DASmirnov@fa.ru Алексей Алексеевич Заворыкин — соискатель, специалист по учебно-методической работе 1-й кате-
гории Департамента налогов и налогового администрирования Факультета налогов, аудита и биз-
нес-анализа, Финансовый университет, Москва, Россия AAZavorykin@fa.ru REFERENCES Disadvantages and prospects for improving the modern classification of fiscal
instruments of the economic mechanism of environmental protection. Russian Economic Internet journal. 2018;(4):42. (In Russ.). 14. Withana S., ten Brink P., Kretschmer B., Mazza L., Hjerp, P., Sauter R. Evaluation of environmental tax
reforms: international experiences, A report by the Institute for European Environmental Policy (IEEP)
for the State Secretariat for Economic Affairs (SECO) and the Federal Finance Administration (FFA) of
Switzerland. Final Report. Brussels; 2013. 73 р. 15. Charveriat C. and Holme C. European Green Deal Barometer 2021. Institute for European Environmental
Policy and GlobeScan, Brussels and Paris; 2021. 38 p. 16. Kopsieker L., Gerritsen E., Stainforth T., Lucic A., Costa Domingo G., Naumann S., Röschel L. and Davis
Mc. “Nature-based solutions and their socio-economic benefits for Europe’s recovery: Enhancing the
uptake of nature-based solutions across EU policies”. Policy briefing by the Institute for European
Environmental Policy (IEEP) and the Ecologic Institute; 2021. 23 p. 17. Alice Pirlot, Exploring the Impact of EU Law on Environmental Taxation. Research Handbook in European
Union Taxation Law. Edward Elgar Publishing: 2020:359–388. 18. Zavorykin A. A. The “multiple win” hypothesis from the use of fiscal instruments for environmental
protection. Russian Economic Internet magazine. 2018;(3):31 (In Russ.). 19. Zavorykin A. A., Smirnov D. A. Greening the Russian tax system: prerequisites, risks, opportunities. Priority areas of tax and customs-tariff policy of Russia under modern conditions. Collection of 19. Zavorykin A. A., Smirnov D. A. Greening the Russian tax system: prerequisites, risks, opportunities. Priority areas of tax and customs-tariff policy of Russia under modern conditions. Collection of www.accounting.fa.ru 30 Д. А. Смирнов, А. А. Заворыкин scientific articles by postgraduates and undergraduates of the Department of Tax Policy and Customs
and Tariff Regulation of the Financial University under the Government of the Russian Federation. Goncharenko L. I., Savina O. N., eds. Moscow: Dashkov and K; 2017:53–59. (In Russ.). scientific articles by postgraduates and undergraduates of the Department of Tax Policy and Customs
and Tariff Regulation of the Financial University under the Government of the Russian Federation. Goncharenko L. I., Savina O. N., eds. Moscow: Dashkov and K; 2017:53–59. (In Russ.). 20. Smirnov D. A., Zavorykin A. A. Fiscal instruments in the sphere of regulation of environmental protection
and ecological, activity: foreign experience. Economics. Taxes. Law. 2016;6(9):122–128. (In Russ.). 20. Smirnov D. A., Zavorykin A. A. Учет. Анализ. Аудит • Т. 8, № 6’2021 scientific articles by postgraduates and undergraduates of the Department of Tax Policy and Customs
and Tariff Regulation of the Financial University under the Government of the Russian Federation.
Goncharenko L. I., Savina O. N., eds. Moscow: Dashkov and K; 2017:53–59. (In Russ.). ABOUT THE AUTHORS Denis A. Smirnov — Dr. Sci. (Econ.), Professor, Associate Professor of the Department of Taxes and Tax
Administration of the Faculty of Taxes, Audit and Business Analysis, Financial University, Moscow, Russia
DASmirnov@fa.ru Denis A. Smirnov — Dr. Sci. (Econ.), Professor, Associate Professor of the Department of Taxes and Tax
Administration of the Faculty of Taxes, Audit and Business Analysis, Financial University, Moscow, Russia
DASmirnov@fa.ru Alexsey A. Zavorikin — postgraduate student, specialist of the 1st category in educational and
methodological work of the Department of Taxes and Tax Administration of the Faculty of Taxes, Audit
and Business Analysis, Financial University, Moscow, Russia
AAZavorykin@fa ru Alexsey A. Zavorikin — postgraduate student, specialist of the 1st category in educational and
methodological work of the Department of Taxes and Tax Administration of the Faculty of Taxes, Audit
and Business Analysis, Financial University, Moscow, Russia
AAZavorykin@fa.ru Статья поступила в редакцию 01.10.2020; после рецензирования 22.10.2020; принята к публикации
01.12.2021. Статья поступила в редакцию 01.10.2020; после рецензирования 22.10.2020; принята к публикации
01.12.2021. вторы прочитали и одобрили окончательный вариант рукописи. Авторы прочитали и одобрили окончательный вариант рукописи. у
The article was submitted on 01.10.2020; revised on 22.10.2020 and accepted for publication on 01.12.2021. The authors read and approved the final version of the manuscript. he authors read and approved the final version of the manuscript. The authors read and approved the final version of the manuscript. Учет. Анализ. Аудит • Т. 8, № 6’2021 Учет. Анализ. Аудит • Т. 8, № 6’2021
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First detection of foot-and-mouth disease virus O/Ind-2001d in Vietnam
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RESEARCH ARTICLE First detection of foot-and-mouth disease
virus O/Ind-2001d in Vietnam Le T. Vu1, Ngo T. Long1, Barbara Brito2,3, Carolina Stenfeldt2,3, Nguyen T. Phuong1, Bui
H. Hoang1, Steven J. Pauszek2, Ethan J. Hartwig2, George R. Smoliga2, Pham P. Vu1, Le T. V. Quang1, Vo V. Hung1, Nguyen D. Tho4, Pham V. Dong4, Phan Q. Minh4,
Miranda Bertram2,3, Ian H. Fish2,3, Luis L. Rodriguez2, Do H. Dung4*, Jonathan Arzt2* 1 Regional Animal Health Office No. 6, Department of Animal Health, Ministry of Agriculture and Rural
Development, Ho Chi Minh City, Vietnam, 2 Foreign Animal Disease Research Unit, Plum Island Animal
Disease Center, ARS, USDA, Orient Point, NY, United States of America, 3 Oak Ridge Institute for Science
and Education, PIADC Research Participation Program, Oak Ridge, TN, United States of America,
4 Department of Animal Health, Ministry of Agriculture and Rural Development, Hanoi, Vietnam a1111111111
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a1111111111 * Jonathan.Arzt@ars.usda.gov (JA); dung.dah@gmail.com (DHD) Editor: Ulrich Melcher, Oklahoma State University,
UNITED STATES Received: January 13, 2017
Accepted: April 17, 2017
Published: June 9, 2017 Received: January 13, 2017
Accepted: April 17, 2017
Published: June 9, 2017 Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose. The work is made available under the Creative
Commons CC0 public domain dedication. Data Availability Statement: Data are available on
GenBank with accession numbers KY399464,
KY399465, KY399466, and KY399468. OPEN ACCESS Citation: Vu LT, Long NT, Brito B, Stenfeldt C,
Phuong NT, Hoang BH, et al. (2017) First detection
of foot-and-mouth disease virus O/Ind-2001d in
Vietnam. PLoS ONE 12(6): e0177361. https://doi. org/10.1371/journal.pone.0177361 Editor: Ulrich Melcher, Oklahoma State University,
UNITED STATES Abstract In recent years, foot-and-mouth disease virus (FMDV) serotype O, topotype Middle East-
South Asia (ME-SA), lineage Ind-2001d has spread from the Indian subcontinent to the
Middle East, North Africa, and Southeast Asia. In the current report, we describe the first
detection of this lineage in Vietnam in May, 2015 in Đắk Noˆng province. Three subsequent
outbreaks caused by genetically related viruses occurred between May–October, 2015
after which the virus was not detected in clinical outbreaks for at least 15 subsequent
months. The observed outbreaks affected (in chronological order): cattle in Đắk Noˆng prov-
ince, pigs in Đắk Lắk province and Đắk Noˆng province, and cattle in Ninh Thuận province. The clinical syndromes associated with these outbreaks were consistent with typical FMD in
the affected species. Overall attack rate on affected premises was 0.85 in pigs and 0.93 in
cattle over the course of the outbreak. Amongst 378 pigs at risk on affected premises, 85
pigs died during the outbreaks; there were no deaths among cattle. The manner in which
FMDV/O/ME-SA/Ind-2001d was introduced into Vietnam remains undetermined; however,
movement of live cattle is the suspected route. This incursion has substantial implications
for epidemiology and control of FMD in Southeast Asia. * Jonathan.Arzt@ars.usda.gov (JA); dung.dah@gmail.com (DHD) FMDV O/Ind-2001d in Vietnam virus was first described in the 1980s in the Indian subcontinent and subsequently dissemi-
nated widely throughout all Southern Asia, the Middle East, and East and Southeast Asia, with
some incursions into South Africa and European countries [5]. In Asia there are three defined
virus pools: Pool 1 includes FMDV strains circulating within East Asia and Southeast Asia,
Pool 2 includes strains from Southern Asia or the Indian subcontinent (excluding Pakistan),
and Pool 3 includes viruses found within the Middle East and West Eurasia [6–8]. virus was first described in the 1980s in the Indian subcontinent and subsequently dissemi-
nated widely throughout all Southern Asia, the Middle East, and East and Southeast Asia, with
some incursions into South Africa and European countries [5]. In Asia there are three defined
virus pools: Pool 1 includes FMDV strains circulating within East Asia and Southeast Asia,
Pool 2 includes strains from Southern Asia or the Indian subcontinent (excluding Pakistan),
and Pool 3 includes viruses found within the Middle East and West Eurasia [6–8]. USDA, ARS Office of International Research
Programs and the Cooperative Biological
Engagement Program of the U.S. Department of
Defense, Defense Threat Reduction Agency. Barbara Brito, Carolina Stenfeldt, Ian Fish, and
Miranda Bertram are the recipients of a Plum
Island Animal Disease Center Research
Participation Program fellowship, administered by
the Oak Ridge Institute for Science and Education
(ORISE) through an interagency agreement
between the USDOE and USDA. None of the
funders had any role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Incidence and serotype-specificity of FMDV varies across East and Southeast Asian coun-
tries. Vietnam and surrounding countries have high reported disease incidence throughout
the year [8]. Three different lineages of serotype O have been circulating in Vietnam during
recent years; O/CATHAY, O/ME-SA/PanAsia, and O/SEA/Mya-98 [9, 10]. Additionally, sero-
type A lineage SEA-97 is endemic in Vietnam, while serotype Asia 1 has not been detected in
Vietnam since 2007 [2]. The O/ME-SA/Ind-2001 lineage of FMDV was detected and first defined in 2001 [11] and
has become the dominant lineage of serotype O within the Indian subcontinent since 2008
[12]. Other FMDV strains collected before the initial characterization of the lineage (between
1995–2002) in Jordan, Israel, Bahrain, Kuwait, Saudi Arabia, the United Arab Emirates
(UAE), Oman, and the Palestinian Autonomous Territories were later classified within the
Ind-2001 lineage [13]. Introduction Foot-and-mouth disease (FMD) is one of the most important diseases of livestock worldwide,
and is endemic in several countries in Asia and Africa [1, 2]. FMD affects animal production
at various levels including large commercial operations, individual household subsistence
units, and countries’ access to international markets [3]. Within the seven serotypes of FMD
virus (FMDV), the distribution of phylogenetically related viral strains and lineages demon-
strates that distinct geographical regions share the same viral lineages and that the spread of
those lineages beyond their defined regions only occurs on limited occasions [4]. However,
there are known exceptions to this trend including the intercontinental spread of FMDV sero-
type O topotype Middle East-South Asia (ME-SA) lineage PanAsia (O/ME-SA/PanAsia). This Funding: This research was funded in part by U.S. Department of Agriculture, Agricultural Research
Service CRIS Project 1940-32000-061-00D and
through an interagency agreement with the Science
and Technology Directorate of the U.S. Department
of Homeland Security under Award Number
HSHQDC-12-X-0060. Additional funding was
provided by the U.S. Department of State,
Biosecurity Engagement Program through the 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 Materials and methods
Permissions and ethics Permissions and ethics Competing interests: The authors have declared
that no competing interests exist. From 2008–2009, there were isolated introductions of FMDV O/ME-SA/Ind-2001 into
UAE and Iran [14]. There were no further (known) incursions of this lineage outside the
Indian subcontinent until 2013, when outbreaks in Libya and Saudi Arabia were reported [13,
15]. Additional outbreaks in Sri Lanka caused by this virus were reported in 2013 and 2014
[16]. In 2014 and 2015 O/ME-SA/Ind-2001 caused several outbreaks in Morocco, Algeria, and
Tunisia, as well as in the UAE and Bahrain [13, 17, 18]. The FMDV O/ME-SA/Ind-2001 lineage has been further classified as Ind-2001a, b, c and d
sublineages based upon phylogenetic analyses. All outbreaks outside of the Indian subconti-
nent that have involved the O/ME-SA/Ind-2001 lineage since 2013 have been caused by the
sublineage Ind-2001d [12]. In 2015, this lineage was detected for the first time in Southeast
Asia in cases reported from Vietnam and Laos [17]. Subsequent outbreaks of O/ME-SA/Ind-
2001 lineage occurred in Myanmar in 2015 and 2016 [19]. In Vietnam, cattle and buffalo are commonly vaccinated against FMDV serotype O twice
annually with varying vaccine formulations, based upon current knowledge of circulating
strains. Population-level coverage of vaccination, and thus herd immunity, is regionally vari-
able. Pigs and small ruminants are typically not vaccinated. When outbreaks occur, suppres-
sive vaccination is often deployed to affected and surrounding premises. Additional outbreak
response practices include regional restriction of animal movement and slaughter, disinfection
of premises, and establishment of temporary quarantine posts for enforcement until 21 days
from the last clinical case. The purpose of the current report is to document and provide basic descriptive characteri-
zation of the first known incursion of FMDV O/ME-SA/Ind-2001d sublineage into Vietnam. This information is critical for consideration of FMDV surveillance and control programs in
Vietnam, throughout Southeast Asia, and globally. Virus isolation Virus isolation was performed following a protocol previously described [20], with minor
modifications. In brief, approximately 0.5–1 g of tissue samples were mechanically disrupted
and resuspended in minimal essential media (Gibco-Invitrogen, Carlsbad, CA) containing 25
mM HEPES (Gibco-Invitrogen, Carlsbad, CA). Samples were clarified by centrifugation and
250 μl of sample suspensions were inoculated onto BHK-21 cell monolayers. After 1 h of
adsorption, 5 ml of media with 1% serum were added to the flasks. The occurrence of cyto-
pathic effect was observed within 48–72 hours post inoculation. Detection of FMDV Upon report of clinical cases of vesicular disease, provincial field veterinarians collected sam-
ples of vesicular tissues, which were transported on cold packs to the RAHO 6 facility for anal-
ysis. Upon arrival, samples were immediately processed and analyzed by virus isolation and
subsequent antigen ELISA for serotype confirmation. Permissions and ethics The field outbreak investigations described herein were conducted by federal staff of the
Regional Animal Health Office (RAHO) 6, Department of Animal Health (DAH) and provin-
cial Sub-DAH offices in Vietnam as part of their official duties. All cases described herein 2 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 FMDV O/Ind-2001d in Vietnam occurred spontaneously in domestic livestock with no experimental inoculation or treatment
of live animals. No animals were euthanized for the purpose of this study. Sample collection
was performed as part of routine field outbreak investigations; samples were subsequently
compiled for the sake of the current investigation. Therefore, ethics approval was not required
for the work presented herein. Field investigations Field teams were deployed to outbreak sites within 24–48 hours of notification from regional
sources. Upon arrival at outbreak premises, interviews of herd managers and animal owners
were performed. All animals on affected premises were examined and epithelial samples were
collected for virus characterization. Distinct FMDV isolates were obtained from five distinct
samples of the multiple field specimens collected. For each outbreak premise, “attack rate” was defined as: total number of new cases / popu-
lation at risk over the course of the outbreak. Similarly, “Case fatality proportion” was defined
as total number of deaths / number of diseased animals over the course of the outbreak. PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 FMDV antigen ELISA Clarified virus isolation supernatants were analyzed by FMDV serotype O antigen ELISA (kit
number R5605, The Pirbright Institute, UK) per manufacturer’s recommended procedure. This
kit is based on an indirect sandwich ELISA technique [21, 22]. In brief, rabbit antisera specific for
FMDV serotype O was adsorbed to polystyrene microwell plates. Coated plates were washed in
phosphate-buffered saline and 50 μl of clarified virus isolation supernatant or diluted control
reagents (provided in kit) were added to each well. All samples were analyzed in duplicate. Plates
were incubated for 1 hour at room temperature and were subsequently washed in phosphate-buff-
ered saline. Detection antibody consisting of guinea pig antisera with the same specificity as the
rabbit antisera used for trapping was added to the wells and plates were incubated for 1 hour at
35˚C. After an additional wash step, horseradish peroxidase-conjugated rabbit anti-guinea pig
immunoglobulin was added to wells and plates were incubated at 35˚C for 45 minutes. After
washing, substrate/chromogen solution consisting of ortho-phenylenediamine (3.3mM) and
hydrogen peroxide (4.4mM) were added to the wells. The color reaction was stopped by addition
of 1.25 M sulphuric acid after 15 minutes of incubation. Optical density values were read at 492
nm. Mean OD values >0.1 above background values were considered positive. PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 3 / 11 FMDV O/Ind-2001d in Vietnam Sequence acquisition and analysis Subsequent to confirmation of presence of FMDV by virus isolation and antigen ELISA,
RNA extraction and RT-PCR were conducted as previously described [5]. In brief, RNA was
extracted from tissue suspensions using silica membrane RNeasy spin columns (Qiagen, cata-
log number 74104) according to the manufacturer’s instructions. Three primer combinations
were used for the RT-PCR of serotype O isolates: FMDV-ARS4-F (ACCAACCTCCTTGATGTG
GCT) /EUR-2B52R (GACATGTCCTCCTGCATCTGGTTGAT), O-1C244F (GCAGCAAAACACA
TGTCAAACACCTT)/EUR-2B52R, and O-1C272F (TBGCRGNCTYGCCCAGTACTAC)/EUR-
2B52R. The following thermal profile was used: 42˚C for 30 min for reverse transcription;
94˚C for 5 min for denaturing; 35 cycles of 94˚C for 60 s for denaturing; 60˚C for 60 s for
primer annealing; and 72˚C for 90 s for extension; followed by a final extension of 72˚C for 5
min. RT-PCR amplicons were visualized on an agarose gel and purified. The purified FMDV
amplicons were processed through the 454 sequencing pipeline in three main steps: (1) Rapid
Library Preparation (Roche Diagnostics; GS FLX Titanium Rapid Library Preparation Kit;
Cat. No. 05608228001), (2) emPCR amplification (Roche Diagnostics; GS emPCR Kit III Cat. No. 04 891 392 001), and (3) sequencing on Genome Sequencer FLX (Roche Diagnostics), as
previously described [23]. The FMDV sequences from Vietnam which are described herein
have been submitted to GenBank (Table 1). Phylogenetic analysis A total of 99 FMDV reference sequences from ME-SA topotype, most belonging to the O/
ME-SA/Ind-2001 lineages, as well as the sequences collected from the outbreaks described
herein, were used to reconstruct a phylogeny using Bayesian phylogenetic methods. The analy-
sis was carried out in BEAST 1.8.2 using a lognormal uncorrelated relaxed clock and a Bayes-
ian skyline tree prior [24] with 200,000,000 iterations. Mixing and convergence was assessed
using Tracer 1.6 [25] making sure that all parameters reached >200 effective sample size. The
maximum clade credibility tree was annotated and graphed in Figtree 1.4 [26]. p
y
aCattle on surrounding farms had been recently vaccinated with FMDV-O. Cattle on surrounding farms had been recently vaccinated with FMDV O.
bSuppressive vaccination with FMDV-O was administered to surrounding cattle and buffalo. *Number of animals reported from the index farm only. *Number of animals reported from the index farm only.
aCattle on s rro nding farms had been recentl
accinated
ith FMDV O y
FMDV-O was administered to surrounding cattle and buffalo. mber of animals reported from the index farm only.
tle on surrounding farms had been recently vaccinated with FMDV-O. from the index farm only.
had been recently vaccinated with FMDV-O. https://doi.org/10.1371/journal.pone.0177361.t001 *Number of animals reported from the index farm only. *Number of animals reported from the index farm only.
aCattle on surrounding farms had been recently vaccinated with FMDV-O. Field investigations Outbreak 1 was reported to RAHO 6 on May 26th, 2015 as acute and rapid onset of vesicular
disease in cattle on a subsistence farm in Ðắk Noˆng province (Table 1). A single sample of Table 1. Outbreaks of FMDV-O in Vietnam during May–October 2015. Out-
break
Date
(2015)
Location
Virus Detected
(Genbank#)
No. Farms
Species
No. Animals
Affected
Total No. Animals
Attack
Rate
Case Fatality
Proportion
Vaccine
1
May 26
Đắk Noˆng
O/Ind-2001d
(KY399464)
2
Cattle
12*
15*
80%*
0*
Nonea
2
Sept 10
Đắk Lắk
O/Ind-2001d
(KY399465)
1
Pig
45
50
90%
16%
Nonea
3
Sept 28
Đắk Noˆng
O/Ind-2001d
(KY399466)
1
Pig
306
328
93%
25%
Ob
4
Oct 20
Ninh
Thuận
O/Ind-2001d
(KY399468)
17
Cattle
5*
5*
100%*
0*
Ob Table 1. Outbreaks of FMDV-O in Vietnam during May–October 2015. PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 4 / 11 FMDV O/Ind-2001d in Vietnam Fig 1. Clinical lesions during outbreaks of FMDV O/Ind-2001d in Vietnam. A) Outbreak 1, interdigital cleft lesion, front hoof of a cow. The
vesicle has ruptured and there is mild hemorrhage (lesion age is estimated as 2–3 days after clinical onset). B) Outbreak 3, intact vesicular lesions
(arrows) and sloughed epithelium (arrow heads), tongue of a pig. Some vesicles are still intact suggesting lesion age of approximately 1–2 days
after clinical onset). C) Outbreak 3, sloughed hooves on front feet of pig from same farm as animal shown in panel B. The sloughing and granulation
tissues suggests these lesions are approximately 4–5 days after clinical onset. Green coloration from animal-marking paint. D) Outbreak 4, dental
pad erosion and sloughed epithelium on tongue of a cow in Ninh Thuận province. Early re-epithelialization and yellow discoloration from secondary
bacterial contamination suggests these lesions are approximately 2–3 days after clinical onset. https://doi.org/10.1371/journal.pone.0177361.g001 Fig 1. Clinical lesions during outbreaks of FMDV O/Ind-2001d in Vietnam. A) Outbreak 1, interdigital cleft lesion, front hoof of a cow. The Fig 1. Clinical lesions during outbreaks of FMDV O/Ind-2001d in Vietnam. A) Outbreak 1, interdigital cleft lesion, front hoof of a cow. The
vesicle has ruptured and there is mild hemorrhage (lesion age is estimated as 2–3 days after clinical onset). B) Outbreak 3, intact vesicular lesions
(arrows) and sloughed epithelium (arrow heads), tongue of a pig. Some vesicles are still intact suggesting lesion age of approximately 1–2 days
after clinical onset). PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 Field investigations C) Outbreak 3, sloughed hooves on front feet of pig from same farm as animal shown in panel B. The sloughing and granulation
tissues suggests these lesions are approximately 4–5 days after clinical onset. Green coloration from animal-marking paint. D) Outbreak 4, dental
pad erosion and sloughed epithelium on tongue of a cow in Ninh Thuận province. Early re-epithelialization and yellow discoloration from secondary
bacterial contamination suggests these lesions are approximately 2–3 days after clinical onset. https://doi.org/10.1371/journal.pone.0177361.g001 https://doi.org/10.1371/journal.pone.0177361.g001 vesicular epithelium was submitted to RAHO 6 and confirmed to be FMDV serotype O by
virus isolation and antigen ELISA. On May 28th, 2015, a field outbreak investigation team was
deployed to the field site. The owner reported having recently purchased 14 cattle from Nghệ
An province. Over the course of the outbreak, 12/15 cattle (80%) on this farm developed clini-
cal signs of FMD (Fig 1). During the outbreak, the owner sold 3 of the affected cattle to a PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 5 / 11 FMDV O/Ind-2001d in Vietnam neighbor, whereby the outbreak spread to a second premise which was not further investigated
due to limited resources in the field. As cattle in the region had recently been vaccinated, no
suppressive vaccination was administered to surrounding farms. The outbreak did not spread
further. neighbor, whereby the outbreak spread to a second premise which was not further investigated
due to limited resources in the field. As cattle in the region had recently been vaccinated, no
suppressive vaccination was administered to surrounding farms. The outbreak did not spread
further. Outbreak 2 was reported to RAHO 6 on September 10th, 2015 as acute and rapid onset of
vesicular disease in pigs in Ðắk Lắk province and was confirmed to be FMDV serotype O by
antigen ELISA. The outbreak investigation team was deployed the following day and deter-
mined that 45/50 pigs (90%) had clinical signs of FMD. Over the course of the outbreak, 7 pigs
in the 12 week-old cohort died. Some pigs were observed to collapse and rapidly die with no
prodromal signs of cardiac involvement. As cattle in the region had recently been vaccinated,
no suppressive vaccination was administered to surrounding farms. The outbreak did not
spread further. Field investigations h Outbreak 3 was reported to RAHO 6 on September 28th, 2015 as acute and rapid onset of
febrile vesicular disease in pigs at a high intensity piggery in Ðắk Noˆng province. The cause of
the syndrome was determined to be FMDV serotype O by virus isolation and antigen ELISA. Across all age groups, 306/328 (93%) of pigs were affected. Overall case fatality proportion was
25% (78/306) with pigs within 3 age strata dying: Piglet (<8 weeks; 13%), Piglet (8–10 weeks;
20%), Fattening (>16 weeks; 45%). No breeding sows died during the outbreak despite 100%
attack rate in this group. Suppressive vaccination (FMDV-O) was delivered to cattle and buf-
falo on surrounding farms. Outbreak 4 was reported to RAHO 6 as vesicular disease with lameness in cattle in Ninh
Thuận province on October 20th, 2015. On October 22nd, 2015 FMDV-O was confirmed by
virus isolation and antigen ELISA. As of that date, 5/5 (100%) of cattle at the Outbreak 4 index
premise were affected and there were unconfirmed reports of additional cases at nearby farms. Suppressive vaccination with FMDV-O monovalent vaccine was immediately initiated in cat-
tle and buffalo throughout the region. Additional clinical cases continued for approximately
14 days affecting at least 17 premises. The precise extent of this outbreak is not known as the
speed and extent of spread surpassed the reserve capacity of field veterinarians. This fact, along
with the likelihood of under-reporting, confound precise accounting of affected premises and
incidence. Discussion In this report, we have described the first detected outbreaks caused by FMDV O/ME-SA/Ind-
2001 lineage in Vietnam. The specific sources of these outbreaks remain undetermined. How-
ever, the purported transfer of cattle from NghệAn to Ðắk Noˆng just before the first outbreak
is suggestive of the source of incursion. NghệAn borders Laos where outbreaks of FMDV/
Ind-2001d had been reported in June 2015 [28]. Although temporal and geographic associa-
tions between the outbreaks in Vietnam and Laos suggest that these events are epidemiologi-
cally related, the specific source and manner of introduction of FMDV/Ind-2001d to Vietnam
remains undetermined. The clinical, epidemiological, and pathological presentations of cattle and pigs infected with
these strains of FMDV/Ind-2001d were consistent with typical descriptions of FMD [29–31]. Although lesions have been described associated with abortions due to FMDV/Ind-2001d
[32], to our knowledge, no previous publication has demonstrated or described characteristics
of lesions in adult animals infected with viruses of this sublineage. Sheep and goats were not
observed to be affected in any of the outbreaks despite large numbers of these species present
within the affected regions. The lack of observation of clinical disease in unvaccinated goats
and sheep in close proximity to affected cattle and pigs suggests that small ruminants may be
unlikely to become infected or clinically affected by these viruses. However, this negative find-
ing is insufficient to make definitive conclusions about host range or species-specific clinical
presentation. Such hypotheses would be more definitively addressed by active surveillance of
small ruminants in the field or controlled exposure experiments in a containment laboratory
setting. There has been no additional detection of this lineage in Vietnam subsequent to the out-
breaks reported herein. The lack of further evidence of circulation or reintroduction of the O/
ME-SA/Ind-2001d lineage in Vietnam suggests that the outbreaks reported herein may repre-
sent one or more isolated introductions with limited expansion. However, subclinical infec-
tions and under-reporting of outbreaks commonly occur such that O/ME-SA/Ind-2001 may
be more prevalent than indicated by reported cases. In 2016, the O/ME-SA/Ind-2001d lineage was reported in Myanmar associated with an
apparently separate introduction of this specific lineage, without relation to the viruses found
in 2015 in Laos and Vietnam [19]. Additionally, samples collected recently in 2016 in Thailand
were also classified as FMDV O/ME-SA/Ind-2001d [16]. Sequence acquisition and phylogenetic analysis The phylogenetic tree reconstructed using FMDV VP1 protein coding segment reference
sequences demonstrated that all serotype O viruses collected from the 4 outbreaks belonged to
the O/ME-SA/Ind-2001d sublineage (Fig 2, S1 Fig). The estimated year to the most recent
common ancestor (MRCA) for the Vietnamese viruses was 2014 (95% high posterior density–
HPD = 2014–2015). These viruses formed a clade most closely related to O/ME-SA/Ind-2001
viruses previously reported from India, Nepal, Bangladesh and the UAE in 2013–2014, and
time to MRCA of the Vietnamese viruses and the closest reference viruses was estimated at
2012 (95%HPD 2011–2012). The second closest cluster that included sequences from Saudi
Arabia, India, Libya, collected in 2013, as well as a sequence collected from Morocco in 2015
had a MRCA estimated at 2011 (95%HPD 2010–2012) (Fig 2). Sequences collected from the 4 outbreaks in Vietnam were highly similar, differing by 1–3
and 0–2 nucleotides and amino acids, respectively, in their VP1 coding segment. Nucleotide
and amino acid differences between the first Vietnamese outbreak (O/Bovine/DakNong/15-
9595/RAHO6/2015) and selected sequences from the closest genetic cluster (Fig 2) KM921878
UAE/2/2014, KJ825804 IND298(634)/2013 were 12 nucleotides (1 amino acid) and 11 nucleo-
tides (2 amino acids), respectively (not shown). 6 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 FMDV O/Ind-2001d in Vietnam Fig 2. Maximum clade credibility tree depicting the phylogeny of FMDV serotype O ME-SA India-2001. Names of the viruses
collected in Vietnam are highlighted in red. Viruses from Vietnam are closely related to other Ind-2001 collected in India, Nepal and
Bangladesh collected in 2013 and 2014. O#1, #2, #3, and #4 correspond to outbreak numbers 1–4 described in text and Table 1. Collapsed clades (*) are expanded in full detail in S1 Fig. https://doi.org/10.1371/journal.pone.0177361.g002 Fig 2. Maximum clade credibility tree depicting the phylogeny of FMDV serotype O ME-SA India-2001. Names of the viruses
collected in Vietnam are highlighted in red. Viruses from Vietnam are closely related to other Ind-2001 collected in India, Nepal and
Bangladesh collected in 2013 and 2014. O#1, #2, #3, and #4 correspond to outbreak numbers 1–4 described in text and Table 1. Collapsed clades (*) are expanded in full detail in S1 Fig. Sequence acquisition and phylogenetic analysis https://doi.org/10.1371/journal.pone.0177361.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 7 / 11 FMDV O/Ind-2001d in Vietnam The mean substitution rate computed for the Ind-2001 lineage was 5.02 x10-3 (95%
HPD = 3.83x10-3, 6.25x10-3) substitutions per site per year in the VP1 protein-coding region,
similar to previous estimates of Ind-2001 and PanAsia lineages estimates [12, 27]. PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 Author Contributions Conceptualization: JA DHD LLR LTV. Resources: DHD JA LLR PVD. Resources: DHD JA LLR PVD. Software: PVD PQM IHF. Supervision: JA DHD PVD. Validation: JA DHD IHF. Validation: JA DHD IHF. Visualization: JA CS BB. Writing – original draft: JA BB CS. Writing – review & editing: JA BB CS IHF DHD. Discussion Continued monitoring of the varying, ongoing trends of
endemic FMDV in Southeast Asia will ultimately elucidate the significance of this lineage for
regional control and eradication of FMD. Supporting information S1 Fig. Maximum clade credibility tree depicting the phylogeny of FMDV serotype O
ME-SA India-2001. The tree indicates all sequences used for phylogenetic reconstruction, and
the 95% high posterior density of the nodes. This figure shows the details of the clades col-
lapsed in Fig 2. (TIFF) Discussion These recent outbreaks and contin-
ued detection of O/ME-SA/Ind-2001d in previously unaffected geographical regions suggests
continued incursions of this virus should be expected in Vietnam and other countries. Limited spread of the reported outbreaks also suggests that FMDV vaccines currently used
in Vietnam may be at least partially protective against O/ME-SA/Ind-2001d. Antigenic charac-
terization of viruses of this lineage has predicted sufficient cross-reactivity with the antigen O/
IND/R2/75 currently used in the Indian vaccines [12, 33]. However, antigenic matching of
recently detected O/ME-SA/Ind-2001 viruses to viruses used for vaccine production for use in
Southeast Asia is critical to ensure adequate protection. Substantial movements of large ruminants occur from the Indian Subcontinent into South-
east Asia in association with trade [34, 35]. However, even though the O/ME-SA/Ind-2001
virus has been the dominant FMDV lineage in the Indian Subcontinent since 2008, this
FMDV lineage was never detected in Southeast Asia before 2015. It is possible that undetected PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
June 9, 2017 8 / 11 FMDV O/Ind-2001d in Vietnam clinical and subclinical incursions may have occurred on previous occasions. Ongoing retro-
spective and prospective genetic characterizations of the lineage may clarify this possibility. clinical and subclinical incursions may have occurred on previous occasions. Ongoing retro-
spective and prospective genetic characterizations of the lineage may clarify this possibility. clinical and subclinical incursions may have occurred on previous occasions. Ongoing retro-
spective and prospective genetic characterizations of the lineage may clarify this possibility. The outbreaks reported herein represent the first confirmed incursion of FMDV/O/
ME-SA/Ind-2001 into Southeast Asia. It is still uncertain if this lineage will become a domi-
nant strain within Southeast Asia. Continued monitoring of the varying, ongoing trends of clinical and subclinical incursions may have occurred on previous occasions. Ongoing retro-
spective and prospective genetic characterizations of the lineage may clarify this possibility. The outbreaks reported herein represent the first confirmed incursion of FMDV/O/
ME-SA/Ind-2001 into Southeast Asia. It is still uncertain if this lineage will become a domi-
nant strain within Southeast Asia. Continued monitoring of the varying, ongoing trends of
endemic FMDV in Southeast Asia will ultimately elucidate the significance of this lineage for
regional control and eradication of FMD. The outbreaks reported herein represent the first confirmed incursion of FMDV/O/
ME-SA/Ind-2001 into Southeast Asia. It is still uncertain if this lineage will become a domi-
nant strain within Southeast Asia. PLOS ONE | https://doi.org/10.1371/journal.pone.0177361
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Análisis de perfiles de difracción de rayos X de una aleación Cu-8% en peso de Cr obtenida por medio de aleado mecánico
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Matéria
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cc-by
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Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009
http://www.materia.coppe.ufrj.br/sarra/artigos/artigo11077
ISSN 1517-7076
Análisis de perfiles de difracción de rayos X de una aleación Cu-8% en
peso de Cr obtenida por medio de aleado mecánico
AGUILAR, C.I; ROJAS, P.A.II; ORDOÑEZ, S.III; GUZMAN, D.IV
I
Instituto de Materiales y Procesos Termomecánicos - Facultad de Ciencias de la Ingeniería - Universidad
Austral de Chile – Av. General Lagos 2086 – casilla 567, Valdivia, Chile.
e-mail: ceaguilar@uach.cl
II
Escuela de Ingeniería Mecánica - Facultad de Ingeniería - Pontificia Universidad Católica de Valparaíso Av. Los Carrera 01567- Casilla 4059, Quilpué, Chile.
e-mail: paula.rojas.s@ucv.cl
III
Departamento de Ingeniería Metalúrgica - Facultad de Ingeniería - Universidad de Santiago de Chile - Av.
L. Bernardo O’Higgins 3363 – Casilla 1033, Santiago, Chile
e-mail: stella.ordonez@usach.cl
IV
Departamento de Metalurgia, Facultad de Ingeniería - Universidad de Atacama - Av. Copayapu 485 –
Casilla 240, Copiapó, Chile
e-mail: danny.guzman@uda.cl
RESUMEN
El presente trabajo estudia la evolución de la estructura de una mezcla de polvos Cu-8% peso de Cr,
aleada mecánicamente, por medio del análisis de perfiles de difracción de rayos X. Se encontró que al
aumentar el tiempo de molienda, el tamaño de cristalitas y la energía de falla de apilamiento disminuyeron, y
los valores de microdeformación y probabilidad de falla de apilamiento aumentaron. La distribución de
cristalitas se desplazo hacia valores menores de tamaño de cristalitas al incrementar el tiempo de molienda.
Después de 50 horas de molienda, los datos indicaron que se ha producido una solución sólida de cromo en
cobre.
Palabras claves: aleado mecánico, Cu-Cr, tamaño cristalita, falla apilamiento.
X-Ray profile analysis of Cu-8wt.%Cr processed by mechanical alloying
ABSTRACT
This work studies the evolution of the microstrucutre of a mechanically alloyed Cu-8 wt. % Cr
powder mixture by X-Ray profiles analysis. The process was carried out using a SPEX8000D upon mix with
stainless steels containers and balls, keeping a constant powder to balls weight ratio of 10. Increasing the
milling time, crystallite size and stacking fault energy decreased and microstrain and stacking fault
probability increased. The crystallite size distribution shifts to lower crystallite size values by increasing the
milling time. After a milling time of 50 hours, the data showed that a solid solution of chromium in copper
was produced.
Keywords: mechanical alloying, Cu-Cr, crystallite size, stacking fault.
1
INTRODUCCIÓN
Actualmente existe un gran interés en desarrollar nuevos materiales debido a los continuos avances
y exigencias tecnológicas. En este contexto, la atención ha sido en parte concentrada en sintetizar materiales
de alto rendimiento, tales como, intermetálicos, aleaciones metálicas amorfas y materiales nanocristalinos
(MNC) [1]. Los dos últimos materiales mencionados son de gran interés debido a que exhiben una inusual
combinación de propiedades como, resistencia, ductilidad, tenacidad a la fractura, resistencia a la corrosión y
propiedades magnéticas, en comparación con sus similares con tamaños de granos micrométricos. Los
materiales amorfos y nanocristalinos son obtenidos por medio de procesos de no equilibrio, tales como:
solidificación rápida [1], deposición física y química de vapor, y aleado mecánico (AM) [2]. Dentro de los
Autor Responsável: AGUILAR, C.
Data de envio: 11/04/2008
Data de aceite: 09/06/2009
AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
procesos señalados, el AM se ha posesionado como un método sencillo y versátil para desarrollar materiales
amorfos y nanocristalinos [2].
El AM se puede definir como una técnica de no equilibrio donde las partículas son sometidas a
severas deformaciones, repetidas fracturas y soldaduras en frío debido a las colisiones entre los medios de
molienda y/o con las paredes del contenedor. En partículas metálicas la intensa deformación plástica sufrida
por las partículas aumenta la densidad de defectos cristalinos, tales como, fallas de apilamiento, maclas,
dislocaciones, vacancias y bordes de grano, principalmente [3].
Dentro de las ventajes que presenta el AM se puede mencionar que puede ser empleado para
manufacturar aleaciones que son difíciles o imposibles de obtener por medio de los métodos convencionales
de fusión y colada, (se obtienen aleaciones por microforja), y también es posible extender los límites de
solubilidad al estado sólido en sistemas con entalpía de mezcla positiva [4, 5].
La estructura de los materiales procesados por AM puede ser adecuadamente estudiada y
caracterizada por medio del análisis de perfiles de difracción de rayos X, debido a que, posición y forma de
los perfiles de difracción están relacionados con los diferentes defectos cristalinos presentes en el material [6,
7]. Una completa información puede ser obtenida de la estructura de los materiales, tal como, tamaño de
cristalitas, microdeformación, probabilidad de falla de apilamiento, distribución de tamaño de cristalitas y
cambio de parámetro de red [6].
Por tanto, el objetivo del presente trabajo es caracterizar la estructura de la aleación Cu-8% en peso
de Cr obtenida a través de aleado mecánico de polvos elementales de Cu y Cr por medio del análisis de
perfiles de difracción de rayos X.
2
2.1
MATERIALES Y MÉTODOS
Materiales
Para la preparación de las aleaciones de Cu-8% peso Cr se utilizaron polvos de Cu con un tamaño de
partícula entre –88 +33 μm con una pureza de 99,9% y polvos de Cr con una granulometría de –43 μm con
pureza 99,0%. Las pruebas se realizaron en un molino de alta energía SPEX 8000D con una velocidad de
rotación de 1725 r.p.m. Las mezclas de polvos fueron cargadas en contenedores de acero inoxidable
(volumen de 25 ml), al interior de una cámara de guantes, Nitrogen Dry Box 850-NB con atmósfera
controlada de argón. Se utilizaron medios de molienda de acero inoxidable de 6.35 mm de diametro. Se uso 8
ml de alcohol para evitar la soldadura en frío. La razón de carga en peso bola/polvos utilizada fue 10. Los
tiempos de molienda a que se sometieron los polvos estuvieron entre 0,5 y 50 horas. Las difracciones de
rayos X se llevaron a cabo en un equipo Siemens D5000, con radiación CuKα, λ=0,15406 nm y con paso de
0,02º/s. El rango de barrido de (2θ) fue entre 40 y 100º. Los perfiles de difracción se ajustaron a una curva
tipo Lorentz.
2.2
Métodos
Los perfiles de difracción pueden ser representados en el espacio de Fourier según la expresión 1
[8].
+∞
⎧
s − s0 ⎞
s − s0 ⎫
⎛
f ( s ) = K ∑ ⎨ A(n) cos⎜ 2π L(
) ⎟ + B(n) sen(2π L(
))⎬
λ
λ
⎝
⎠
−∞ ⎩
⎭
(1)
Donde, s0 y s son el haz incidente y refractado de rayos X, respectivamente, L=na3 (distancia normal
a los planos reflectantes hkl), a3=λ/2(sen(θ2)-sen(θ1)), (θ2-θ1) el rango angular del perfil de difracción
medido, n el número armónico, λ longitud de onda utilizada, A(n), B(n) los coeficientes de Fourier y K una
constante propia de la difracción. Si el perfil de difracción es simétrico B(n)=0 [9]. A(n) es igual al producto
de A(n)SA(n)D, donde A(n)S esta relacionado con el tamaño de cristalitas y A(n)D con el valor de
microdeformación. Warren y Averbach [10] propusieron un método eficiente de separación de ambos efectos
en la segunda mitad del siglo pasado.
Ungár y Bórbely [11] propusieron una modificación al clásico método de Warren-Averbach que
considera la influencia de los factores de contraste (<C>) y la presencia de deformación anisotrópica para
separar ambos efectos. La ecuación propuesta por ellos se entrega en la ecuación 2.
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
ln A(L) ≅ −(L / d) − ρBL2 ln(
(
)
Re 2
) g < C > + QB2L4 ln(R1 / L)ln(R2 / L)(g2 < C >)2
L
(2)
Donde; d es el tamaño de cristalita, B=πb2/2 (b vector de Burger), ρ la densidad de dislocaciones,
Re el radio de acción de dislocaciones, g el vector de difracción (g=2senθ/λ), R1 y R2 parámetros auxiliares,
Q una constante, <C> el factor de contraste promedio. Los factores de contraste toman en cuenta la
deformación anisotrópica sufrida por los materiales y que es causada por la presencia de dislocaciones y
pueden ser encontrados en varios trabajos [12, 13]. La ecuación anterior se resuelve gráficamente
presentando lnA(L) versus g2<C> [11] y ajustando los datos a una curva parabólica.
Ungár y Bórbely [11] también propusieron una nueva metodología para la ecuación de WilliamsonHall, denominada Williamson-Hall modificada que toma en cuenta los efectos de deformación anisotrópica
por medio de los factores de contraste de dislocaciones, ecuación 3.
1/ 2
Δg =
0.9 ⎛ π A2b 2 ⎞
+⎜
⎟
D ⎝ 2 ⎠
⎛ π A ' b 2 ⎞ 1/ 2 2
⎟ Q ( g < C >)
⎝ 2 ⎠
ρ 1/ 2 ( g < C >1/ 2 ) + ⎜
(3)
Donde; Δg=cos(θ)(Δθ)/λ, D es el tamaño de cristalita y A, A’ son parámetros determinados por Re.
Las metodologías de Warren-Averbach y Williamson-Hall modificadas deben ser usadas en forma
complementaria para obtener toda la información para caracterizar la estructura de un material.
Se ha encontrado que la distribución de tamaño de cristalitas (ϕ) en materiales nanocristalinos sigue
una distribución log-normal [14, 15, 16] dada por la expresión 4, donde m y σ son constantes que describen
la media y varianza de la distribución, respectivamente.
1 ⎛ ln( x / m ) ⎞
⎟
σ
⎠
2
− ⎜
1
2⎝
ϕ( x ) =
e
1/ 2
( 2π ) xσ
(4)
La probabilidad de falla de apilamiento (α), puede ser determinada a partir del desplazamiento que
sufren los perfiles de difracción usando la ecuación 5 [10], donde θhkl es el ángulo de Bragg del plano hkl. Se
utilizan los ángulos correspondientes a los planos {111} y {200} porque presentan desplazamientos
contrarios en un difractograma de una estructura fcc, así se evitan errores introducidos por otras fuentes que
originan desplazamiento en los perfiles de difracción. Los sub índices cw y ann corresponden a los
especimenes deformados enfrío (muestra estudiada) y recocido (muestra patrón), respectivamente.
°
(2θ200 − 2θ111 )°cw − (2θ200 − 2θ111 )ann
=
−45 3α
π2
( tanθ200 + 1 / 2 tanθ111 )
(5)
Una dislocación perfecta puede disociarse en dos dislocaciones parciales con un área intrínseca de
falla entre ellas. Esta región cambia su secuencia de apilamiento, en el caso de cobre, desde fcc en el plano
{111} a hcp. La energía de falla de apilamiento (EFA) es la energía por unidad de área necesaria para
producir la falla. Un equilibrio se alcanza cuando las fuerzas repulsivas entre dislocaciones parciales son
balanceadas con la fuerza atractiva producto de la energía de EFA. Cuando EFA es baja, la falla ocurrirá
frecuentemente y el área fallada será extensa. Caso contrario, sucede cuando el valor de EFA es alto. El valor
de EFA puede ser estimado con la expresión propuesta por Reed y Schramm [17], ecuación 6.
γ=
K111ω0G111a0
π 3
A−0.37
2
< ε 50
>111
α
(6)
Donde, K111ω0 ha sido determinado como 6,6 +/- 0,1 [17], G111 es el modulo de corte en el plano
{111}, <ε502>111 es la microdeformación promediada sobre una distancia de 50 A, en la dirección <111>, α la
probabilidad de falla de apilamiento, a0 de parámetro de red y A la constante de anisotropía de Zener
(A=2c44/(c11-c12), donde, cij son los módulos de elasticidad, (para cobre son c11=168.4 GPa, c12=121.4 GPa y
c44= 75.4 GPa [18]).
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
3
RESULTADOS Y DISCUSION
El patrón de difracción de rayos X completo de la aleación estudiada fue medido para un rango
angular de 2θ entre 40 y 100°, pero sólo se muestra la parte del patrón de difracción de rayos X con los picos
más intensos de Cu y Cr en la Figura 1 (para un rango de 2θ entre 41.5 y 45.5°). Los difractogramas
presentan el comportamiento típico de metales procesados mediante AM [2, 19, 20, 21], aumento del ancho
medio, desplazamiento de los perfiles de difracción y desaparición de líneas de difracción principales del
elemento soluto. Se ha puesto como referencia el patrón de difracción de cobre sin moler a modo de
comparación. El pico con la intensidad más fuerte de cromo es {110}, el cual difracta a 2θ= 44.39° [22].
Desde la Figura 1 se observa que el pico más intenso de cromo levemente se puede apreciar hasta 8 horas de
molienda, desapareciendo completamente a 50 horas de molienda. Este hecho se puede asumir que átomos de
cromo han entrado en solución sólida de cobre.
El tamaño de cristalita de cobre puro inicial se calculó en 440 nm. Desde la Figura 2 se observa que
en la medida del aumento del tiempo de molienda, el tamaño de cristalitas disminuye, alcanzando un valor de
7 nm para 50 horas de molienda, indicando que la aleación ha alcanzado una estructura manométrica. Para
cobre puro procesado por AM se ha reportado un valor de tamaño de cristalita mínimo de 14 nm [23]. El
valor logrado en el presente trabajo se explica debido a que, cuando los átomos de soluto entran en solución
sólida, las aleaciones son más duras y resistentes en comparación con el metal puro en que se basan, así hay
un aumento en la tendencia a la fragmentación, favoreciendo la disminución en el tamaño de cristalita.
Similares resultados obtuvieron Eckert et al. [24], quienes reportaron para la molienda de polvos de Cu y Fe
que el tamaño de cristalita mínimo decrece en función del contenido de Fe.
La evolución de la microdeformación cuadrática media a una distancia de 50 Å <ε250>1/2, se entrega
en la Figura 3. Se observa que los valores de <ε250>1/2 aumentan cuando se incrementa el tiempo de molienda,
lo que está en acuerdo con lo reportado en otros trabajos de aleado mecánico [2, 25]. A 50 horas de molienda
los valores de <ε250>1/2 presentan una leve disminución, lo cual se explica por el hecho que el tamaño de
cristalita ha alcanzado un valor de saturación. Un mayor tiempo de molienda no produce más dislocaciones,
debido a la dificultad de generar más dislocaciones en tamaños de granos muy pequeños (en materiales
nanoestructurados el tamaño de cristalita coincide con el tamaño de grano [26]). En este estado, un aumento
del tiempo de molienda no generará más dislocaciones, sino que las dislocaciones presentes serán arregladas
y algunas de ellas serán aniquiladas, decreciendo el valor de la microdeformación, como fue reportado de
igual forma por Lucks et al. [27].
Figura 1: Patrón de difracción de rayos X aleación Cu-8% en peso de Cr en función del tiempo de molienda.
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
Figura 2: Evolución del tamaño de cristalitas en función del tiempo de molienda.
Figura 3: Evolución de la microdeformación en función del tiempo de molienda.
Se encontró que la probabilidad de falla (α) de apilamiento incrementa cuando aumenta el tiempo de
molienda, Figura 4. Los valores calculados de α son similares a los reportados en la literatura, la Tabla 1
muestra una comparación entre los valores obtenidos y los reportados en literatura. Por ejemplo, Gayle y
Biancaniello [28] entregaron valores del orden de α ≈7.5x10-3 para una aleación de cobre con 5 % en peso de
cobalto procesada por AM. También observaron el incremento de los valores de α en la medida del
incremento del porcentaje de cobalto en la aleación. Kapoor et al. [29] verificaron el aumento de α para
aleaciones de Cu-1 % en peso de Cr-0.1 % en peso Zr, reportando un valor máximo de α ≈5.6x10-3. Sahu et
al. [30] reportaron valores para α de 7.05x10-3 para aleaciones de Cu-15 % en peso Ni-5 % en peso Sn.
Finalmente se puede mencionar que en los primeros estudios de mediciones de α por medio del análisis de
perfiles de difracción, se reportaron valores del orden de 10-2-10-3 en aleaciones de cobre [31]. El valor
reciproco de probabilidad de falla de apilamiento, (1/α=número de planos cristalinos), representa una falla de
apilamiento sobre n planos cristalinos de átomos en el plano {111} en la dirección <111> en estructuras
cristalinas fcc [10]. Por tanto, desde la Figura 4 se puede calcular que para 0.5 y 50 horas de molienda, el
número de planos cristalinos son alrededor de 400 y 50, respectivamente. Se puede decir que el aumento de
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
los valores de <ε250>1/2 y α (disminución de la distancia de planos entre fallas de apilamiento) muestran que
los polvos han sufrido una fuerte deformación plástica producto de las continuas colisiones de las bolas.
Tabla 1: Comparación de valores de probabilidad de falla de apilamiento calculados en este trabajo con
valores reportados en literatura.
Valor de α
-3
Autor
7.5x10
(Cu-5%
en peso Co)
Gayle
y
Biancaniello [28]
5.6x10-3 (Cu-1 %
en peso de Cr-0.1
% en peso Zr)
Kapoor et al. [29]
-3
7.05x10
(Cu-15
% en peso Ni-5 %
en peso Sn)
Sahu et al. [30]
-3
8x10 (Cu-2% en
peso Cr-6% en
peso de Mo)
Aguilar et al. [32]
Con los valores de <ε250>1/2 y α y usando la expresión 6 se puede calcular la energía de falla de
apilamiento (EFA). Los valores encontrados para la aleación en estudio disminuyen en función del tiempo
hasta valores cercanos a 20 mJ/m2, Figura 5. Similares resultados han sido reportados para aleaciones
binarias de Cu-x % en peso de Cr (x=1 y 3) [25] y en aleaciones ternarias de Cu-2% en peso de Cr-6 % en
peso de Mo procesadas por AM. La disminución de la EFA en función del tiempo de molienda apoya el
hecho que átomos de cromo han entrado en solución sólida en cobre, pues en estructuras cristalinas tipo fcc
la EFA disminuye cuando los átomos de soluto entran en solución [33]. Ha sido demostrado que EFA ejerce
una importante influencia en la estructura y propiedades mecánicas de metales y aleaciones con estructuras
fcc [34]. Para materiales con bajos valores de EFA la separación entre dislocaciones parciales es grande y la
fuerza requerida para la recombinación es alta. En forma contraria, cuando γ es alta, la fuerza necesaria para
recombinar es pequeña. La disminución de EFA trae consigo dos aspectos importantes: por una parte, el área
fallada entre planos compactos es mayor, debido a que las dislocaciones parciales están más separadas y por
otra parte, las dislocaciones tienden a disociarse porque el deslizamiento cruzado se ve restringido, producto
de los obstáculos al movimiento de las dislocaciones que permanecen con un alto nivel de tensión, de
acuerdo a lo esperado [18]. Así, por tanto, el coeficiente de endurecimiento se incrementa.
Figura 4: Evolución de los valores de α en función del tiempo de molienda.
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
Figura 5: Evolución de la energía de falla de apilamiento.
La tendencia general que se observa en la distribución de tamaño de cristalitas es un desplazamiento
de las curvas hacia menores valores de tamaño de cristalitas cuando el tiempo de molienda aumenta, como se
observa en la Figura 6. Por tanto, se desprende que en la medida que el tiempo de molienda aumenta, la
estructura de la aleación se refina, alcanzando un carácter nanométrico a 50 horas de molienda como fue
mencionado anteriormente.
Figura 6: Función distribución de tamaño de cristalitas en función del tiempo de molienda.
El parámetro de red del cobre fue calculado por medio del método propuesto por Cohen [35]. Se ha
puesto el eje del tiempo en escala logarítmica y a objeto de lograr una mejor comparación, se ha incluido en
la gráfica el valor del parámetro de red de cobre puro sin moler, señalado con un asterisco a un tiempo de 10-4
horas. Los resultados muestran una disminución del parámetro de red del cobre en función del tiempo de
molienda. En una primera aproximación se podría pensar en usar la ley de Vegard para estimar la cantidad de
cromo que ha entrado en solución, pero su uso genera incertidumbres debido a que la ley de Vegard
considera sólo una variación lineal del volumen de la celda unitaria con respecto a la composición. En otras
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AGUILAR, C.; ROJAS, P.A.; ORDOÑEZ, S.; GUZMAN, D; Revista Matéria, v. 14, n. 2, pp. 777 – 786, 2009.
palabras al usar la ley de Vegard se considera un modelo de solución ideal entre los componentes. Los radios
atómicos del cobre (0.128 nm) [36] y del cromo (0.127 nm) [36] son similares, por tanto, cuando átomos de
cromo entran en solución sólida, lo hacen de forma substitucional. Por otra parte el radio atómico de cromo
es levemente menor con respecto al radio atómico de cobre, por eso, al entrar en solución los átomos de
cromo, el parámetro de red de cobre experimenta una disminución, como se observa claramente en la Figura
7.
Figura 7: Variación parámetro de red del cobre en función del tiempo de molienda.
4
CONCLUSIONES
Al aumentar el tiempo de molienda aumenta la densidad de defectos cristalinos, tales como,
dislocaciones y fallas de apilamiento y microdeformación.
El parámetro de red de cobre y la energía de falla de apilamiento disminuyen cuando se incrementa
el tiempo de molienda. Estos hechos pueden ser tomados como una prueba que átomos de cromo han entrado
en solución sólida en cobre.
Se verifica un refinamiento de la estructura hasta alcanzar niveles nanométricos a 50 horas de
molienda.
5
AGRADECIMIENTOS
Los autores desean agradecer el apoyo económico a CONICYT, Fondo de Desarrollo Científico y
Tecnológico de Chile, FONDECYT, proyecto Nº 11070052.
6
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Drosophila CENP-A Mutations Cause a BubR1- Dependent Early Mitotic Delay without Normal Localization of Kinetochore Components
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PLOS genetics
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cc-by
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Drosophila CENP-A Mutations Cause a BubR1-
Dependent Early Mitotic Delay without Normal
Localization of Kinetochore Components Michael D. Blower1, Tanya Daigle2, Thom Kaufman3, Gary H. Karpen1,4*
1 Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America, 2 Department of Anesthesiology, University of
Washington, Seattle, Washington, United States of America, 3 Department of Biology, Indiana University, Bloomington, Indiana, United States of America, 4 Department of
Genome Biology, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America Michael D. Blower1, Tanya Daigle2, Thom Kaufman3, Gary H. Karpen1,4*
1 Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America, 2 Department of Anesthesiology, University of
Washington, Seattle, Washington, United States of America, 3 Department of Biology, Indiana University, Bloomington, Indiana, United States of America, 4 Department of
Genome Biology, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome
movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which
delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic
spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a
functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null
mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the
DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component
BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle
disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the
absence of sustained kinetochore localization. Citation: Blower MD, Daigle T, Kaufman T, Karpen GH (2006) Drosophila CENP-A mutations cause a BubR1-dependent early mitot
kinetochore components. PLoS Genet 2(7): e110. DOI: 10.1371/journal.pgen.0020110 Citation: Blower MD, Daigle T, Kaufman T, Karpen GH (2006) Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of
kinetochore components. PLoS Genet 2(7): e110. DOI: 10.1371/journal.pgen.0020110 outer kinetochore proteins, including some SAC components
[9–11]. In contrast, disruptions of components of the SAC
result in premature entry into anaphase, rather than mitotic
arrest or delay. Drosophila CENP-A Mutations Cause a BubR1-
Dependent Early Mitotic Delay without Normal
Localization of Kinetochore Components These results suggest that continuous
kinetochore localization of SAC components may not be
necessary to signal the cell to delay anaphase onset. However,
incomplete depletion of the inner kinetochore proteins could
produce partially functional kinetochores in which some
outer proteins localized properly, while others were mis-
localized [8,11,12]. A partially dysfunctional kinetochore
could recruit sufficient amounts of SAC proteins to generate
an effective checkpoint signal, consistent with a requirement
for SAC component recruitment to kinetochores. PLoS Genetics | www.plosgenetics.org Introduction Proper kinetochore assembly and function is essential for
the faithful transmission of chromosomes during both mitosis
and meiosis. One critical function of the kinetochore is to
serve as the site of the mitotic spindle attachment checkpoint
(SAC), which monitors kinetochore microtubule attachment
prior to anaphase onset [1,2]. It is hypothesized that the SAC
monitors the attachment of kinetochore microtubules during
prometaphase and metaphase, and inhibits anaphase pro-
gression until all chromosomes have achieved bipolar spindle
attachment. The current model for SAC function suggests
that unattached kinetochores recruit checkpoint proteins
(such as components of the MAD, BUB, and ZW10/ROD
protein complexes), and that these proteins are modified by
unattached kinetochores to generate a diffusible signal that
delays the onset of anaphase. It has recently been demon-
strated that defects in SAC function result in organismal
lethality and dominant haploinsufficiency defects. Haploin-
sufficiency for mouse MAD2, BUB3, or RAE1 results in
elevated rates of chromosome missegregation, defects in SAC
function, and a predisposition to cancer [3–5], demonstrating
the fundamental importance of this checkpoint. A key component of the inner kinetochore is the
centromere-specific histone H3-like protein CENP-A [13]. Several recent studies have demonstrated that CENP-A
proteins are present in all eukaryotes, and that these proteins
are essential for both cell and organismal viability [14–20]. CENP-A proteins replace both copies of histone H3 in The role of kinetochore localization of SAC components in
the generation of the anaphase delay [6] has come into
question recently with the published disruption of several
inner kinetochore proteins. Disruption of Nuf2 or Ndc80/
Hec1 in human cells results in a mitotic arrest, despite the
fact that several outer kinetochore components (including
Mad2) are unable to sustain kinetochore localization [7,8]. Similarly, disruption of human or chicken CENP-H or CENP-
I (the homolog of Schizosaccharomyces pombe Mis6) arrests cells
in mitosis for hours, despite the mislocalization of a variety of A previous version of this article appeared as an Early Online Release on June 5,
2006 (DOI: 10.1371/journal.pgen.0020110.eor). DOI: 10.1371/journal.pgen.0020110 Copyright: 2006 Blower 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. Abbreviations: SAC, spindle attachment checkpoint Abbreviations: SAC, spindle attachment checkpoint * To whom correspondence should be addressed. E-mail: karpen@fruitfly.org * To whom correspondence should be addressed. Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of
America Received November 9, 2005; Accepted May 31, 2006; Published July 14, 2006 Synopsis CENP-A
proteins are at or near the top of the kinetochore assembly
pathway, and are required for the localization of nearly all
other kinetochore proteins examined to date, including all
tested SAC components [13]. Therefore, cells lacking CENP-A
would be expected to contain chromosomes with severely
compromised kinetochores, which would be incapable of
generating the SAC signal. In later stages of development (stages 13–15), high levels of
CID staining were observed in heterozygous siblings, whereas
in cid homozygous mutant embryos, most cells in mitotically
active tissues had no visible CID signal (Figure 1C and 1D). The PROD protein binds a satellite DNA near the Chromo-
somes 2 and 3 centromeres [28], and its localization is not
dependent on the presence of CID [17]. Comparison of the
levels of CID and PROD staining in homozygous mutant and
heterozygous control embryos suggest that approximately
90%–100% of CID was depleted in stage 15 cid mutants
(Figure S1, see Materials and Methods). Thus, some cells
retain small amounts of maternal CID, and these alleles
behave as nulls with respect to functional zygotic protein, as
predicted from the early stop codons present in the
mutations. Here we report that mutations in the Drosophila CENP-A
family member (CID, for ‘‘Centromere Identifier’’) [24] result
in an early mitotic delay. Furthermore, cid mutants have an
intact SAC response to microtubule disruption despite the
absence of kinetochore localization of SAC components ROD
and BUBR1. We present data that suggest that the DNA
damage/repair checkpoint is not responsible for the CID-
mediated early mitotic delay. In contrast, the mitotic delay of
cid mutants is partially suppressed by mutation of the SAC
component bubr1. We discuss models for the role of SAC
proteins in monitoring aspects of kinetochore assembly early
in mitosis. Homozygous cid null embryos displayed few defects
associated with gross morphological patterning or develop-
ment (unpublished data). However, defects were associated
with the organization of the developing nervous tissue,
consistent with the fact that few other cell types are actively
dividing in later stage embryos, and that the most severe
defects are also associated with the nervous tissue in other
mitotic mutants that die during embryogenesis [29]. These
later stage, terminal embryos displayed a high degree of
disorganization of the developing nervous tissue, with
obvious micronuclei, large presumably polyploid nuclei, very
few true metaphase plates, and few anaphases and telophases. Synopsis Synopsis Normal inheritance of genetic traits from one cell or organismal
generation to the next depends on accurate chromosome
replication and segregation. Defective chromosome segregation is
associated with birth defects and cancer. The centromere is a single
site on the chromosome that is responsible for assembling the
kinetochore, which mediates chromosome attachment to the
microtubule spindle and all chromosome movements. In addition,
the spindle assembly checkpoint (SAC) ensures normal inheritance
by delaying entry into anaphase when chromosome–spindle
attachments are defective. Previous studies suggested that SAC
function required kinetochore localization of key components. This
study shows that elimination of a centromere-specific histone (CID)
results in an early mitotic delay. Although this delay occurs earlier
than the established time of SAC function (at the metaphase–
anaphase transition), it depends on the presence of an essential SAC
protein (BUBR1). Furthermore, the CID-mediated early mitotic delay
occurs in the absence of kinetochore formation or localization of key
SAC proteins. These results suggest that the fidelity of kinetochore–
microtubule attachment is also monitored early in mitosis, and in
the absence of kinetochore formation and localization of SAC
components. To examine the phenotypic consequences of cid disruption,
crosses were made between parents heterozygous for a cid
mutation and a balancer that contained an ElaV-LacZ fusion
construct, which is expressed in the developing nervous
system [25]. We collected embryos from these crosses and
stained them for CID, LacZ, histone H3 phosphorylation at
serine 10 (PH3) [26], and DNA (DAPI). cid null and
heterozygous embryos were unambiguously distinguished by
the absence or presence (respectively) of ElaV-LacZ expres-
sion. cid null mutant embryos died around stage 15 of
embryogenesis, and displayed a phenotypic series that
correlated with the temporal disappearance of maternal
CID protein and the absence of newly synthesized zygotic
protein. At embryonic stages 9–10, cid null embryos displayed
lagging chromosomes during anaphase and unresolved
chromatin bridges during telophase, which were not observed
in heterozygous controls (Figure 1A and 1B). These pheno-
types are the result of partial loss of CID protein; staining
with anti-CID antibodies demonstrated that maternally
derived CID is still present in stage 9–10 embryos, albeit at
reduced levels (Figure 1A and 1B). Lagging chromosomes and
chromatin bridges are entirely consistent with the pheno-
types we observed after partial disruption of CID by RNAi or
antibody injection [17,27]. centromeric nucleosomes, and physically and genetically
interact with all other core histones [14,21–23]. Synopsis The overall nuclear density was much lower in the cid mutants
(;1/2 of heterozygous controls), which is also consistent with
the aneuploidy that results from failures in chromosome
segregation and cell division. These phenotypic character-
istics are very similar to a recently described mutation in
Drosophila CENP-C [30], suggesting that these defects result
from disruption of the inner kinetochore. PLoS Genetics | www.plosgenetics.org Introduction E-mail: karpen@fruitfly.org 1025 July 2006 | Volume 2 | Issue 7 | e110 cid-Mediated Mitotic Delay Requires SAC unpublished observations). All of these alleles are lethal when
homozygous, in trans-heterozygous combinations, and over a
deficiency for the region (unpublished data); thus, the cid gene
is essential for Drosophila development. Results CID Null Mutants Display Embryonic Lethality CID Null Mutants Display Embryonic Lethality
In a previous study, we found that anti-CID antibody
injections into syncitial embryos resulted in phenotypes
expected for kinetochore disruption (failure to congress in
prometaphase, metaphase arrest, and anaphase segregation
defects), but also produced unusual phenotypes (interphase
and prophase arrests). However, it was unclear if these
phenotypes were the consequence of loss of CID function, an
artifact of antibody binding to CID, or a consequence of the
specialized nature of the syncitial nuclear divisions. There-
fore, we examined the phenotypic consequences of cid null
mutations in Drosophila embryos. The alleles examined were
T11–2 (Q51 to stop), T12–1 (Q83 to stop), T21–3 (Q94 to
stop), and T22–4 (Q102 to stop) (J. Cecil and T. Kaufman, PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 1026 cid-Mediated Mitotic Delay Requires SAC Figure 2. cid Null Mutants Exhibit a G2/Prophase Delay. Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes. CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different
stages of development were monitored to evaluate mitotic progression
and segregation defects. (A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic
defects and robust CID staining at kinetochores (inset). (B) cid null animals (trans-heterozygous for different cid alleles, see
Materials and Methods) exhibited lagging chromosomes during ana-
phase. Some CID staining was still visible at this stage, demonstrating
that these phenotypes resulted from partial loss of CID function, due to
the presence of maternal CID protein. (C) cid/CyO stage 14–15 embryos show normal mitotic progression and
normal CID staining at kinetochores (inset). (D) cid null animals exhibited an elevated mitotic index, lower nuclear
density, and little detectable CID staining in some cells at stage 14–15. The strong depletion of CID staining suggests that these phenotypes are
the result of complete loss of zygotic cid function. cid null animals have a
large number of presumably polyploidy cells (inset) suggesting high
levels of aneuploidy due to repeated failures in cell division. Scale bar Figure 2. cid Null Mutants Exhibit a G2/Prophase Delay. Cell cycle progression was monitored in cid/CyO and cid/cid embryos by
staining for PH3, cyclin A, cyclin B, and tubulin. (A) Schematic diagram of the appearance and destruction of various cell
cycle regulatory factors and markers. ANA, anaphase; META, metaphase;
PRO, prophase; PRO-META, prometaphase; TELO, telophase. Results (B) cid null animals (trans-heterozygous for different cid alleles, see
Materials and Methods) exhibited lagging chromosomes during ana-
phase. Some CID staining was still visible at this stage, demonstrating
that these phenotypes resulted from partial loss of CID function, due to
the presence of maternal CID protein. p
p
p
(C) cid/cid animals had a 2-fold higher number of cyclin A and B positive
cells than cid/cid controls. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g002 p
p
(C) cid/CyO stage 14–15 embryos show normal mitotic progression and
normal CID staining at kinetochores (inset). (D) cid null animals exhibited an elevated mitotic index, lower nuclear
density, and little detectable CID staining in some cells at stage 14–15. The strong depletion of CID staining suggests that these phenotypes are
the result of complete loss of zygotic cid function. cid null animals have a
large number of presumably polyploidy cells (inset) suggesting high
levels of aneuploidy due to repeated failures in cell division. Scale bar
indicates 15 lm. in prophase and prometaphase, as judged by chromosome
and spindle morphology (Figure 2B). Third, very few cells
progressed to anaphase in cid mutants, suggesting that cid
mutants were delayed prior to the metaphase–anaphase
transition (Figure 2B). Thus, complete depletion of CID in
embryos results in a mitotic delay, predominantly in
prophase and prometaphase. l
DOI: 10.1371/journal.pgen.0020110.g001 PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 Results (B) cid/cid animals displayed an elevated mitotic index, and an increased
number of cells in prophase and prometaphase, compared to cid/CyO
controls. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM,
prometaphase; T, telophase. (C) cid/cid animals had a 2-fold higher number of cyclin A and B positive
cells than cid/cid controls. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g002 Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes. CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different
stages of development were monitored to evaluate mitotic progression
and segregation defects. (A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic Figure 2. cid Null Mutants Exhibit a G2/Prophase Delay. Cell cycle progression was monitored in cid/CyO and cid/cid embryos by
staining for PH3, cyclin A, cyclin B, and tubulin. Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes. CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different
stages of development were monitored to evaluate mitotic progression
and segregation defects. (A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic
defects and robust CID staining at kinetochores (inset). Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes. CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different
stages of development were monitored to evaluate mitotic progression
and segregation defects. Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes. CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different
stages of development were monitored to evaluate mitotic progression
and segregation defects. Cell cycle progression was monitored in cid/CyO and cid/cid embryos by
staining for PH3, cyclin A, cyclin B, and tubulin. (A) Schematic diagram of the appearance and destruction of various cell
cycle regulatory factors and markers. ANA, anaphase; META, metaphase;
PRO, prophase; PRO-META, prometaphase; TELO, telophase. (B) cid/cid animals displayed an elevated mitotic index, and an increased
number of cells in prophase and prometaphase, compared to cid/CyO
controls. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM,
prometaphase; T, telophase. (C) cid/cid animals had a 2-fold higher number of cyclin A and B positive
cells than cid/cid controls. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g002 (A) Schematic diagram of the appearance and destruction of various cell
cycle regulatory factors and markers. ANA, anaphase; META, metaphase;
PRO, prophase; PRO-META, prometaphase; TELO, telophase. (A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic
defects and robust CID staining at kinetochores (inset). Inactivation of the DNA Damage Checkpoint Does Not
Abrogate the cid-Mediated Mitotic Delay DOI: 10.1371/journal.pgen.0020110.g003 decreased mitotic index in cid heterozygotes demonstrates
that a DNA damage-induced cell cycle delay results in a
fundamentally different (opposite) phenotype from the
increased mitotic index observed in untreated cid null
embryos. ment and regulates the metaphase to anaphase transition. However, the cid-mediated mitotic delay appears temporally
and phenotypically distinct from SAC-mediated cell cycle
effects. When the SAC is activated (e.g., by the addition of
microtubule polymerization inhibitors), cyclin A is degraded,
but not cyclin B [31,36] (compare to Figure 2), and cells arrest
in prometaphase/metaphase with condensed but unaligned
chromosomes. Finally, the absence of normal kinetochore
formation in all cases in which CENP-A proteins have been
depleted or mutated [15–20,37] suggests that cid null mutant
cells should not have an intact SAC, and that SAC
components should not play a role in the cid-mediated
mitotic delay. We also performed the reciprocal experiment, to deter-
mine if an intact DNA damage checkpoint was necessary for
the cid-mediated early mitotic delay. A central component of
the DNA damage response, the MEI-41 ATR kinase [35], was
inhibited by treating cid mutant and heterozygous embryos
with 2 mM caffeine [34]. We found that caffeine treatment of
Drosophila embryos phenocopied mei-41 and grapes maternal
affect mutations, and is likely to completely inactivate the
DNA damage checkpoint (unpublished data). Inactivation of
MEI-41 by caffeine treatment did not suppress the cid-
mediated mitotic delay. The mitotic index of cid mutants
remained nearly twice that of heterozygous controls, and
most of the mitotic cells were found in prophase or
prometaphase, with very few cells progressing to later stages
of mitosis (Figure 3D–3F). These results demonstrate that
inactivation of the DNA damage checkpoint does not
abrogate the cid-mediated mitotic delay, and confirms that
this delay is not the result of DNA damage induced by cid
mutations. y
To directly test for the presence of an SAC response to
microtubule disruption in cid mutant animals, we treated
stage 15 cid homozygous and heterozygous animals with the
microtubule depolymerizing agent colcemid. We found that
both homozygous and heterozygous cells were delayed in
response to colcemid treatment (Figure 4), consistent with the
observation that the Saccharomyces cerevisiae CENP-A homolog
Cse4 is not required for SAC function [38]. First, both
genotypes displayed a nearly 2-fold increase in mitotic index
after 1 h of treatment (Figure 4B). Inactivation of the DNA Damage Checkpoint Does Not
Abrogate the cid-Mediated Mitotic Delay The appearance of H3 phospho-serine 10 (PH3), destruc-
tion of the mitotic cyclins, and mitotic spindle morphology
can be used to discriminate different stages of G2 and mitosis
(Figure 2A). Cyclins A and B begin to accumulate in S and G2
phases [31], and PH3 begins to appear in late G2, and is used
as a general marker for mitotic index [32]. Subsequently,
cyclin A destruction is observed during prometaphase, cyclin
B destruction occurs at the metaphase to anaphase (M:A)
transition, and PH3 staining is gradually lost from chromo-
somes at the end of telophase. The mitotic delay observed in homozygous cid mutant
embryos suggested that cid depletion and failure to form a
kinetochore activated a cell cycle checkpoint. A recent study
in Xenopus suggested that DNA damage and repair may be
involved in CENP-A assembly at centromeres [33]. Therefore,
incomplete kinetochore chromatin assembly or chromosome
segregation errors caused by cid mutation could result in
DNA damage and activation of the DNA damage checkpoint,
which would mediate the early mitotic cell cycle delay. To
determine whether DNA damage phenocopies the cid null
mutations, we treated cid mutant and heterozygous embryos
with doxorubicin, a topoisomerase II inhibitor known to
generate dsDNA breaks [34]. We found that doxorubicin
treatment dramatically decreased the mitotic index of cid
heterozygous embryos (Figure 3A–3C), consistent with pre-
vious studies of the effects of DNA damage on cell cycle
progression [35]. Doxorubicin treatment had little effect on
the mitotic index of cid homozygous mutant embryos, which
likely reflects the fact that these cells were already delayed in
mitosis at the time of drug addition. Importantly, the To determine the effects of cid depletion on cell cycle
progression, the number of cells in specific stages of mitosis
was determined by staining homozygous and heterozygous
mutant stage 15 embryos for tubulin, cyclin A, cyclin B, and
PH3. Three observations demonstrated that cid mutants were
delayed early in mitosis, predominantly in prophase/prom-
etaphase. First, cid mutants displayed a 2.4-fold higher mitotic
index (p , 0.01) and a 2-fold higher number of cells positive
for cyclin A (p 0.01) and cyclin B (p 0.01), in comparison
to heterozygous control siblings (Figure 2B and 2C). Second,
cid mutants showed a marked increase in the number of cells 1027 July 2006 | Volume 2 | Issue 7 | e110 cid-Mediated Mitotic Delay Requires SAC Figure 3. Inactivation of the DNA Damage Checkpoint Does Not
Abrogate the cid-Mediated Mitotic Delay Second, the increased
mitotic index was accompanied by a large increase in the
number of cells accumulated in prometaphase in both cid null
and heterozygous animals. We conclude that cid mutant cells
retain an intact SAC response to microtubule disruption;
thus, SAC components could play a role in the cid-mediated
early mitotic delay. In addition, the fact that some cid/cid cells
accumulated in prometaphase/metaphase after colcemid cid Mutant Cells Have an Intact SAC Response to
Microtubule Disruption PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 Inactivation of the DNA Damage Checkpoint Does Not
Abrogate the cid-Mediated Mitotic Delay DNA Damage Is Not Responsible for the cid-Mediated Mitotic Delay
The effect of DNA damage on cell cycle progression was determined by treating stage 15 cid null and heterozygous embryos with the topoisomerase II
inhibitor doxorubicin. (A–C) cid/CyO cells dramatically decreased entry into mitosis in response to DNA damage, whereas cid/cid cells were unaffected by doxorubicin (Dox.)
treatment. (D–F) The MEI-41/ATR kinase was inhibited by treating cid homozygous and heterozygous embryos with 2 mM caffeine. Inactivation of the DNA
damage checkpoint did not suppress the cid-mediated mitotic delay, as the mitotic index of cid mutants remained double that of controls, with the
majority of the mitotic cells delayed in prophase or prometaphase. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g003 tic Dela Figure 3. DNA Damage Is Not Responsible for the cid-Mediated Mitotic Delay
The effect of DNA damage on cell cycle progression was determined by treating stage 15 cid null and heterozygous embryos with the topoisomerase II
inhibitor doxorubicin. (A–C) cid/CyO cells dramatically decreased entry into mitosis in response to DNA damage, whereas cid/cid cells were unaffected by doxorubicin (Dox.)
treatment. (D–F) The MEI-41/ATR kinase was inhibited by treating cid homozygous and heterozygous embryos with 2 mM caffeine. Inactivation of the DNA
damage checkpoint did not suppress the cid-mediated mitotic delay, as the mitotic index of cid mutants remained double that of controls, with the
majority of the mitotic cells delayed in prophase or prometaphase. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g003 Figure 3. DNA Damage Is Not Responsible for the cid-Mediated Mitotic Delay
The effect of DNA damage on cell cycle progression was determined by treating stage 15 cid null and heterozygous embryos with the topoisomerase II
inhibitor doxorubicin. (A–C) cid/CyO cells dramatically decreased entry into mitosis in response to DNA damage, whereas cid/cid cells were unaffected by doxorubicin (Dox.)
treatment. (D–F) The MEI-41/ATR kinase was inhibited by treating cid homozygous and heterozygous embryos with 2 mM caffeine. Inactivation of the DNA
damage checkpoint did not suppress the cid-mediated mitotic delay, as the mitotic index of cid mutants remained double that of controls, with the
majority of the mitotic cells delayed in prophase or prometaphase. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. Scale bars indicate 15 lm. cid Mutant Cells Have an Intact SAC Response to
Microtubule Disruption (B) bubr1 suppressed the high mitotic index and high number of cells
delayed in prophase and prometaphase observed in cid single mutants
(compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic
index; P, prophase; PM, prometaphase; T, telophase. DOI: 10.1371/journal.pgen.0020110.g005 (A) cid bubr1 double mutants were examined for mitotic progression by
staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear
density and number of anaphases compared to cid single mutants. Scale
bar indicates 15 lm. cid null and heterozygous embryos were treated with colcemid to
determine if they have an intact SAC response to spindle disruption. Both cid/CyO and cid/cid cells were able to delay the cell cycle in
response to spindle disruption (A), as evidenced by an approximately 2-
fold increase in mitotic index and an accumulation of cells in
prometaphase (B). l
(B) bubr1 suppressed the high mitotic index and high number of cells
delayed in prophase and prometaphase observed in cid single mutants
(compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic
index; P, prophase; PM, prometaphase; T, telophase. DOI: 10.1371/journal.pgen.0020110.g005 p
p
A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM,
prometaphase; T, telophase. p
p
p
Scale bars indicate 15 lm. l
DOI: 10.1371/journal.pgen.0020110.g004 only comparing homozygous double mutants to heterozygous
double mutants (see Materials and Methods for a more
detailed discussion). We conclude that inactivation of a
component of the SAC relieves the cid-mediated mitotic
delay, suggesting that at least one component of the SAC is
involved in delaying cell cycle progression in the absence of
CID. treatment indicates that cells can eventually overcome the
prophase delay, and that prometaphase is likely to be the
terminal arrest point, similar to an SAC-mediated cell cycle
arrest. PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 cid Mutant Cells Have an Intact SAC Response to
Microtubule Disruption The other major cell cycle checkpoint that could be
responsible for the cell cycle delay observed in cid mutants
is the SAC, which monitors kinetochore microtubule attach- July 2006 | Volume 2 | Issue 7 | e110 1028 cid-Mediated Mitotic Delay Requires SAC only comparing homozygous double mutants to heterozygous
Figure 5. A bubr1 Mutation Partially Suppresses the cid-Mediated Mitotic
Delay
(A) cid bubr1 double mutants were examined for mitotic progression by
staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear
density and number of anaphases compared to cid single mutants. Scale
bar indicates 15 lm. (B) bubr1 suppressed the high mitotic index and high number of cells
delayed in prophase and prometaphase observed in cid single mutants
(compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic
index; P, prophase; PM, prometaphase; T, telophase. DOI: 10.1371/journal.pgen.0020110.g005
Figure 4. cid Mutants Retain an Intact SAC Response to Microtubule
Depolymerization
cid null and heterozygous embryos were treated with colcemid to
determine if they have an intact SAC response to spindle disruption. Both cid/CyO and cid/cid cells were able to delay the cell cycle in
response to spindle disruption (A), as evidenced by an approximately 2-
fold increase in mitotic index and an accumulation of cells in
prometaphase (B). A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM,
prometaphase; T, telophase. Scale bars indicate 15 lm. DOI: 10.1371/journal.pgen.0020110.g004 Figure 5. A bubr1 Mutation Partially Suppresses the cid-Mediated Mitotic
Delay
(A) cid bubr1 double mutants were examined for mitotic progression by
staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear
density and number of anaphases compared to cid single mutants. Scale
bar indicates 15 lm. (B) bubr1 suppressed the high mitotic index and high number of cells
delayed in prophase and prometaphase observed in cid single mutants
(compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic
index; P, prophase; PM, prometaphase; T, telophase. DOI: 10.1371/journal.pgen.0020110.g005 Figure 5. A bubr1 Mutation Partially Suppresses the cid-Mediated Mitotic
Delay Figure 4. cid Mutants Retain an Intact SAC Response to Microtubule
Depolymerization (A) cid bubr1 double mutants were examined for mitotic progression by
staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear
density and number of anaphases compared to cid single mutants. Scale
bar indicates 15 lm. CENP-C and the SAC Components BUBR1 and ROD Are
Unable to Localize to Kinetochores in cid Mutants To directly examine the role of the SAC in the cid-mediated
mitotic delay, we determined if a mutation that inactivates
the SAC can restore normal cell cycle progression. cid bubr1
double mutants were generated, and homozygous and
heterozygous double mutant embryos were monitored for
cell cycle progression by staining for PH3. Surprisingly, bubr1
mutations partially suppressed most of the cell cycle
phenotypes associated with cid mutation (Figure 5A and 5B). The mitotic index in cid bubr1 double mutants was nearly the
same as observed in heterozygous controls (1.1-fold, p ¼ 0.6,
cid bubr1/cid bubr1 compared to cid bubr1/CyO), compared to the
2.4-fold increase observed for cid/cid mutants over controls
(see above). The number of cells delayed in prophase and
prometaphase also decreased dramatically in cid bubr1 double
mutants and was comparable to heterozygous controls,
whereas the number of cells in anaphase showed a corre-
sponding increase and was greater that controls. Note that cid
bubr1 double mutants had a mitotic index nearly double that
of cid single mutants, for reasons that are unclear at this time. We eliminated bias that could arise from this difference by It has been proposed that the APC (anaphase-promoting
complex) inhibitory signal is generated by the rapid turnover
of SAC proteins at unattached kinetochores [39–43]. We
previously demonstrated that all tested outer kinetochore
proteins (ROD, BUBR1, Cenp-meta, and POLO) fail to
localize to kinetochores in CID-depleted tissue culture cells
and CID antibody-injected embryos [17]. CENP-A disruptions
in Caenorhabditis elegans, mouse, and human cells also result in
failure to properly localize kinetochore components, includ-
ing SAC proteins [15,17–19,37]. Disruption of kinetochore
formation and SAC protein localization in cid/cid embryos was
determined by staining for inner and outer kinetochore
proteins. We found that the inner kinetochore protein CENP-
C [30] was absent in most cid/cid cells, and occasionally was
mislocalized in a diffuse pattern throughout the cell,
consistent with studies in other organisms and with a severe
disruption of kinetochore assembly (Figure 6). Consistent
with these results, the SAC components ROD and BUBR1
were unable to localize to kinetochores in stage 15 cid null 1029 July 2006 | Volume 2 | Issue 7 | e110 cid-Mediated Mitotic Delay Requires SAC Figure 6. Inner and Outer Kinetochore Protein Localizations Are
Disrupted in cid Mutant Embryos
Kinetochore localization of CENP-C, ROD, and BUBR1 were determined in
stage 15 embryos. CENP-C and the SAC Components BUBR1 and ROD Are
Unable to Localize to Kinetochores in cid Mutants In cid/CyO control embryos (left), all three proteins
were localized to the centromere/kinetochore during interphase (CENP-
C) or the early stages of mitosis (ROD and BUBR1). All three proteins were
absent from centromeres/kinetochores in cid/cid animals (right); in some
cases, CENP-C was mislocalized in a diffuse pattern. Scale bars indicate 5
lm. DOI: 10.1371/journal.pgen.0020110.g006 However, these studies did not determine when the delay
occurred in mitosis, whether the mitotic delays involved the
SAC or the DNA repair checkpoint, or whether similar
responses to CENP-A depletion occurred in animals. We addressed the possible involvement of two known cell
cycle checkpoints in the CID-mediated early mitotic delay,
specifically the DNA damage and SACs. A recent study
suggested that DNA damage and repair may be involved in
CENP-A assembly in Xenopus [33], raising the possibility that
elimination of CID alters centromeric chromatin, resulting in
DNA damage at the centromere. We addressed this hypoth-
esis in two complementary ways. First, we compared the cid
mutant mitotic delay phenotypes to the behavior of cells after
inducing general DNA damage with doxorubicin. Induction
of DNA damage resulted in a reduced mitotic index, not the
increased mitotic index observed in the cid mutant embryos. Second, we disrupted the DNA damage checkpoint in cid
mutant embryos using caffeine treatment, which inhibits
MEI-41 (ATR), an essential component of the DNA damage
response [34,35]. Caffeine treatment did not abrogate the cid-
mediated mitotic delay. We conclude that DNA damage does
not appear to be the signal that induces the cid-mediated
early mitotic delay, and that this delay does not require an
intact DNA damage checkpoint. These results led us to address the possible involvement of
the SAC in the CID-mediated early mitotic delay in animals. The SAC monitors microtubule attachments to the kinet-
ochore; if normal bipolar attachments are not formed,
activation of the SAC blocks entry into anaphase, resulting
in a prometaphase/metaphase arrest [34,35]. The fact that the
CID-mediated delay occurred earlier in mitosis than expected
for activation of the SAC suggested that this checkpoint
might not be involved. However, we observed that cid null
mutant cells retained an intact SAC response to microtubule
disruption by colcemid, which is similar to the response of
Cse4 mutants in Sa. cerevisiae [38]. In addition, mutating an
essential SAC component (BUBR1) resulted in abrogation of
the CID-mediated delay. Previous studies suggested that
kinetochore localization of SAC proteins (e.g. CENP-C and the SAC Components BUBR1 and ROD Are
Unable to Localize to Kinetochores in cid Mutants MAD2, BUBR1,
ROD, and CENP-E) is absolutely required for SAC function. Nevertheless, we observed that BUBR1 and ROD, and the
inner kinetochore protein CENP-C, lacked kinetochore
localization in cid mutant embryos. These results suggest that
the CID-mediated early mitotic delay involves the SAC, and
that BUBR1 is serving a kinetochore-independent role in
delaying mitotic progression, as suggested by recent studies in
human and yeast cells [12,44,45]. Figure 6. Inner and Outer Kinetochore Protein Localizations Are
Disrupted in cid Mutant Embryos Kinetochore localization of CENP-C, ROD, and BUBR1 were determined in
stage 15 embryos. In cid/CyO control embryos (left), all three proteins
were localized to the centromere/kinetochore during interphase (CENP-
C) or the early stages of mitosis (ROD and BUBR1). All three proteins were
absent from centromeres/kinetochores in cid/cid animals (right); in some
cases, CENP-C was mislocalized in a diffuse pattern. Scale bars indicate 5
lm. l Kinetochore localization of CENP-C, ROD, and BUBR1 were determined in
stage 15 embryos. In cid/CyO control embryos (left), all three proteins
were localized to the centromere/kinetochore during interphase (CENP-
C) or the early stages of mitosis (ROD and BUBR1). All three proteins were
absent from centromeres/kinetochores in cid/cid animals (right); in some
cases, CENP-C was mislocalized in a diffuse pattern. Scale bars indicate 5
lm. DOI 10 1371/j
l
0020110 006 l
DOI: 10.1371/journal.pgen.0020110.g006 animals, whereas BUBR1 and ROD were localized to
kinetochores during all stages of mitosis in heterozygous
controls (Figure 6). We conclude that cid null mutations delay
cells in early mitosis in the absence of sustained kinetochore
localization of essential components of the SAC, despite the
requirement for at least one of these SAC components
(BUBR1). Why Do cid Mutants Display an Early Mitotic Delay That Is
BUBR1-Dependent? PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 Discussion SAC Components Affect Cell Cycle Progression in the Absence of Kinetochore Localization
In normal cells, CENP-A chromatin assembly is followed by the recruitment of inner and outer kineto
kinetochore assembly is complete, free SAC components may be responsible for cell cycle inhibition (earl
kinetochore assembly, SAC components delay anaphase until all chromosomes have achieved bipolar s
inner and outer kinetochore proteins are free, resulting in a SAC-dependent early mitotic delay tha
components to kinetochores. DOI: 10.1371/journal.pgen.0020110.g007 Figure 7. SAC Components Affect Cell Cycle Progression in the Absence of Kinetochore Localization Cell Cycle Progression in the Absence of Kinetochore Loc In normal cells, CENP-A chromatin assembly is followed by the recruitment of inner and outer kinetochore proteins [17]. We propose that until
kinetochore assembly is complete, free SAC components may be responsible for cell cycle inhibition (early activation of the SAC). Upon completion of
kinetochore assembly, SAC components delay anaphase until all chromosomes have achieved bipolar spindle attachment. In cid null mutants, both
inner and outer kinetochore proteins are free, resulting in a SAC-dependent early mitotic delay that does not depend on localization of SAC
components to kinetochores. DOI: 10 1371/journal pgen 0020110 g007 monitoring kinetochore-microtubule attachments [12,45]. Furthermore, recent studies in Drosophila have revealed a role
for Bub3 in G2 and early mitosis in promoting the accumu-
lation of mitotic cyclins [47], suggesting that components can
ensure normal mitotic progression by inhibiting the APC in a
kinetochore-independent manner. This interpretation is also
consistent with recent studies that demonstrate that SAC
proteins play multiple roles in cell cycle regulation [48–50]. For example, mutations in Drosophila bubr1 have been shown to
bypass the SAC, and are also able to suppress mutations that
activate both the DNA damage and SAC in early embryos
[48,51]. Furthermore, it has recently been demonstrated that
SAC components are responsible for mediating a mitotic
arrest in response to DNA damage in vertebrate cells [34], and
the mitotic arrest in response to spindle malorientation in Sc. pombe [52]. These results strengthen the conclusion that the
SAC can respond to more than bipolar kinetochore micro-
tubule attachment, and suggest multiple roles for SAC
components in cell cycle regulation. Therefore, the most
likely explanation for the cid-mediated mitotic delay is that
inhibitory SAC complexes can be formed in the absence of
kinetochore localization (Figure 7). Discussion Based on the previous observation of interphase/prophase
arrest after CID antibody injection into embryos, we
proposed that cells monitor kinetochore assembly early in
mitosis, in addition to monitoring the presence of bipolar
attachments later in mitosis [17]. It is also possible that cid
null kinetochores may be able to recruit normal levels of SAC
components at early stages of mitosis, but are unable to retain
functional levels later in mitosis, as observed for disruption of
human Hec1 and Nuf2 and DT-40 CENP-A [7,20]. We have shown that null mutations in the Drosophila
member of the CENP-A protein family result in embryonic
lethality after depletion of maternal CID protein. CID-
depleted embryonic cells display an early mitotic delay,
consistent with cell cycle defects observed after CID antibody
injection [17], suggesting the involvement and activation of a
cell cycle checkpoint. This result is similar to a recent
knockout of CENP-A in chicken DT-40 cells and a CENP-C
mutation in Drosophila, both of which resulted in a mitotic
delay in the absence of kinetochore assembly [20,30]. Alternatively, mitotic arrest may occur in the absence of
kinetochore localization of SAC components. Consistent with
this hypothesis, the cid-mediated early mitotic delay requires 1030 July 2006 | Volume 2 | Issue 7 | e110 PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 cid-Mediated Mitotic Delay Requires SAC Figure 7. SAC Components Affect Cell Cycle Progression in the Absence of Kinetochore Localization
In normal cells, CENP-A chromatin assembly is followed by the recruitment of inner and outer kinetochore proteins [17]. We propose that until
kinetochore assembly is complete, free SAC components may be responsible for cell cycle inhibition (early activation of the SAC). Upon completion of
kinetochore assembly, SAC components delay anaphase until all chromosomes have achieved bipolar spindle attachment. In cid null mutants, both
inner and outer kinetochore proteins are free, resulting in a SAC-dependent early mitotic delay that does not depend on localization of SAC
components to kinetochores. DOI: 10.1371/journal.pgen.0020110.g007 Figure 7. PLoS Genetics | www.plosgenetics.org Materials and Methods Cytology. cid mutant embryos were collected from interallelic
crosses and stained as described using either a formaldehyde or
MeOH:EGTA fixation. Trans-heterozygous combinations of the
different cid alleles were generated in order to eliminate phenotypic
effects of other lethal mutations present on each of the cid mutant
chromosomes (unpublished data). All of the data presented were
obtained for crosses between cid11–2 and cid 22–4, although crosses
between other alleles produced identical phenotypes. Antibodies
used were cyclin A [31], cyclin B [31], LacZ (Sigma, St. Louis, Missouri,
United States), tubulin (Sigma), ROD [53], BUBR1 [51], and CID [17]. For quantitation of mitotic index and cyclin abundance, all cells
within the developing central nervous system were counted from at
least five mutant and five control embryos. The ratios presented are
the number of PH3- or cyclin-positive cells divided by total cells, in
order to normalize for the lower nuclear density present in cid
mutant embryos. Quantification of the stages of mitosis was
performed by costaining embryos for PH3 and tubulin. The
distinction between prophase and prometaphase was made as follows:
Prophase was classified as chromosomes with incomplete condensa-
tion (i.e., round PH3þ nucleus), in which no individual chromosomes
or chromosome arms were visible and DNA was not obviously
aligning at the metaphase plate. Prophase tubulin staining showed
bright centrosomal signals with little or no obvious microtubules
interacting with the chromosomes. Prometaphase was classified as
chromosomes with complete condensation (i.e., clearly visible
individual chromosomes and chromosome arms) in which the
chromosomes were clearly in the process of aligning at the metaphase
plate. Tubulin staining showed a focused bipolar microtubule array
that was clearly interacting with the chromosomes. Examination of cid bubr1 heterozygous mutants. During the course
of scoring the mitotic parameters of cid bubr1 double mutants, we
noticed that cidbubr1 single mutants had a mitotic index nearly twice
as high as cid single mutants alone, which prompted us to investigate
these heterozygous mutants further for possible haploinsufficiency
effects. We found no incidence of chromosome segregation defects in
cid bubr1/CyO embryos despite their elevated mitotic index. We also
examined mitotic tissue of cid bubr1/CyO third instar larval brains
because this tissue allows a more precise karyotypic analysis and
could reveal subtle defects not seen in embryonic tissue. We found
that cid bubr1/CyO animals had a higher mitotic index than cid/CyO
animals (1.10 [n ¼ 315 fields] vs. 2.
Rieder CL, Maiato H (2004) Stuck in division or passing through: What
happens when cells cannot satisfy the spindle assembly checkpoint. Dev
Cell 7: 637–651. Discussion at least one SAC component (BUBR1), yet occurs without
sustained kinetochore localization of multiple, essential
components of the SAC (reported here and in [17]). The
finding that defects in kinetochore assembly lead to a BUBR1-
dependent early mitotic delay is supported by several recent
studies. Disruption of chicken CENP-A, CENP-H, or CENP-I,
all inner kinetochore proteins, delays cells in mitosis for
hours [9–11,20]. These results suggest that the SAC is able to
respond to multiple types of signals and inhibit cell cycle
progression. How could SAC components contribute to cell cycle delay
early in mitosis, prior to their well-established role in
monitoring bipolar attachments in prometaphase/metaphase? Loss of CENP-A proteins blocks kinetochore assembly, which
may generate ‘‘free’’ (non-kinetochore localized) SAC com-
plexes capable of inhibiting mitotic progression (Figure 7). Since the active inhibitory complex for the SAC is present
throughout the cell cycle [46], the complete absence of
kinetochore assembly, or the presence of ‘‘free’’ SAC
components, could block cells early in mitosis by chronically
activating the SAC. It has recently been shown that both
BUBR1 and MAD2 function in a kinetochore-independent
manner to regulate the length of mitosis, in addition to 1031 July 2006 | Volume 2 | Issue 7 | e110 July 2006 | Volume 2 | Issue 7 | e110 cid-Mediated Mitotic Delay Requires SAC The genes in this genomic region, proximal to distal, are cnn
(centrosomin/CG4832), Cbs (centrosomin’s beautiful sister/CG4840), arr
(arrow/CG5912), cbc (crowded by cid/CG5970), cid (centromere identifier/
CG13329), bbc (b-b in a boxcar/CG6016), and drk (downstream of receptor
kinase/CG6033). The initial characterization of this region included
screening of cDNA libraries and expressed sequence tag (EST)
collections to produce transcript profiles for each of these genes, and
saturation mutagenesis screens to recover recessive lethal and sterile
mutations. Breakpoint-associated mutations, principally deletions,
and complementation analyses were used to localize each of the
newly recovered mutations to the individual molecularly defined and
computationally identified transcription units. Using primers de-
signed from genomic and cDNA sequences the mutant alleles of each
locus were sequenced and the genetic localization of the comple-
mentation groups confirmed. This screen resulted in the recovery of
the four alleles of cid reported in this paper: cidt11–2 Q51 to stop,
cidt12–1 Q83 to stop, cidt21–3 Q94 to stop, and cidt22–4 Q102 to stop. Materials and Methods 0.76 [n ¼ 400 fields]), yet we did not
find any evidence for aneuploidy or mitotic defects in any of the
mitotic figures examined. We then determined whether cid bubr1 and
cid heterozygous animals had a normal response to colcemid
treatment by incubating brains with colcemid for 1 h. We found
that cid bubr1 and cid heterozygous mutants had a normal response to
colcemid treatment (cid/CyO mitotic index increased from 0.86 to 2.17
[n ¼ 551 fields], and cid bubr1/CyO mitotic index increased from 1.10 to
2.37 [n ¼ 264 fields]). From this data we conclude that cid and cid bubr1
mutants do not have a haploinsufficient effect on mitosis, and that
there are likely to be other factors in the genetic background that
lead to the increased mitotic index of cid bubr1 double mutants. To
avoid interpretation artifacts that might be caused by this difference,
in all cases we only compared data from cid bubr1 homozygotes to cid
bubr1 heterozygotes, and cid homozygotes to cid heterozygotes. y
g
For quantification of CID levels in mitotically active cells in mutant
and control embryos (Figure S1), the sum of pixel values for both CID
and PROD immunofluorescence from five to seven embryos was
obtained using the two-dimensional polygon finding tool in
softWoRx (Applied Precision, Issaquah, Washington, United States). The pixel values were summed and presented as a ratio of CID:PROD,
to provide a rough estimate of the amount of CID depletion in each
embryo. Based on these ratios, 90%–100% of CID was depleted in
stage 15 cid/cid mutants, relative to heterozygous controls, suggesting
retention of a small amount of maternal protein in some cells. For all
quantitations, standard deviations were calculated per embryo, and
data were compared using the Student t test. Note that the amount of
CID depletion in cid homozygotes is likely to be an underestimate (up
to 2-fold) with respect to wild-type embryos, since cid mutant
heterozygotes were used as the quantitation controls. Supporting Information Figure S1. Quantification of the Amount of CID Depletion in cid
Mutant Embryos Figure S1. Quantification of the Amount of CID Depletion in cid
Mutant Embryos Stage 15 cid/cid and cid/CyO embryos were stained for CID and PROD. PROD was present in a punctate pattern in both genotypes. Estimation of the amount of CID depletion was preformed by
comparing the ratio of total CID staining to PROD staining in five
different mutant and heterozygous embryos. From this analysis we
estimate that 90%–100% of CID protein is depleted in mutant
embryos. yg
q
All images were acquired using a DeltaVision workstation (Applied
Precision) and analyzed using softWoRx software, as described
previously [17]. All images were acquired using a DeltaVision workstation (Applied
Precision) and analyzed using softWoRx software, as described
previously [17]. Found at DOI: 10.1371/journal.pgen.0020110.sg001 (4.0 MB PDF). Found at DOI: 10.1371/journal.pgen.0020110.sg001 (4.0 MB PDF). Drug treatments. cid mutant and heterozygous embryos were
bleach dechorionated and incubated in a 1:1 mixture of Schneider’s
medium (þ10% heat-inactivated FBS) and octane as described in [54]. Colcemid was used at a concentration of 3 lg/ml for 1 h, caffeine was
used at a concentration of 2 mM for 2 h, and doxorubicin was used at
a concentration of 2 lM for 2 h. After drug treatment, embryos were
fixed using formaldehyde and processed for immunofluorescence as
described above. Discussion The role of the kinetochore in cell cycle progression and
the functions of SAC components are clearly more complex
than previously thought. Future studies should focus on
identifying the components and mechanisms responsible for
the cid-mediated mitotic delay, and determining if this
complex is identical to the standard SAC inhibitory complex. Acknowledgments We thank the following for providing antibodies: Barbara Mellone
(CENP-C), David Glover (cyclins A and B), Tibor Torok (PROD),
Claudio Sunkel (BUBR1), and Roger Karess (ROD). Author contributions. MDB and GHK conceived and designed the
experiments. MDB and TD performed the experiments. MDB and
GHK analyzed the data. MDB and TK contributed reagents/materials/
analysis tools. MDB and GHK wrote the paper. Genetics. The bubr1 allele used was k03113, and was obtained from
the Bloomington Stock Center (Bloomington, Indiana, United States). cid bubr1 double mutants were generated by recombination using
standard methods. Funding. This research was supported by NIH R01 GM066272 to
GK. Funding. This research was supported by NIH R01 GM066272 to
GK. Mutations in cid (centromere identifier/CG13329) were recovered in
genetic screens designed to isolate new mutant alleles of cnn
(centrosomin) [55]. The cid locus is tightly linked to cnn in the 50A
region of the right arm of the second chromosome in D. melanogaster. Competing interests. The authors have declared that no competing
interests exist. Competing interests. The authors have declared that no competing
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31. Whitfield WG, Gonzalez C, Maldonado-Codina G, Glover DM (1990) The A- . Whitfield WG, Gonzalez C, Maldonado-Codina G, Glover DM ( PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e110 July 2006 | Volume 2 | Issue 7 | e110 1033
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Qualitative Accounts of School-Aged Children’s Diets during the COVID-19 Pandemic in Rural, Central, Kenya
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Nutrients
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cc-by
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Brief Report
Qualitative Accounts of School-Aged Children’s Diets during
the COVID-19 Pandemic in Rural, Central, Kenya enriette Zeidler 1, Laura Shapiro 1
, Rebecca Clarke 1
, Haatembo Mooya 2
,
de 2, Danielle Matthews 3
, Grace Koteng 4, Pamela Wadende 4,*
and Claire Farrow 1,* Megan Jarman 1, Henriette Zeidler 1, Laura Shapiro 1
, Rebecca Clarke 1
, Haatembo Mooya 2
,
Barnabas Simatende 2, Danielle Matthews 3
, Grace Koteng 4, Pamela Wadende 4,*
and Claire Farrow 1,* 1
School of Psychology, College of Health and Life Sciences, Aston University, Birmingham B4 7ET, UK;
m.jarman@aston.ac.uk (M.J.); hzeidler@gmail.com (H.Z.); L.R.Shapiro@aston.ac.uk (L.S.);
clarker3@aston.ac.uk (R.C.) 1
School of Psychology, College of Health and Life Sciences, Aston University, Birmingham B4 7ET, UK;
m.jarman@aston.ac.uk (M.J.); hzeidler@gmail.com (H.Z.); L.R.Shapiro@aston.ac.uk (L.S.);
clarker3@aston.ac.uk (R.C.) 2
Department of Psychology, Humanities and Social Sciences, University of Zambia, Lusaka 32379, Zambia;
haatembo.mooya@unza.zm (H.M.); lifeisgoodregardless@gmail.com (B.S.) y
g
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3
Department of Psychology, University of Sheffield, Sheffield S10 2TN, UK; Danielle.matthews@sheffield.ac.uk
4
Department of Educational Psychology, School of Education and Human Resource Development,
Ki ii U i
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4
Department of Educational Psychology, School of Education and Human Resource Development,
Kisii University, Kericho 20200, Kenya; g.koteng@gmail.com y
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*
Correspondence: pamela.wadende@gmail.com (P.W.); Claire.farrow@aston.ac.uk (C.F.); y
y
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*
Correspondence: pamela.wadende@gmail.com (P.W.); Claire.farrow@aston.ac.uk (C.F.);
Tel.: +254-(0)-704928984 (P.W.); +44-(0)-1212045384 (C.F.) p
p
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(
)
Tel.: +254-(0)-704928984 (P.W.); +44-(0)-1212045384 (C.F.) Abstract: The COVID-19 pandemic has caused disruption to food security in many countries,
including Kenya. However, the impact of this on food provision to children at an individual level is
unknown. This small study aimed to provide a qualitative snapshot of the diets of children during
the COVID-19 pandemic. During completion of 24-h food recalls, with 15 families with children aged
5–8 years, caregivers were asked about changes they had made to foods given to their children due
to the pandemic. Food recalls were analysed to assess nutrient intakes. Qualitative comments were
thematically analysed. Most of the families reported making some changes to foods they provided to
their children due to COVID-19. Reasons for these changes fell into three themes, inability to access
foods (both due to formal restriction of movements and fear of leaving the house), poorer availability
of foods, and financial constraints (both decreases in income and increases in food prices).
Citation: Jarman, M.; Zeidler, H.;
Shapiro, L.; Clarke, R.; Mooya, H.;
Simatende, B.; Matthews, D.; Koteng,
G.; Wadende, P.; Farrow, C. Qualitative Accounts of School-Aged
Children’s Diets during the
COVID-19 Pandemic in Rural,
Central, Kenya. Nutrients 2021, 13,
3543. https://doi.org/10.3390/
nu13103543 Keywords: qualitative; nutrient intakes; COVID-19; rural; Kenya; children Brief Report
Qualitative Accounts of School-Aged Children’s Diets during
the COVID-19 Pandemic in Rural, Central, Kenya The
COVID-19 pandemic has affected some foods parents in rural Kenya can provide to their children. nutrients nutrients nutrients nutrients 2.2.2. Demographics The Kenyan Demographics and Health Survey Tool (https://dhsprogram.com/ ac-
cessed on 1 June 2020), education and poverty indicators were adapted and used to gain
information about maternal and paternal education and family demographics. 2.2. Procedure Researchers used existing contacts to local primary schools, whose preschool teach-
ers verbally invited eligible families to take part. Where families were interested in the
study, they were given more details about the procedures by local researchers who shared
information sheets and consent forms with participants, reading out and explaining items
whenever necessary. The study team have a long-standing relationship with these com-
munities which aided participation. Participating parents gave written informed consent,
either by signing or providing a thumb print, depending on literacy levels. For the mea-
sures reported in this study, parents provided details in interviews, prior to local COVID-19
lockdown restrictions, or over the telephone after lockdown restrictions. Child anthropo-
metric data were collected in schools by trained teachers and research assistants after the
lockdown restrictions were lifted (March 2021). 2.2.1. Qualitative Statements Parents were asked open-ended questions about the impact of the pandemic on the
child’s food provision. In particular, as part of each 24-h recall, parents were asked whether
the child’s food was typical on that day (and if not, what was different and why), and for
each food/drink item whether there was any specific impact of COVID-19 on what the
child had consumed (i.e., on the food which the family was able to provide). Data were
entered into NViVo version 12 [5] for analysis. 2.1. Participants Participants included 15 families of children aged between 4.8 and 7.6 years. Families
were recruited from two rural communities in Laikipia East in central Kenya. Both commu-
nities (Chuma and Matanya) are located about 15 km south-west of Nanyuki, the nearest
town. Participants were recruited as part of a larger ongoing study funded by the UKRI
Global Challenges Research Fund (via ESRC, see funding sources), with 80 families taking
part in multiple mealtime observations at home and school in Kenya and Zambia. 1. Introduction Access and availability to affordable nutritious foods has often been a challenge in
rural Kenya, as highlighted by studies reporting that the prevalence of food insecurity is as
high as 63–76% [1,2]. In March 2020, the COVID-19 global pandemic reached Kenya, and
in response, the government, in line with guidance from the World Health Organisation,
implemented restrictions to reduce the spread of the virus. Such regulations included
boarder closures, social distancing, movement restrictions and closure of non-essential
services [3]. These restrictions are likely to have had unintended effects on food access and
availability via the disruption of food systems, and reduced incomes of families who are
no longer able to find work [4] Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Whilst rapid analyses have been conducted to estimate the financial effects on fami-
lies [3], there has been little consideration of food availability and access issues, and impact
on the dietary intakes of children in rural Kenya at the individual level. As part of a
project already taking place with families in rural, central Kenya, we were able to collect
qualitative data from families to provide a snapshot of some issues parents faced when
feeding their children during the COVID-19 pandemic. In this short report, we aim to
describe the challenges these families faced and changes they made to foods they fed their
children at this time. 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/). https://www.mdpi.com/journal/nutrients Nutrients 2021, 13, 3543. https://doi.org/10.3390/nu13103543 Nutrients 2021, 13, 3543 2 of 7 2.2.3. Dietary Recall Data The dietary recall procedure was adapted to local standards in line with recommen-
dations from the GloboDiet-Africa team [6] and extensively piloted with a team of local
preschool teachers and research assistants. Parents completed the 24 h recalls on behalf
of their child. Parents completed a 24-h dietary recall over the telephone with a local,
trained, researcher. Recalls were completed on three separate days (two weekdays, and one
Sunday) in the course of two weeks. Three-repeated 24-h recalls has been shown to yield
similar nutrient intakes as three days of prospective weighed food diaries in children in
rural Kenya [7], furthermore a review of use of 24 h recalls in low-income countries shows
that fewer days of reporting is needed due to the limited variability in individual dietary
intakes [8]. Parents provided information about all foods that their child had consumed in
the 24-h period preceding the interview. In addition to the information concerning the time
and location of snacks and/or meals, the foods consumed (including foods, beverages,
condiments, sauces and spreads), brand information, preparation methods, and portion
sizes were collected. Grams/day of each food item was calculated for each child and the
Food Composition Tables from Kenya [9] were then used to calculate mean daily nutrient
intakes. These were compared to nutrient intake recommendations as outlined in the
joint FAO/WHO 2002 report [10] and each child was identified as having an adequate
or inadequate intake for their age category. The joint FAO/WHO 2004 report on energy Nutrients 2021, 13, 3543 3 of 7 requirements [11], was used to determine those with adequate or inadequate energy (kcal)
intakes, based on age, sex and weight. requirements [11], was used to determine those with adequate or inadequate energy (kcal)
intakes, based on age, sex and weight. 2.2.4. Anthropometrics Child height and weight were collected in schools by a team of teachers/research
assistants trained in using measuring materials. The scales used were Ramtons RM304,
which were re-calibrated before each measurement. Height was measured using a yardstick
after marking the child’s height on a wall. All measures were taken by one team member
and confirmed by a second member of the team. Children were weighed in their school
uniforms with shoes removed. Child height and weight were converted to weight for height
for age Z scores (WHZ), and height for age (HFA) Z scores, using the WHO AnthroPlus
software version 1.0.4 [12]. Children were classed as underweight if their WHZ z scores
were ≤−2SD. Having overweight or obesity were defined as >2SD and >3SD, respectively. Stunting was defined as HFA ≤−2SD. 2.3. Analysis The qualitative data were collected by local, trained fieldworkers in the language
requested by the participant (either Kikuyu, Kiswahili, or English). The fieldworkers
were fluent in all three languages, and they translated the data from the local language
into English. The qualitative data were then analysed by two UK researchers (RC and
MJ) through a process of data familiarisation, independent and inductive coding, and
grouping of codes into themes. Codes and themes emerging from the data were discussed
with researchers in Kenya (PW and HZ) to ensure that results were not biased by cultural
assumptions. Consensus on coding and theme generation was reached through discussion. Reports of particular food/drink items that were altered in the diet were extracted from
the statements to produce a list of commonly excluded foods. The summary statistics of
the quantitative data (n, percentages, mean (sd)) were analysed in Stata version 14.0 [13]. 3.2. Qualitative Analyses In total, 11 of the 15 participants discussed making changes to foods usually provided
to their child due to the COVID-19 pandemic. The most commonly reduced or excluded
food items were fruits, tomatoes, meat and sugar. Qualitative analysis generated three core
themes which reflect why changes were made due to the COVID-19 pandemic: (1) barriers
to access, (2) financial changes and (3) limited availability. 3.1. Quantitative Analyses Participants were 15 children: seven girls and eight boys. Fourteen of the children
had siblings. Eight children were recruited from Chuma village and seven from the
Matanya village. The main caregiver of 12 of the children was the mother, for two it was
the father and for one the great-grandmother. Children’s mothers reported an average
of 9.9 (SD 2.5) years in school and fathers reported an average of 9.8 (SD 2.4) years (all
numbers include two years of preschool). Thirteen children lived in timber houses, one
stone and one iron-sheet housing. Six children had access to electricity at home (three had
solar panels and three had Kenya Power supplied energy). Only one child’s home had a
private water source, and for the others the average walking distance to the water supply
was 27 minutes walking (SD 14). One child presented as underweight, one overweight and one with obesity. All other
children (n = 12) fell within the healthy WHZ category. Furthermore, no children in this
cohort were classified as wasted or stunted. Median nutrient intakes, and the number of children with inadequate intakes over
the three days of reporting are shown in Table 1. Ten of the 15 children had inadequate
calcium intakes and nine children had inadequate niacin intakes. Whilst some children had
inadequate intakes of the other nutrients, fewer than half of the sample were inadequate in
this regard. Nutrients 2021, 13, 3543 4 of 7 Table 1. Description of nutrient intakes over three days of reporting during the COVID-19 pandemic. Nutrient
Median (Range)
N Inadequate Intake
Kcal
1613.7 (868.1–2638.2)
6
Calcium (mg)
506.7 (266.1–1090.4)
10
Iron (mg)
11.2 (5.7–30.4)
1
Magnesium (mg)
274.0 (128.8–420.6)
0
Zinc (mg)
6.9 (4.2–9.9)
2
Selenium (mcg)
24.9 (9.9–35.6)
0
Vitamin A Retinol Equivalents (mcg)
246.1 (134.6–464.7)
6
Thiamin (mg)
0.7 (0.3–2.3)
5
Riboflavin (mg)
1.3 (0.6–2.1)
2
Niacin (mg)
7.8 (4.3–17.9)
9
Folate (mcg)
331.4 (171.2–573.7)
3
Vitamin B12 (mcg)
2.1 (0.9–5.7)
2
Vitamin C (mg)
55.1 (17.1–105.2)
5 3.2.2. Financial Changes Changes to families’ financial circumstances caused by a reduction in family income
due to job loss, and an increase in the cost of the foods at the market, led to alterations
in diet: “In times like this when we receive a visitor, we could add some meat to the stew but it
has become hard to get some wage and casual works due to the effect of the virus” (Participant 15) “Yes, the amount of milk used to prepare the tea has reduced since the virus broke. This is
because we cannot afford to buy more because jobs have become scarce and we rely on
wage work” (Participant 8) “We used to buy fruits for the child but this has become hard since movements to the
market have been minimized” (Participant 6) “When cooking meat stew we usually include tomatoes but we did not because they have
turned to be expensive” 3.2.1. Barriers to Access Parents revealed that accessing food was difficult as they were either afraid to travel to
the market in case of catching the virus, or the restriction of movement meant that visiting
the market was not allowed: “Yes, we were unable to get some ingredients because of the fear to get to the market due
to covid-19” “Yes, we were unable to get some ingredients because of the fear to get to the market due
to covid-19” (Participant 7) “We would have wished to have maybe fruits in their meal but it has become hard to get
to the market due to the fear of being infected” c map showing th
4. Discussion 4. Discussion
In this short report, we aimed to present a snapshot of the food provision to some
school-aged children in rural, central, Kenya during the COVID-19 pandemic. We found
that most of the families we spoke to had to make changes to the food they provided their
children, either due to reduced household budgets, and/or a rise in the cost of some foods,
poorer access to the market places, and/or poorer availability of foods. Furthermore,
whilst this small cohort were mostly of a healthy weight for their height and age, there
were some important micronutrients lacking from the diets of these children over the
In this short report, we aimed to present a snapshot of the food provision to some
school-aged children in rural, central, Kenya during the COVID-19 pandemic. We found
that most of the families we spoke to had to make changes to the food they provided
their children, either due to reduced household budgets, and/or a rise in the cost of some
foods, poorer access to the market places, and/or poorer availability of foods. Furthermore,
whilst this small cohort were mostly of a healthy weight for their height and age, there
were some important micronutrients lacking from the diets of these children over the same
time-period. were some important micronutrients lacking from the diets of these children over the
same time-period. In the FAO policy briefing describing the impact of COVID-19 on the food and nu-
trition security of families in Kenya, the availability and access to foods was discussed [4]. At a national level it was reported that there was no impact on food availability with “su-
permarkets and food stores not running out of stock”[4]. Whilst there was mention of a
possible impact on market availability, this was focused on those in an urban or peri-ur-
ban setting. However, at a rural level, some of the families in our study had noticed a
reduction in the availability of some foods, citing the ban on movement as a potential
reason for food vendors having limited stock. In line with the policy briefing however,
was the discussion of limited food access and financial constraints, with food prices rising
in conjunction with income losses. 3.2.3. Limited Availability The summary of the themes is shown in the thematic map (Figure 1). f
p
g
f
The summary of the themes is shown in the thematic map (Figur The summary of the themes is shown in the thematic map (Figure 1). f
p
g
f
The summary of the themes is shown in the thematic map (Figur Access to food Financial Constraints Dietary changes Poor food availability Figure 1. Thematic map showing the main themes and subthemes of reasons for making dietary changes (Participant 9) (Participant 9) Nutrients 2021, 13, 3543 5 of 7 3.2.3. Limited Availability
Participants identified that travel restrictions caused by the COVID-19 pandemic
limited the availability of some foods:
“Yes, we were not able to add tomatoes to the food as the pandemic has made it hard for
the vendors to transport them together with other commodities . . . because of various
restrictions”
(Participant 6)
“We are forced to take whatever is available as the pandemic has changed life”
(Participant 2)
The summary of the themes is shown in the thematic map (Figure 1). s 2021, 13, x FOR PEER REVIEW
5 of 7
“We are forced to take whatever is available as the pandemic has changed life” (Participant 2)
The summary of the themes is shown in the thematic map (Figure 1.)
Figure 1 Thematic map showing the main themes and subthemes of reasons for making dietary changes
Dietary changes
Access to food
Financial Constraints
Poor food availability
Fear of going out
Formal restrictions on
movement
Lower income
Increased food prices
Figure 1. Thematic map showing the main themes and subthemes of reasons for making dietary changes. 3.2.3. Limited Availability
Participants identified that travel restrictions caused by the COVID-19 pandemic
limited the availability of some foods: c map showing th
4. Discussion The informal work sector accounts for 83.7% of em-
ployment in Kenya [5], and in our study sample two thirds of families reported relying on
‘wage work’ or ‘selling local produce’ A recent study focusing on the financial impact of
p
In the FAO policy briefing describing the impact of COVID-19 on the food and
nutrition security of families in Kenya, the availability and access to foods was discussed [4]. At a national level it was reported that there was no impact on food availability with
“supermarkets and food stores not running out of stock” [4]. Whilst there was mention of a
possible impact on market availability, this was focused on those in an urban or peri-urban
setting. However, at a rural level, some of the families in our study had noticed a reduction
in the availability of some foods, citing the ban on movement as a potential reason for food
vendors having limited stock. In line with the policy briefing however, was the discussion
of limited food access and financial constraints, with food prices rising in conjunction with
income losses. The informal work sector accounts for 83.7% of employment in Kenya [5],
and in our study sample two thirds of families reported relying on ‘wage work’ or ‘selling
local produce’. A recent study focusing on the financial impact of COVID-19 on food
security in Kenyan families also documented the double-edged sword of increasing food Nutrients 2021, 13, 3543 6 of 7 cost with decreasing incomes [5]. Seventy-three respondents reported lower regular income
due to COVID-19 and 40% reported changing their dietary patterns as a result, although
which foods were reduced/eliminated was not reported; they employed the use of social
media to collect data, and therefore had a more affluent sample with internet access, and
were mainly from urban areas. Our study has also suggested that limited food access was
not only due to financial constraints and formal restrictions on movement, but participants
also reported fear of becoming infected with COVID-19 as a reason for limiting travel to
the marketplace. A reduction in going out due to fear of the pandemic was highlighted in
an article considering impacts on physical activity [14], but to the best of our knowledge
the effect of fear on limiting food acquisition has not previously been considered in the
literature. c map showing th
4. Discussion In a recent (2020) debate piece, highlighting potential impacts of COVID-19
on dietary and physical behaviours related to non-communicable diseases in Urbanising
countries such as Kenya, the authors state that the measures employed to curb the spread
of COVID-19 would likely have a multipronged effect on diets through impacting family
incomes, food availability, price, and access [15]. Although the Kenyan government aimed
to limit food insecurity by auditing the supply and prices of food staples and prioritising
vulnerable families, our empirical data showed that food access and availability were still
impacted for some rural families. Our participants reported that fruits, tomatoes, meats and sugar were among the
foods most commonly reduced or removed from the diet due to the COVID-19 pandemic. More perishable foods, such as fruits and tomatoes, may have been more difficult for
market vendors to acquire, thus limiting their availability and increasing their price. In
contrast, although meats and sugar were still readily available, these may be considered
more expendable when families food budgets are reduced. Over half the participants had
inadequate niacin intakes, which may partly reflect the lack of meat intake in most of their
diets. Inadequate calcium intakes were also noted, which could be a result of low dairy
intakes; whilst every child consumed a little milk every day in tea, they did not have other
forms of dairy in their diet (data not shown). The sample size is a clear limitation of our study, although qualitative studies do not
intend to be representative of whole populations. Instead, we aimed to provide qualitative
insights into school-aged children’s diets during the COVID-19 pandemic in two villages in
rural Kenya. It is a strength that we were able to collect data with rural families, which was
due to the long-standing, trusting, relationship which had already been forged between our
study team and the communities. These communities tend to be underserved in studies. Funding: This work was funded by a grant from the Global Challenges Research Fund via the
Economic and Social Research Council. Grant number ES/T004959/1. 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. References 1. Shinsugi, C.; Matsumura, M.; Karama, M.; Tanaka, J.; Changoma, M.; Kaneko, S. Factors associated with stunting among children
according to the level of food insecurity in the household: A cross-sectional study in a rural community of Southeastern Kenya. BMC Public Health 2015, 15, 441. [CrossRef] [PubMed] 2. Nagata, J.M.; Fiorella, K.J.; Salmen, C.R.; Hickey, M.D.; Mattah, B.; Magerenge, R.; Milner, E.M.; Weiser, S.D.; Bukusi, E.A.; Cohen,
C.R. Around the Table: Food Insecurity, Socioeconomic Status, and Instrumental Social Support among Women Living in a Rural
Kenyan Island Community. Ecol. Food Nutrition. 2015, 54, 358–369. [CrossRef] [PubMed] y
y
3. Kansiime, M.K.; Tambo, J.A.; Mugambi, I.; Bundi, M.; Kara, A.; Owuor, C. COVID-19 implications on household income and food
security in Kenya and Uganda: Findings from a rapid assessment. World Dev. 2021, 137, 105199. [CrossRef] [PubMed] 4. Demeke, M.; Kariuki, J.; Wanjiru, M. Assessing the Impact of COVID-19 on Food and Nutrition Security and Adequacy of
Responses in Kenya. FAO Policy Briefing May 2020. Available online: https://evidencefrontiers.com/wp-content/uploads/2020
/05/Policy-Brief_Assessing-the-Impact-of-COVID_19-on-Food-and-Nutrition-Security-1.pdf (accessed on 6 August 2021). /
/
y
_
g
p
_
y
p
(
g
)
5. QSR International Pty Ltd. NVivo (Version 12). 2018. Available online: https://www.qsrinternational.com/nvivo-qualitative-
data-analysis-software/home (accessed on 1 June 2021). 6. Aglago, E.K.; Landais, E.; Nicolas, G.; Margetts, B.; Leclercq, C.; Allemand, P.; Aderibigbe, O.; Agueh, V.D.; Amuna, P.;
Annor, G.A.; et al. Evaluation of the international standardized 24-h dietary recall methodology (GloboDiet) for potential
application in research and surveillance within African settings. Glob. Health 2017, 13, 35. [CrossRef] [PubMed] 7. Kigutha, H.N. Assessment of dietary intake in rural communities in Africa: Experiences in Kenya. Am. J. Clin. Nutr. 1997, 65
(Suppl. 1), 1168S–1172S. [CrossRef] [PubMed] 8. Gibson, R.S.; Charrondiere, U.R.; Bell, W. Measurement errors in dietary assessment using self-reported 24-hour recalls in
low-income countries and strategies for their prevention. Adv. Nutr. 2017, 8, 980–991. [CrossRef] [PubMed] g
p
f Kenya. Kenya Food Composition Tables. Nairobi. 2018. 254. Available online: http://www.fao.org/3/I912
ccessed on 1 August 2021). g
9. FAO/Government of Kenya. Kenya Food Composition Tables. Nairobi. 2018. 254. Available online: http
0EN/i9120en.pdf (accessed on 1 August 2021). 10. World Health Organisation. Food and Agriculture Organisation of the United Nations. In Vitamin and Mineral Requirements in
Human Nutrition, 2nd ed.; WHO and FAO of the United Nations: Bankok, Thailand, 2004; pp. 17–299. pp
11. United Nations University; World Health Organization. Food and Agriculture Organization of the United Nations. 5. Conclusions The COVID-19 pandemic may have adversely effected foods that were provided
to children in some rural locations in Kenya. Barriers to food provision caused by the
pandemic included reduced access to marketplaces, both due to fear of infection and formal
movement restrictions, lower family incomes, increasing food prices, and poor availability
of produce on offer. These provide some insight into some of the immediate implications of
the pandemic, highlighting which types of foods tend to be reduced when hardship occurs. This may be taken into consideration in future studies of food-related relief procedures
during crises, in particular how such events may exacerbate food availability and access in
rural communities. Author Contributions: L.S., C.F., P.W., D.M., H.M., H.Z., B.S., M.J. and G.K. conceived the study idea
and obtained study funding. L.S. is overall P.I. on project, P.W. is local P.I. in Kenya. C.F., L.S., P.W. and H.Z. prepared and oversaw ethical approval. H.Z. and P.W. oversaw data collection. M.J., R.C. and C.F. prepared analyses and results. M.J. led on writing the paper. All authors have read and
agreed to the published version of the manuscript. Funding: This work was funded by a grant from the Global Challenges Research Fund via the
Economic and Social Research Council. Grant number ES/T004959/1. Nutrients 2021, 13, 3543 7 of 7 Institutional Review Board Statement: The Study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by Baraton University of Eastern Africa, Kenya, Research
Ethics Committee protocol code UEAB/REC/03/10/2020 on a 9-10-2020. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data that support the findings of this study are openly available in
the UK Data Archive at http://reshare.ukdataservice.ac.uk/855241/ accessed on 10 September 2021. Data Availability Statement: The data that support the findings of this study are openly available in
the UK Data Archive at http://reshare.ukdataservice.ac.uk/855241/ accessed on 10 September 2021. Acknowledgments: The authors would particularly like to thank the parents, children, and teachers
of Chuma and Matanya Primary Schools who supported our work, and the two research assistants
who collected all food recall data: Stephen Kuom Elondo and Caroline Wanjiru Maina. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References In Human
Energy Requirements: Report of a Joint FAO/WHO/UNU Expert Consultation; FAO: Rome, Italy, 2004. 12. World Health Organistion AnthroPlus Software. Version 1.0.4. 2007. Available online: https://www.who.int/tools/growth-
reference-data-for-5to19-years/application-tools (accessed on 5 August 2021). 13. Stata Statistical Software. Release version 14.0; StataCorp, College Station, Texas USA. 2015. Available online: https://www.stata. com/stata14/ (accessed on 10 July 2021). 14. Ben Hassen, T.; El Bilali, H.; Allahyari, M.S. Impact of COVID-19 on Food Behavior and Consumption in Qatar. Sustainability
2020, 12, 6973. [CrossRef] 15. Oni, T.; Micklesfield, L.K.; Wadende, P.; Obonyo, C.O.; Woodcock, J.; Mogo, E.R.; Odunitan-Wayas, F.A.; Assah, F.; Tatah,
L.; Foley, L.; et al. Implications of COVID-19 control measures for diet and physical activity, and lessons for addressing other
pandemics facing rapidly urbanising countries. Glob. Health Action 2020, 13, 1810415. [CrossRef] [PubMed]
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Adjustment in the Composition and Organization of Proteus mirabilis Lipids during the Swarming Process
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International journal of molecular sciences
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Citation: Stolarek, P.; Bernat, P.;
Ró˙zalski, A. Adjustment in the
Composition and Organization of
Proteus mirabilis Lipids during the
Swarming Process. Int. J. Mol. Sci. 2023, 24, 16461. https://doi.org/
10.3390/ijms242216461 Keywords: Proteus mirabilis; swarmer cells; rods morphology; phospholipids; fatty acids; lipid rafts Academic Editor: Tino Krell Paulina Stolarek 1,*
, Przemysław Bernat 2
and Antoni Ró˙zalski 1 1
Department of Biology of Bacteria, Faculty of Biology and Environmental Protection, University of Lodz,
B
h 12/16 90 237 L d
P l
d
t
i
l ki@bi l
i l d
l 1
Department of Biology of Bacteria, Faculty of Biology and Environmental Protection, University of Lodz,
Banacha 12/16, 90-237 Lodz, Poland; antoni.rozalski@biol.uni.lodz.pl
2
Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection,
University of Lodz, Banacha 12/16, 90-237 Lodz, Poland; przemyslaw.bernat@biol.uni.lodz.pl
*
Correspondence: paulina.stolarek@biol.uni.lodz.pl; Tel.: +48-42-635-43-24; Fax: +48-42-665-58-18 Abstract: Proteus mirabilis, an opportunistic pathogen of the urinary tract, is known for its dimorphism
and mobility. A connection of lipid alterations, induced by the rods elongation process, with enhanced
pathogenicity of long-form morphotype for the development of urinary tract infections, seems
highly probable. Therefore, research on the adjustment in the composition and organization of P. mirabilis lipids forming elongated rods was undertaken. The analyses performed using the ultra-
high performance liquid chromatography with tandem mass spectrometry showed that drastic
modifications in the morphology of P. mirabilis rods that occur during the swarming process are
directly related to deprivation of the long-form cells of PE 33:1 and PG 31:2 and their enrichment
with PE 32:1, PE 34:1, PE 34:2, PG 30:2, PG 32:1, and PG 34:1. The analyses conducted by the gas
chromatography-mass spectrometry showed negligible effects of the swarming process on fatty
acids synthesis. However, the constant proportions between unsaturated and saturated fatty acids
confirmed that phenotypic modifications in the P. mirabilis rods induced by motility were independent
of the saturation of the phospholipid tails. The method of the Förster resonance energy transfer
revealed the influence of the swarming process on the melting of ordered lipid rafts present in the
short-form rods, corresponding to the homogeneity of lipid bilayers in the long-form rods of P. mirabilis. Confocal microscope photographs visualized strong Rhod-PE fluorescence of the whole
area of swarmer cells, in contrast to weak membrane fluorescence of non-swarmer cells. It suggested
an increased permeability of the P. mirabilis bilayers in long-form rods morphologically adapted to
the swarming process. These studies clearly demonstrate that swarming motility regulates the lipid
composition and organization in P. mirabilis rods. International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences Article
Adjustment in the Composition and Organization of Proteus
mirabilis Lipids during the Swarming Process Paulina Stolarek 1,*
, Przemysław Bernat 2
and Antoni Ró˙zalski 1 1. Introduction Received: 16 October 2023
Revised: 7 November 2023
Accepted: 15 November 2023
Published: 17 November 2023 The dimorphic, motile, Gram-negative, and facultative anaerobic bacterium Proteus
mirabilis is commonly found in soil and water, as well as in the normal human intestinal
flora. This opportunistic pathogen has been known to cause serious infections in hu-
mans, although the vast majority of P. mirabilis-mediated infections occur in patients with
anatomical or functional abnormalities of the urinary tract and long-term urinary catheter-
ized patients. Urinary tract infections (UTIs) continue to be a global problem affecting
approximately 150 million people each year [1]. 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/). P. mirabilis is frequently mentioned in terms of its swarming motility. The swarming
phenomenon is a surface-induced differentiation process of short (1–2 µm) single cells with
a few peripheral flagella into elongated (20–80 µm), hyperflagellated, multinucleated, and
non-septate forms arranged in rafts, which enables rapid movement of the cell population
across solid surfaces in a highly coordinated manner. P. mirabilis motility is possible on both
biotic (i.e., urethra epithelium) and abiotic surfaces (i.e., catheters, intravenous lines, and https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 16461. https://doi.org/10.3390/ijms242216461 Int. J. Mol. Sci. 2023, 24, 16461 2 of 18 2 of 18 other medical equipment) [2]. Research proves that uropathogens are able to migrate over
all-silicone catheters, silicone-coated latex catheters, and hydrogel-coated latex catheters [3]. It has been shown that cell migration across urethral catheters enables P. mirabilis swarmers
to gain entry to the mouse urinary tract [4]. Moreover, it has been suspected that elongated
and hyperflagellated cells could be well suited in an ascending route of infection in non-
catheterized patients. Allison et al. [5] demonstrated the ability of P. mirabilis swarmer cells
to establish the ascending infection of the mouse kidney. On the other hand, more recent
studies have shown that in mouse models of ascending urinary tract infection, short rods
of uropathogens dominate instead of long ones [6], which undermined the crucial role of
swarming motility in ascending UTIs and pyelonephritis. Significance of swarming in the
pathogenesis of P. mirabilis still remains to be assessed. p
g
As mentioned before, motility is an important virulence factor because it could allow
for the microorganisms to reach the site of colonization and then spread to other areas
of the urinary tract. It has been documented that non-swarming or weakly swarming
strains of P. mirabilis are characterized by a reduced ability of cell adhesion to the bladder
epithelial cell line 5637 [7], weakened capacity to colonize the mice bladder and kidneys [8],
and limited ability of form biofilm on abiotic surfaces [9]. Moreover, structural changes
occurring in the rods during the swarming process resulting in a modified cell phenotype
undoubtedly influence other aspects of P. mirabilis pathogenesis. It has been documented
that swarmer cells demonstrate higher activity of urease, IgA metaloprotease ZapA and
HpmA hemolysin, and intensified flagellin synthesis, in comparison to swimmer cells [2,10]. Interestingly, recently published research indicates that motile cells of P. mirabilis induce
low levels of proinflammatory cytokines IL-6, IL-8, and IL-1β, as well as the antimicrobial
peptide HBD-2, while stimulating high concentrations of the anti-inflammatory cytokine
TGF-β in infected epithelial cells [9]. These morphotypes of P. mirabilis rods upregulate the
intrinsic pathway factors, especially BCL-2, recognized as an anti-apoptotic factor which
indicates the lowest degree of induced apoptosis in human prostatic adenocarcinoma PC-
3 [11]. Taking into account the above, it seems reasonable to assume that the elongated cell
type plays an important role in some stages of the bacterial infection. yp p y
p
g
Initiation of the P. mirabilis rods differentiation has been quite well recognized. The
process requires inhibition of flagellar rotation plus accumulation of extracellular putrescine
and O-antigen interactions with a surface [12]. It could be a response to specific nutrients,
as well as environmental signals, and is regulated by quorum sensing [13]. It has been
documented that proteins for flagellar biosynthesis and rotation, surface elongation, control
and coordination of multicellular motility, lipopolysaccharide (LPS) and peptidoglycan
production, and cell division are needed for successful transformation of swimmer cells
to swarmer cells [2]. Studies conducted at the transcriptome level showed that during
drastic morphological changes occurring in the bacterial cells, 541 genes were upregulated
in short-form cells and only 9 genes were upregulated in long-form cells. Genes involved in
processes such as flagellar biosynthesis, amino acid import and metabolism, oligopeptide
transport, and cell division were upregulated in short-form rods [14]. The authors suggest
that during the consolidation phase, P. mirabilis prepares for the next wave of swarming. A
study which was focused on changes in peptidoglycan composition occurring during cell
differentiation demonstrated that the level of peptidoglycan O-acetylation was lowering
from 51% to 29% in the process of P. mirabilis rods elongation. It was accompanied by a
simultaneous rise in the content of anhydromuropeptides and a reduction in the amount of
Lys-Lys-muropeptides arising from bound lipoprotein. The latter pointed to the shortening
of the muropeptides chain length in swarmer cells [15]. Interestingly, to understand
biological systems where the spatial dynamics depend on the local physiological structure,
Ayati et al. [16] developed composite hyperbolic-parabolic partial differential equations
to model P. mirabilis swarm colony expansion. Despite the fact that swarming motility of
this urinary tract pathogen has been extensively studied, modifications in the lipidome of
P. mirabilis have been pushed to the background. In a paper published in the late 1970s,
similarities rather than differences in the phospholipid composition between swimming Int. J. Mol. Sci. 2023, 24, 16461 3 of 18 3 of 18 and swarming cells were presented [17]. On the other hand, more current data indicate
significant quantitative changes in the amounts of fatty acids (FAs) (lipids components) [18]
and phospholipids (PLs) in the elongated rods of P. mirabilis [19]. However, unique spectral
signals produced by infrared microspectroscopy were unable to specify the alterations in
the bacterial phospholipid composition [19]. p
p
p
p
Cell membranes, composed of thousands of different lipid molecules, are dynamically
responsive and form stable or transient structures which may be used by various proteins as
platforms for their activity [20]. A vast majority of amphiphilic lipids located in membranes
are glycerophospholipids, composed of a glycerol molecule, a phosphate group, two
fatty acids, and a variable head group such as choline, glycerol, ethanolamine, serine,
or inositol [21]. Phospholipids are recognized as factors of membrane-protein topology,
required for proper transport of solutions and electrons, the initiation of DNA replication,
and cell division [22]. Fatty acids, the acyl constituents of phospholipids, are listed among
the most important cellular components. Alongside their structural functions, fatty acids
serve as storage materials and energy suppliers in cells, and their derivatives are involved
in cell signaling. For instance, cis-2-unsaturated fatty acids with different chain lengths and
branching patterns are cell-to-cell signals which modulate bacterial activities in response to
environmental conditions [23]. The adaptation to environmental conditions (sometimes
extreme) allows bacteria to survive. This process may involve changes in their protein,
sterol, hopanoid, and carotenoid contents proceeded by alterations in the composition of
membrane lipids [24]. Modifications of the structure of glycerolipid acyl chains allow for
maintaining passive permeability for hydrophobic molecules, active solute transport, and
protein–protein interactions [24], minimizing energy expenditure and optimizing bacterial
growth [22]. Considering the above, an assumption that phospholipids play an important
role in the process of P. mirabilis cells differentiation seems to be proper. 2. Results
2.1. Expans
S 2.1. Expansion Rate, Behavior, and Visual Appearance of the P. mirabilis Swarm Colony
Swarming motility of the elongated P. mirabilis rods (panels A1 and A2 in Fig
tifid b
t
ti
t
f th
l
i
LB Swarming motility of the elongated P. mirabilis rods (panels A1 and A2 in Figure 1)
was quantified by a systematic measurement of the swarm colony sizes on an LB plate. Examined uropathogens differed significantly in the dynamics of swarming growth (a
graph in Figure 1). The lag time for the 1984 strain took almost twofold longer than for the
reference strain. As a result, the swarming zone formed in a motility plate by the P. mirabilis
ATCC 29906 achieved 90.50 mm ± 4.50 mm after 24 h, while that formed by the 1984 strain
reached 58.70 mm ± 0.56 mm. Based on this data, colony expansion rates were estimated
at 3.77 mm h−1 and 2.45 mm h−1 for the ATCC 29906 and 1984 strains, respectively. was quantified by a systematic measurement of the swarm colony sizes on an LB plate. Examined uropathogens differed significantly in the dynamics of swarming growth (a
graph in Figure 1). The lag time for the 1984 strain took almost twofold longer than for the
reference strain. As a result, the swarming zone formed in a motility plate by the P. mirabilis ATCC 29906 achieved 90.50 mm ± 4.50 mm after 24 h, while that formed by the
1984 strain reached 58.70 mm ± 0.56 mm. Based on this data, colony expansion rates were
estimated at 3.77 mm h−1 and 2.45 mm h−1 for the ATCC 29906 and 1984 strains,
respectively. Figure 1. Swarming zones of the P. mirabilis measured over time. The error values equal the standard
deviations. Statistical significance is indicated as * (p-value ≤ 0.05), ** (p-value ≤ 0.01), *** (p-value ≤
0.001), and ns means not significant (p-value > 0.05). Panels A show images of the elongated P. mirabilis rods produced by phase-contrast microscopy (magnification = 600×, scale bar = 5 µm). Panels B show a characteristic swarming pattern, formed by Proteus sp. The ATCC 29906 (1) and
1984 (2) strains are presented in both panels. Figure 1. Swarming zones of the P. mirabilis measured over time. The error values equal the standard
deviations. The present study was focused on the adjustment in the lipid composition and
organization in the P. mirabilis rods induced by the swarming process. Two uropathogenic g
p y
The present study was focused on the adjustment in the lipid composition and or-
ganization in the P. mirabilis rods induced by the swarming process. Two uropathogenic
strains capable of swarming motility were initially assessed for the duration of the lag
phase, speed of expansion, and the visual appearance of the colony. Morphologically
differentiated forms of bacterial cells were compared with each other in terms of their fatty
acid and phospholipid profiles. This step was carried out by applying the gas or liquid
chromatography separation of the cellular components and their identification by mass
spectrometry. Moreover, short-form and elongated rods of the P. mirabilis were examined in
search of co-existing ordered and disordered domains using the Förster resonance energy
transfer (FRET). The research was undertaken due to the highly probable connection of
lipid alterations induced by the elongation of P. mirabilis rods with enhanced pathogenic-
ity of the morphotype developed by the swarmer cells in order to be more effective in
causing UTIs. organization in the P. mirabilis rods induced by the swarming process. Two uropathogenic
strains capable of swarming motility were initially assessed for the duration of the lag
phase, speed of expansion, and the visual appearance of the colony. Morphologically
differentiated forms of bacterial cells were compared with each other in terms of their fatty
acid and phospholipid profiles. This step was carried out by applying the gas or liquid
chromatography separation of the cellular components and their identification by mass
spectrometry. Moreover, short-form and elongated rods of the P. mirabilis were examined
in search of co-existing ordered and disordered domains using the Förster resonance
energy transfer (FRET). The research was undertaken due to the highly probable
connection of lipid alterations induced by the elongation of P. mirabilis rods with enhanced
pathogenicity of the morphotype developed by the swarmer cells in order to be more
effective in causing UTIs. 2. Results p
p
p
Many bacterial lipid-associated virulence factors recognized by the human innate
immune response, such as LPS in Gram-negative bacteria, lipoteichoic acid in Gram-
positive bacteria, and lipoglycans in mycobacteria interact specifically with the host cell
machinery during the bacterial infection. Despite the fact that both Escherichia coli and
Mycobacterium tuberculosis differ in their cell envelope composition and organization, their
cell wall-associated lipid components are considered as virulence factors disrupting host
cellular signaling and finally leading to the development of diseases. A review of these
lipid-derived molecules, active in various stages of host–pathogen interactions, was exten-
sively presented by Dadhich and Kapoor [25]. Recently, information regarding aminoacyl
phospholipids has appeared in the literature. These lipids have proven to significantly
increase bacterial resistance to antibiotics, bacteriocins, and host defense molecules, as
well as contributing to enhanced virulence. Moreover, membrane flexibility and stability
regulated by aminoacyl phospholipids enables bacteria to control processes such as sporu-
lation, conjugation, biofilm formation, and swarming motility [26]. On the other hand, lipid
properties make them an attractive target for pathogens to modulate host cell processes
to allow for their replication and survival. Various strategies which pathogens use for
the modulation of cellular processes by modifying host cellular lipid homeostasis were
summarized by van der Meer-Janssen [27]. Taking into account the connection between
bacterial lipid factors and their cells pathogenicity, the recognition and understanding of
the changes in the rod lipidome occurring during the swarming process are extremely
important in the context of P. mirabilis-mediated UTIs development. Biological membranes are composed of dynamic lipid and protein clusters, rather
than forming a homogeneous mosaic. It has been hypothesized that affinity between lipids
leads to the creation of lipid microdomains with unique compositions, which provide
environments favorable to the activity of certain proteins. Recently, sphingolipid- and
cardiolipin-enriched liquid-ordered domains drifting between liquid-disordered phospho-
lipids, named as lipid rafts, are of particular interest due to their close relation with cell
signaling proteins located both in the inner and outer membrane leaflets [20]. Lipid rafts
or raft-like lipid heterogeneities containing high melting lipids may also determine and Int. J. Mol. Sci. 2023, 24, 16461 4 of 18
also
n has maintain the physical properties of membranes. This kind of domain has been noticed in
some microorganisms such as E. coli, Bacillus subtilis, and Staphylococcus aureus [28,29]. aureus [28,29]. 2. Results
2.1. Expans
S Statistical significance is indicated as * (p-value ≤0.05), ** (p-value ≤0.01), *** (p-value
≤0.001), and ns means not significant (p-value > 0.05). Panels A show images of the elongated
P. mirabilis rods produced by phase-contrast microscopy (magnification = 600×, scale bar = 5 µm). Panels B show a characteristic swarming pattern, formed by Proteus sp. The ATCC 29906 (1) and 1984
(2) strains are presented in both panels. Figure 1. Swarming zones of the P. mirabilis measured over time. The error values equal the standard
deviations. Statistical significance is indicated as * (p-value ≤ 0.05), ** (p-value ≤ 0.01), *** (p-value ≤
0.001), and ns means not significant (p-value > 0.05). Panels A show images of the elongated P. mirabilis rods produced by phase-contrast microscopy (magnification = 600×, scale bar = 5 µm). Panels B show a characteristic swarming pattern, formed by Proteus sp. The ATCC 29906 (1) and
1984 (2) strains are presented in both panels. Figure 1. Swarming zones of the P. mirabilis measured over time. The error values equal the standard
deviations. Statistical significance is indicated as * (p-value ≤0.05), ** (p-value ≤0.01), *** (p-value
≤0.001), and ns means not significant (p-value > 0.05). Panels A show images of the elongated
P. mirabilis rods produced by phase-contrast microscopy (magnification = 600×, scale bar = 5 µm). Panels B show a characteristic swarming pattern, formed by Proteus sp. The ATCC 29906 (1) and 1984
(2) strains are presented in both panels. Macroscopically, bull’s-eye patterns formed by the tested strains appeared to be almost
the same (panels B1 and B2 in Figure 1). However, greater colony spreading, as well as more
visibly separated swarming and consolidation terraces, were characteristic for the pattern
of the ATCC 29906 strain. Moreover, microscopic analyses showed relevant differences in
the visual appearance of the P. mirabilis swarm fronts (Figure 2). Unlike the reference strain
with a smooth front of the outermost migration ring, the 1984 strain was distinguished by
forming finger-shaped projections in this plate region. Int. J. Mol. Sci. 2023, 24, 16461 5 of 18
he
as Figure 2. The time-dependent spreading of the ATCC 29906 (1) and 1984 (2) swarmer cells across an
agar surface presented by a sequence of phase-contrast microscope images. Swarm fronts are shown
in A panels (magnification = 40×; scale bar = 20 µm). 2. Results
2.1. Expans
S These alterations involved the deprivation of the long-form
cells of PE 33:1 and PG 31:2 and their enrichment with PE 32:1, PE 34:1, PE 34:2, PG 30:2,
PG 32:1, and PG 34:1, compared to the short-form cells. g y
p
q
p
the double bond index (DBI) values remained unchanged (Table 1) after the elongation of
the P. mirabilis cells. Constant PG/PE ratios resulted from compensating for the lower
content of several lipid species with higher levels of the other species, within the same
phospholipid class. For instance, the increase in the PE 32:1, PE 34:1, and PE 34:2 total
quantity (~22.42%) was compensated for by the decrease in the PE 33:1 level (~23.23%),
while the increased total content of PG 32:1 and PG 34:1 (~11.69%) was compensated for
by the decreased total level of PG 31:2 and PG 33:1 (~12.14%). Therefore, proportions
between PGs and PEs remained similar after the morphological modifications of the P. mirabilis 1984 rods. Th
d
bl
b
d i d
(T bl
1)
l
l t d f
ll PL
f
d t
th
p
Interestingly, despite quantitative alterations in almost all PL species, the PG/PE and
the double bond index (DBI) values remained unchanged (Table 1) after the elongation of the
P. mirabilis cells. Constant PG/PE ratios resulted from compensating for the lower content
of several lipid species with higher levels of the other species, within the same phospholipid
class. For instance, the increase in the PE 32:1, PE 34:1, and PE 34:2 total quantity (~22.42%)
was compensated for by the decrease in the PE 33:1 level (~23.23%), while the increased total
content of PG 32:1 and PG 34:1 (~11.69%) was compensated for by the decreased total level
of PG 31:2 and PG 33:1 (~12.14%). Therefore, proportions between PGs and PEs remained
similar after the morphological modifications of the P. mirabilis 1984 rods. The double bond index (Table 1), calculated for all PLs, referred to the average
number of double bonds in the fatty acids esterified to polar lipid molecular species,
p
g
The double bond index (Table 1), calculated for all PLs, referred to the average number
of double bonds in the fatty acids esterified to polar lipid molecular species, indicating the
level of PL unsaturation. Similarly to the PG/PE ratios, the DBI values remained constant
for the P. mirabilis rods, independently of their morphotype. 2. Results
2.1. Expans
S Course of the edge of the swarm colony over a
four-minute is shown in B panels (magnification = 200×; scale bar = 20 µm). Figure 2. The time-dependent spreading of the ATCC 29906 (1) and 1984 (2) swarmer cells across an
agar surface presented by a sequence of phase-contrast microscope images. Swarm fronts are shown
in A panels (magnification = 40×; scale bar = 20 µm). Course of the edge of the swarm colony over a
four-minute is shown in B panels (magnification = 200×; scale bar = 20 µm). Figure 2. The time-dependent spreading of the ATCC 29906 (1) and 1984 (2) swarmer cells across an
agar surface presented by a sequence of phase-contrast microscope images. Swarm fronts are shown
in A panels (magnification = 40×; scale bar = 20 µm). Course of the edge of the swarm colony over a
four-minute is shown in B panels (magnification = 200×; scale bar = 20 µm). Figure 2. The time-dependent spreading of the ATCC 29906 (1) and 1984 (2) swarmer cells across an
agar surface presented by a sequence of phase-contrast microscope images. Swarm fronts are shown
in A panels (magnification = 40×; scale bar = 20 µm). Course of the edge of the swarm colony over a
four-minute is shown in B panels (magnification = 200×; scale bar = 20 µm). 2.2. Alterations in the P. mirabilis Phospholipid Profile Occurring during Its Rods Elongation
Th
l
f
d
i
th
lt
hi h
f
li
id
h
t
h
2.2. Alterations in the P. mirabilis Phospholipid Profile Occurring during Its Rods Elongation The analyses performed using the ultra-high performance liquid chromatography
with tandem mass spectrometry (UHPLC–MS/MS) provided important information on
the qualitative and quantitative compositions of phospholipids building morphologically
differentiated cells of the P. mirabilis formed during the swarming process. Phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) represented
The analyses performed using the ultra-high performance liquid chromatography
with tandem mass spectrometry (UHPLC–MS/MS) provided important information on
the qualitative and quantitative compositions of phospholipids building morphologically
differentiated cells of the P. mirabilis formed during the swarming process. Phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) represented
respectively almost 70% and nearly 30% of the total PLs content. The 26 major PL species
identified in the bacterial cells are shown in Table 1. 2. Results
2.1. Expans
S A notable influence of the swarming process on the P mirabilis lipid composition was
g
g p
Phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) represented re-
spectively almost 70% and nearly 30% of the total PLs content. The 26 major PL species
identified in the bacterial cells are shown in Table 1. A notable influence of the swarming process on the P. mirabilis lipid composition was
indicated using our UHPLC–MS/MS generated profiles. Statistically significant changes
in the levels of 23 PLs species, among the 26 identified, were found. The increase in the
amounts of the 32- and 34-carbon phospholipids, as well as strong reduction in the levels
of the 31- and 33-carbon lipids, were specific for all swarmer cells. The first one ranged
from 36% to 600% for the PE 34:1 and PE 34:2 levels, respectively. The second was on the
border of limit detection in most cases (like for PE 31:0, PE 33:0, PE 33:1, PG 31:2, and PG
33:1). It was confirmed that drastic modifications in the morphology of the P. mirabilis rods
that occur during the swarming process were directly related to the changes in the
phospholipid profile of the elongated cells. These alterations involved the deprivation of
the long-form cells of PE 33:1 and PG 31:2 and their enrichment with PE 32:1, PE 34:1, PE
34:2, PG 30:2, PG 32:1, and PG 34:1, compared to the short-form cells. Interestingly, despite quantitative alterations in almost all PL species, the PG/PE and
A notable influence of the swarming process on the P. mirabilis lipid composition was
indicated using our UHPLC–MS/MS generated profiles. Statistically significant changes
in the levels of 23 PLs species, among the 26 identified, were found. The increase in the
amounts of the 32- and 34-carbon phospholipids, as well as strong reduction in the levels of
the 31- and 33-carbon lipids, were specific for all swarmer cells. The first one ranged from
36% to 600% for the PE 34:1 and PE 34:2 levels, respectively. The second was on the border
of limit detection in most cases (like for PE 31:0, PE 33:0, PE 33:1, PG 31:2, and PG 33:1). It was confirmed that drastic modifications in the morphology of the P. mirabilis rods that
occur during the swarming process were directly related to the changes in the phospholipid
profile of the elongated cells. 2. Results
2.1. Expans
S The features are color coded by row with yellow indicating high intensity and purple
indicating low intensity. The underlying numerical data used to generate this figure (along with
statistical significance values) are available in Table 1 and Table 2. t
Figure 3. Heat map generated from quantitation of phospholipids and fatty acids in the short- and
long-form rods of the P. mirabilis. Morphologically differentiated cells are shown on the lower x-axis,
while the lipid analytes are presented on the y-axis. Only the seventeen most important phospholipids
and three most important fatty acids (based on their levels ≥1% and p-values ≤0.05) are displayed. The features are color coded by row with yellow indicating high intensity and purple indicating
low intensity. The underlying numerical data used to generate this figure (along with statistical
significance values) are available in Tables 1 and 2. 2.3. Modifications in the P. mirabilis Fatty Acid Profile Occurring during Its Rods Elongation
Using a gas chromatography-mass spectrometry technique (GC–MS), 12 types of
fatty acids in the morphologically differentiated cells of P. mirabilis formed during the
swarming growth were recognized and quantified. Mass spectra illustrated that acyl
chains of FAs varied in length between 14 and 20 carbon atoms. The major saturated FAs
(SFAs) were hexadecanoic (palmitic) acid (C16:0) and octadecanoic (stearic) acid (C18:0),
Table 1. Relative abundances (%) of phospholipids isolated from the swarmer and non-swarmer cells
of the P. mirabilis strains. PL Species
P. mirabilis ATCC 29906 Cells
P. 2. Results
2.1. Expans
S It was probably related to
the negligible impact of the rods elongation process on the saturation of fatty acids, and a
greater effect on their variable distribution, i.e., attachment to other phospholipid heads,
leading to a formation of different PL species. These results suggested that the changes in
the rods phenotype occurring during swarming motility did not concern the saturation of
the phospholipid acyl chains of the P. mirabilis rods. A visual summary of the above data is presented as a simple heat map in which colors
represent the obtained values (Figure 3). Int. J. Mol. Sci. 2023, 24, 16461 6 of 18 Figure 3. Heat map generated from quantitation of phospholipids and fatty acids in the short- and
long-form rods of the P. mirabilis. Morphologically differentiated cells are shown on the lower x-
axis, while the lipid analytes are presented on the y-axis. Only the seventeen most important
phospholipids and three most important fatty acids (based on their levels ≥ 1% and p-values ≤ 0.05)
are displayed. The features are color coded by row with yellow indicating high intensity and purple
indicating low intensity. The underlying numerical data used to generate this figure (along with
statistical significance values) are available in Table 1 and Table 2. 2 3 Modifications in the P mirabilis Fatty Acid Profile Occurring during Its Rods Elongation
Figure 3. Heat map generated from quantitation of phospholipids and fatty acids in the short- and
long-form rods of the P. mirabilis. Morphologically differentiated cells are shown on the lower x-axis,
while the lipid analytes are presented on the y-axis. Only the seventeen most important phospholipids
and three most important fatty acids (based on their levels ≥1% and p-values ≤0.05) are displayed. The features are color coded by row with yellow indicating high intensity and purple indicating
low intensity. The underlying numerical data used to generate this figure (along with statistical
significance values) are available in Tables 1 and 2. d f
f
h
h l
d
d f
d
h
h
d Figure 3. Heat map generated from quantitation of phospholipids and fatty acids in the short- and
long-form rods of the P. mirabilis. Morphologically differentiated cells are shown on the lower x-
axis, while the lipid analytes are presented on the y-axis. Only the seventeen most important
phospholipids and three most important fatty acids (based on their levels ≥ 1% and p-values ≤ 0.05)
are displayed. 2. Results
2.1. Expans
S mirabilis 1984 Cells
Short-Form
Long-Form
Short-Form
Long-Form
PG 29:2
0.91 ± 0.6
0.10 ± 0.7
ns
0.87 ± 0.0
0.37 ± 0.5
ns
PG 30:0
0.36 ± 0.1
0.98 ± 0.4
*
0.24 ± 0.0
1.02 ± 0.4
**
PG 30:1
0.73 ± 0.1
1.08 ± 0.4
ns
1.23 ± 0.1
1.51 ± 0.6
ns
PG 30:2
5.09 ± 0.3
7.92 ± 0.2
***
4.99 ± 0.6
7.77 ± 0.5
***
PG 31:2
11.0 ± 0.6
0.81 ± 0.2
***
10.9 ± 1.1
0.95 ± 0.4
***
PG 32:1
2.11 ± 0.2
9.98 ± 1.5
***
1.74 ± 0.1
8.14 ± 0.1
***
PG 32:2
2.96 ± 0.3
5.30 ± 0.2
*
3.61 ± 0.4
5.32 ± 0.6
*
PG 33:1
2.94 ± 0.3
0.59 ± 0.3
***
2.84 ± 0.3
0.65 ± 0.3
***
PG 34:0
0.22 ± 0.0
0.85 ± 0.2
***
0.27 ± 0.0
0.89 ± 0.2
**
PG 34:1
1.86 ± 0.1
8.65 ± 1.2
***
2.50 ± 0.1
7.80 ± 1.3
***
PG/PE
ratio
0.43 ± 0.0
0.59 ± 0.2
ns
0.45 ± 0.0
0.54 ± 0.1
ns
DBI
1.17 ± 0.0
1.17 ± 0.1
ns
1.14 ± 0.0
1.18 ± 0.2
ns
Values represent mean percentages of the phospholipid pool. The error values equal the standard deviations. PG:
phosphatidylglycerol, PE: phosphatidylethanolamine, DBI: double bond index. Statistical significance is indicated
as * (p-value ≤0.05), ** (p-value ≤0.01), *** (p-value ≤0.001), and ns means not significant (p-value > 0.05). Table 2. Relative abundances (%) of fatty acids determined in the swarmer and non-swarmer cells of
the P. mirabilis strains. the P. mirabilis strains. FAs
P. mirabilis ATCC 29906 Cells
P. 2. Results
2.1. Expans
S mirabilis 1984 Cells
Short-Form
Long-Form
Short-Form
Long-Form
PE 30:0
4.13 ± 0.4
3.84 ± 0.4
ns
4.02 ± 0.2
3.64 ± 0.6
ns
PE 30:1
1.44 ± 0.1
1.84 ± 0.2
ns
0.88 ± 0.0
1.31 ± 0.2
*
PE 31:0
2.35 ± 0.1
0.18 ± 0.2
***
2.12 ± 0.0
0.51 ± 0.5
**
PE 32:0
2.90 ± 0.1
4.59 ± 0.5
*
3.79 ± 0.2
5.01 ± 1.7
ns
PE 32:1
14.6 ± 0.3
20.6 ± 0.4
*
13.1 ± 0.2
21.1 ± 0.4
**
PE 32:2
0.33 ± 0.1
0.35 ± 0.3
ns
0.25 ± 0.0
0.86 ± 0.2
**
PE 33:0
2.74 ± 0.1
0.11 ± 0.1
***
2.80 ± 0.1
0.09 ± 0.0
***
PE 33:1
23.5 ± 0.2
1.49 ± 0.2
***
23.9 ± 0.5
0.63 ± 0.8
***
PE 34:0
0.85 ± 0.1
1.17 ± 0.1
*
1.05 ± 0.1
1.46 ± 0.2
ns
PE 34:1
7.28 ± 0.7
12.4 ± 1.2
**
9.61 ± 0.5
13.0 ± 1.4
*
PE 34:2
4.78 ± 0.8
11.3 ± 0.7
**
2.20 ± 0.6
13.2 ± 0.5
***
PE 36:0
0.15 ± 0.0
0.94 ± 0.3
**
0.10 ± 0.0
0.65 ± 0.3
***
PE 36:2
0.81 ± 0.2
1.30 ± 0.2
**
0.82 ± 0.1
0.85 ± 0.5
ns
PE 37:2
1.54 ± 0.1
2.34 ± 0.2
**
1.39 ± 0.0
1.95 ± 0.2
**
PE 38:2
2.89 ± 0.3
0.07 ± 0.1
***
3.20 ± 0.0
0.08 ± 0.1
***
PG 28:1
1.59 ± 0.1
1.17 ± 0.4
ns
1.66 ± 0.1
1.24 ± 0.3
* 3. Modifications in the P. mirabilis Fatty Acid Profile Occurring during Its Rods Elongation
Using a gas chromatography-mass spectrometry technique (GC–MS), 12 types of
atty acids in the morphologically differentiated cells of P. mirabilis formed during the
Table 1. Relative abundances (%) of phospholipids isolated from the swarmer and non-swarmer cells
of the P. mirabilis strains. Int. J. Mol. Sci. 2023, 24, 16461 7 of 18 Table 1. Cont. PL Species
P. mirabilis ATCC 29906 Cells
P. 2. Results
2.1. Expans
S mirabilis 1984 Cells
Short-Form
Long-Form
Short-Form
Long-Form
C14:0
1.95 ± 0.3
2.04 ± 0.3
ns
1.95 ± 0.2
1.77 ± 0.1
ns
C15:0
0.88 ± 0.8
1.75 ± 0.2
ns
1.84 ± 0.1
1.47 ± 0.2
ns
iso-C16:0
1.64 ± 0.3
1.83 ± 0.2
ns
1.34 ± 0.3
1.48 ± 0.2
ns
C16:0
36.5 ± 1.1
36.5 ± 4.2
ns
39.0 ± 2.8
36.8 ± 0.7
ns
cis C16:1
2.37 ± 0.4
2.67 ± 0.5
ns
2.43 ± 0.1
2.05 ± 0.4
ns
C17:0
1.68 ± 0.3
1.93 ± 0.2
ns
1.63 ± 0.2
1.63 ± 0.2
ns
cy C17:0
3.01 ± 0.8
1.76 ± 0.3
*
3.89 ± 0.2
1.46 ± 0.2
***
C18:0
44.0 ± 1.6
43.5 ± 4.3
ns
39.9 ± 3.5
46.0 ± 1.9
*
cis C18:1
2.52 ± 0.5
2.45 ± 0.3
ns
2.90 ± 0.4
2.18 ± 0.2
ns
C19:0
1.65 ± 0.3
1.19 ± 1.1
ns
1.39 ± 0.2
1.58 ± 0.2
ns
cy C19:0
1.90 ± 0.4
1.99 ± 0.3
ns
2.13 ± 0.1
1.66 ± 0.2
**
C20:0
1.98 ± 0.4
2.32 ± 0.3
ns
1.64 ± 0.3
1.96 ± 0.2
ns
C16/C18
0.87 ± 0.0
0.89 ± 0.2
ns
1.00 ± 0.2
0.84 ± 0.0
ns
SFA
90.2 ± 1.9
91.1 ± 1.2
ns
88.7 ± 0.7
92.6 ± 0.8
***
UFA
4.89 ± 0.9
5.12 ± 0.7
ns
5.33 ± 0.5
4.24 ± 0.5
*
UFA/SFA
0.05 ± 0.0
0.06 ± 0.0
ns
0.06 ± 0.0
0.05 ± 0.0
ns
CFA
4.91 ± 0.1
3.75 ± 0.5
ns
6.02 ± 0.2
3.12 ± 0.3
***
CFA/UFA
1.00 ± 0.0
0.73 ± 0.0
***
1.13 ± 0.1
0.74 ± 0.1
***
straight/branched-
chain
FAs
9.20 ± 2.2
10.3 ± 1.6
ns
7.81 ± 0.5
12.6 ± 1.5
*
Values represent mean percentages of the total fatty acids pool. The error values equal the standard deviations. SFA: saturated fatty acids, UFA: unsaturated fatty acids, CFA: cyclopropane fatty acids, FAs: fatty acids. Statistical
significance is indicated as * (p-value ≤0.05), ** (p-value ≤0.01), *** (p-value ≤0.001), and ns means not significant
(p-value > 0.05). Values represent mean percentages of the total fatty acids pool. The error values equal the standard deviations. SFA: saturated fatty acids, UFA: unsaturated fatty acids, CFA: cyclopropane fatty acids, FAs: fatty acids. 2.4. Lipid Organization in the P. mirabilis Rods
2.4. Lipid Organization in the P. mirabilis Rods The FRET method was used to reveal the degree of the bacterial membrane order. The
donor (pyrene PE) was distributed into the liquid-ordered phase, while the acceptor (rhod
PE) was located in the liquid-disordered phase [30]. The ratios of donor fluorescence in
the presence of the acceptor to that in its absence (F/Fo) are presented in Figure 4. High
F/Fo values (even 0.94 ± 0.07) were observed in the short-form cells of P. mirabilis. Weak
FRET corresponded to strongly ordered bilayers. The swarming phenomenon led to a
significant reduction in F/Fo values for the bacterial rods. High values of energy transfer
fractions from the donor to the acceptor (even 0.966) corresponded to the homogeneous
lipid bilayers of the elongated P. mirabilis rods. The FRET method was used to reveal the degree of the bacterial membrane order. The donor (pyrene PE) was distributed into the liquid-ordered phase, while the acceptor
(rhod PE) was located in the liquid-disordered phase [30]. The ratios of donor fluorescence
in the presence of the acceptor to that in its absence (F/Fo) are presented in Figure 4. High
F/Fo values (even 0.94 ± 0.07) were observed in the short-form cells of P. mirabilis. Weak
FRET corresponded to strongly ordered bilayers. The swarming phenomenon led to a
significant reduction in F/Fo values for the bacterial rods. High values of energy transfer
fractions from the donor to the acceptor (even 0.966) corresponded to the homogeneous
lipid bilayers of the elongated P. mirabilis rods. Figure 4. Ability to form ordered-state bilayers by the P. mirabilis lipids, as assayed by the Förster
resonance energy transfer technique. The values of fractions of energy transfer are located at the
bottom of the bars. The error bars equal the standard deviations. Statistical significance is indicated
as *** (p-value ≤ 0.001). The confocal microscopy images present short- (A) and long-form cells (B)
of the ATCC 29906 (1) and 1984 (2) strains stained with a membrane probe Rhod PE (magnification
= 1000×; scale bar = 5 µm). Red arrows indicate morphologically differentiated bacterial rods of P. mirabilis labeled with the fluorescent probe
Figure 4. Ability to form ordered-state bilayers by the P. mirabilis lipids, as assayed by the Förster
resonance energy transfer technique. The values of fractions of energy transfer are located at the
bottom of the bars. The error bars equal the standard deviations. Statistical significance is indicated
as *** (p-value ≤0.001). 2. Results
2.1. Expans
S Statistical
significance is indicated as * (p-value ≤0.05), ** (p-value ≤0.01), *** (p-value ≤0.001), and ns means not significant
(p-value > 0.05). 2.3. Modifications in the P. mirabilis Fatty Acid Profile Occurring during Its Rods Elongation Using a gas chromatography-mass spectrometry technique (GC–MS), 12 types of fatty
acids in the morphologically differentiated cells of P. mirabilis formed during the swarming
growth were recognized and quantified. Mass spectra illustrated that acyl chains of FAs
varied in length between 14 and 20 carbon atoms. The major saturated FAs (SFAs) were
hexadecanoic (palmitic) acid (C16:0) and octadecanoic (stearic) acid (C18:0), while the major Int. J. Mol. Sci. 2023, 24, 16461
Int. J. Mol. Sci. 2023, 24, x FOR P 8 of 18
of 17 8 of 18
of 17 unsaturated FAs (UFAs) were 9-hexadecenoic (palmitoleic) acid (C16:1) and 9-octadecenoic
(oleic) acid (C18:1), both in the cis conformation. Two cyclopropane FAs (CFAs) called
9,10-methyl hexadecanoic acid (cy C17:0) and 11,12-methyl octadecanoic acid (cy C19:0)
were also identified, both in the cis conformation (Table 2). The obtained results show that
the 15 PEs and 11 PGs of P. mirabilis were composed mostly of C16:0 and C18:0 (~80% in
total), and to a lesser extent of C16:1 and C18:1 (~5% in total). while the major unsaturated FAs (UFAs) were 9-hexadecenoic (palmitoleic) acid (C16:1)
and 9-octadecenoic (oleic) acid (C18:1), both in the cis conformation. Two cyclopropane
FAs (CFAs) called 9,10-methyl hexadecanoic acid (cy C17:0) and 11,12-methyl
octadecanoic acid (cy C19:0) were also identified, both in the cis conformation (Table 2). The obtained results show that the 15 PEs and 11 PGs of P. mirabilis were composed mostly
of C16:0 and C18:0 (~80% in total), and to a lesser extent of C16:1 and C18:1 (~5% in total). The swarming process affected FA composition of the 1984 strain more than that of
ATCC 29906. As shown in Table 2, the levels of cy C17:0 and cy C19:0 were significantly
decreased (by about 62% and 22%, respectively) in the long-form rods of P. mirabilis. In
effect, the CFA/UFA ratio was reduced from 1.13 to 0.74. Interestingly, the saturation
of fatty acyl chains increased significantly in the elongated 1984 cells, in contrast to the
branched fatty acyl chains, which remained unchanged. On the other hand, the constant
values of UFA/SFA for both P. 2. Results
2.1. Expans
S mirabilis strains were in line with the unchanged DBI values
presented in Table 1, which confirmed that phenotypic modifications in their rods induced
by swarming motility were independent of the saturation of the phospholipid tails. A
vast majority of other FA modifications occurring in bacterial cells during their movement
turned out to be statistically insignificant. It seems that the swarming process has a
negligible effect on fatty acids synthesis but may affect FA distribution for the formation of
the phospholipid species required by the long-form rods. The swarming process affected FA composition of the 1984 strain more than that of
ATCC 29906. As shown in Table 2, the levels of cy C17:0 and cy C19:0 were significantly
decreased (by about 62% and 22%, respectively) in the long-form rods of P. mirabilis. In
effect, the CFA/UFA ratio was reduced from 1.13 to 0.74. Interestingly, the saturation of
fatty acyl chains increased significantly in the elongated 1984 cells, in contrast to the
branched fatty acyl chains, which remained unchanged. On the other hand, the constant
values of UFA/SFA for both P. mirabilis strains were in line with the unchanged DBI values
presented in Table 1, which confirmed that phenotypic modifications in their rods induced
by swarming motility were independent of the saturation of the phospholipid tails. A vast
majority of other FA modifications occurring in bacterial cells during their movement
turned out to be statistically insignificant. It seems that the swarming process has a
negligible effect on fatty acids synthesis but may affect FA distribution for the formation
of the phospholipid species required by the long-form rods. p
p
p
p
q
y
g
A visual summary of the above data is presented in Figure 3. p
p
p
p
q
y
g
A visual summary of the above data is presented in Figure 3. 3. Discussion P. mirabilis, a member of the Morganellaceae, is a urinary tract pathogen which is
problematic for catheterized patients. Complications include not only a catheter blockage
by encrustation but also a risk of ascending infections enabled by uropathogen migration
over the catheter surface. Nevertheless, the role of swarmer cell differentiation in the
virulence of P. mirabilis is still a matter of debate. In laboratory conditions, a P. mirabilis colony spreads on agar forming the bull’s-eye
pattern by consecutive phases of rapid swarming followed by consolidation into shorter
rods [14]. This characteristic pattern was observed during the migration of the tested
uropathogens. However, the consolidation zones of the 1984 strain were tightly clustered,
which meant shorter periods of swarming, compared to those of the ATCC 29906 strain. Consequently, the migration distance of the 1984 strain was shorter by about 32 mm in
24 h of the swarming process, in comparison to the result obtained for ATCC 29906. It is
well known that a number of various factors, such as agar concentration [31], inoculation
density and incubation temperature [32], and specific nutrients and environmental cues [13]
strongly affect the kinetics of the colony area expansion and its appearance. However, it is
not an explanation for differences in spreading rates of the 1984 and ATCC 29906 strains
incubated in the same conditions. Another possible cause could be a twofold extension
of the lag phase of the P. mirabilis 1984 strain. Some reports indicate that the lag phase is
needed for the excretion of biosurfactants which facilitate microbial motility by lowering
the surface tension [33]. Among available papers, only Kannan et al. [34] described P. mirabilis DMTMMK1 as a biosurfactant producer. However, preliminary studies did not
detect the surfactants’ presence in the swarm colony of either the 1984 or ATCC 29906 strain. On the other hand, delayed differentiation of short-form rods to swarmer cells could be
associated with impaired putrescine secretion. Sturgill and Rather [35] indicated that the P. mirabilis PM437 strain with a disrupted speB gene (encoding putrescine) was featured by a
two-hour elongated lag phase, incapability of effective migration across agar surfaces, and
formation of very closely spaced swarming rings. However, confirmation of this hypothesis
would require supplementation with exogenous putrescine or carrying out research on the
1984 and ATCC 29906 strains at the genetic level. Another cause of varied expansion rates
of the P. 2.4. Lipid Organization in the P. mirabilis Rods
2.4. Lipid Organization in the P. mirabilis Rods The confocal microscopy images present short- (A) and long-form cells (B)
of the ATCC 29906 (1) and 1984 (2) strains stained with a membrane probe Rhod PE (magnification
= 1000×; scale bar = 5 µm). Red arrows indicate morphologically differentiated bacterial rods of P. mirabilis labeled with the fluorescent probe. Figure 4. Ability to form ordered-state bilayers by the P. mirabilis lipids, as assayed by the Förster
resonance energy transfer technique. The values of fractions of energy transfer are located at the
bottom of the bars. The error bars equal the standard deviations. Statistical significance is indicated
as *** (p-value ≤ 0.001). The confocal microscopy images present short- (A) and long-form cells (B)
of the ATCC 29906 (1) and 1984 (2) strains stained with a membrane probe Rhod PE (magnification
= 1000×; scale bar = 5 µm). Red arrows indicate morphologically differentiated bacterial rods of P. mirabilis labeled with the fluorescent probe
Figure 4. Ability to form ordered-state bilayers by the P. mirabilis lipids, as assayed by the Förster
resonance energy transfer technique. The values of fractions of energy transfer are located at the
bottom of the bars. The error bars equal the standard deviations. Statistical significance is indicated
as *** (p-value ≤0.001). The confocal microscopy images present short- (A) and long-form cells (B)
of the ATCC 29906 (1) and 1984 (2) strains stained with a membrane probe Rhod PE (magnification
= 1000×; scale bar = 5 µm). Red arrows indicate morphologically differentiated bacterial rods of P. mirabilis labeled with the fluorescent probe. Int. J. Mol. Sci. 2023, 24, 16461 9 of 18 The green channel of the confocal microscope visualized the Rhod PE-stained P. mirabilis membranes. The short-form rods displayed a weak membrane fluorescence,
while the elongated rods showed a strong fluorescence of the whole cells (panels A1–B2
in Figure 4). The imaging confirmed modifications in the arrangement of the bacterial
membrane components occurring during the swarming process. 3. Discussion mirabilis 1984 and ATCC 29906 swarm colonies could be differentiated surface
density of flagella. Tuson et al. [36] proved that cell length has a small effect on motility
but overexpression of FlhD4C2, the major regulator of the flagellar operon in vegetative
cells of P. mirabilis, increases flagellum density resulting in an increased cell velocity. On
the other hand, it is worth mentioning that a delay or a velocity of spreading rods are
individual features of each bacterial strain. For the ATCC 29906 and 1984 strains, colony
expansion rates were 3.77 mm h−1 and 2.45 mm h−1, respectively, for P. mirabilis BB2000
maximum swarming translocation speed was 7 mm h−1 [37], while for P. mirabilis PRM1
it reached 1 mm h−1 [32], and for P. mirabilis HI4320 it was 1.36 mm h−1 [14]. Data report
that even when the growth kinetics in liquid environments and swarmer cell migrations
are quantitatively similar, the minimum length of the lag phase varies by over 40% among
different clinical isolates of P. mirabilis [32]. High-magnification microscopy showed that the leading edge of the P. mirabilis
swarmer population is composed of several stacked cell layers, while the population
inside the swarming ring forms a monolayer. Swarmer cells translocate from the prior ter-
race into the newly formed terrace as increasingly thicker populations [32]. In general, the
edge of the active swarm front is smooth, while the edge of the newly formed consolidation
ring generates finger-like projections [14,36,38]. However, actively swarming colonies of Int. J. Mol. Sci. 2023, 24, 16461 10 of 18 the P. mirabilis ATCC 29906 and 1984 strains had a smooth and a “fingered” appearance
edge, respectively, and both were in vigorous motion. Only Rauprich et al. [32] indicated
that the leading edge of P. mirabilis migrating cells was quite ragged in appearance. How-
ever, the encroaching swarm fronts of the following strains Serratia liquefaciens MG1 [39],
B. subtilis JCM 32,485 [40], Bacillus cereus NCIB 8122 [41], and Vibrio parahaemolyticus
RIMD 2,210,633 [42] also resembled finger-like structures migrating outward towards the
colony border. y
The ability of bacteria to control the biophysical properties of their membrane phospho-
lipids enables them to survive under various physical conditions. Moreover, an adjustment
in composition of bacterial membranes, i.e., homoviscous adaptation, is interpreted as
a mechanism of minimizing energy expenditure and optimizing growth just through
modifications of the phospholipid bilayer permeability [22]. 3. Discussion Therefore, we com-pared
morphologically differentiated forms of the P. mirabilis rods in terms of their phospholipid
and fatty acid profiles to associate phenotypic changes induced by swarming motility with
modifications of these cellular components. Firstly, our results showed that proportions
between phosphatidylethanolamines and phosphatidylglycerols were unchanged when
the P. mirabilis rods were swarming. The same results had been previously described for
the P. mirabilis P11 [17] and Paenibacillus polymyxa ATCC 842 strains [43]. Distribution
of phospholipids across the inner membrane of Gram-negative bacteria may be relevant. Bogdanov et al. [44] demonstrated that distribution of PEs was 75% (cytoplasmic leaflet) to
25% (periplasmic leaflet) in rod-shaped cells and quite opposite in elongated E. coli cells. Unfortunately, the UHPLC–MS/MS profiling of the lipid composition of whole cells did
not provide information on the distribution of these membrane molecules. A dynamic and
shape-dependent arrangement of PEs in the inner membrane of Gram-negative bacteria
is an interesting finding which suggests that PE distribution facilitates bacterial shape
changes or is their consequence. Furthermore, the authors suggested that PE asymmetry is
controlled metabolically to balance the synthesis and translocation of the lipid molecules
which is supposed to ensure the capacity of envelope growth and adaptation of chem-
ical and physical properties of the bilayer [44]. Despite the constant PG/PE ratio, the
23 phospholipid species showed significantly different levels during the formation of the
P. mirabilis 1984 and ATCC 29906 swarmers. It was confirmed that drastic modifications
in the morphology of the P. mirabilis rods were directly related to the deprivation of the
long-form cells of PE 33:1 and PG 31:2 and their enrichment with PE 32:1, PE 34:1, PE 34:2,
PG 30:2, PG 32:1, and PG 34:1, compared to the short-form cells. On the other hand, the DBI
values, indicating the level of PL unsaturation, remained constant for the bacterial rods,
independently of their morphotype. These results suggested that the changes in the cells
phenotype occurring during swarming motility did not concern the saturation of the phos-
pholipid acyl chains of P. mirabilis rods. According to Navas et al. [45], the two-dimensional
organization in the PE/PG membranes appears to be dominated by headgroup interactions
and to be largely independent of the hydrocarbon chain length or degree of saturation. This is a probable explanation of the constant PG/PE and DBI values, whose modifications
caused by P. mirabilis rods elongation would be potentially unnecessary. 3. Discussion [50] reported an increased level of anteiso C15:0 in
the swarmer cells of the Serratia marcescens CH-1. The last modification worth mentioning
is a change in the saturation degree of fatty acids. The phospholipid acyl desaturase in-
troduces a cis double bond into fatty acids, causing flexion in the chain, which leads to a
reduction in transition temperatures and elevation of bilayers permeability. Such regulation
of acyl chains packing, described for Bacillus spp. and Pseudomonas spp., is considered to
be necessary for a suitable bacterial reaction to abrupt temperature change [22]. Lack of
dependences between the levels of saturated and monounsaturated FAs, as well as between
the contents of monounsaturated and double unsaturated FAs containing the same number
of carbon atoms in the acyl chains, seems to exclude the activity of desaturases during
morphological changes occurring in the swarming rods of the P. mirabilis ATCC 29906 and
1984 strains. Gué et al. [18] noticed a reduction in the C16:1 level at the benefit of longer C18
chains of FAs with various degrees of unsaturation in swarmer cells of P. mirabilis WT19. However, proportions between the levels of C18:0 and C18:1, as well C18:1 and C18:2, did
not indicate the action of desaturases during the swarming of the WT19 strain. Ghorbal
et al. [49] showed a significant increase in SFAs accompanied by a significant decrease in
UFAs when P. aeruginosa PAO1 cells were undergoing the swarming process. A higher
level of C18:0 associated with a lower level of C18:1 in the PAO1 swarmers may point to
a reduced ∆-9-desaturase activity during cell differentiation. The activity of this enzyme
in microorganisms belonging to Pseudomonas sp. had been discovered earlier by Garba
et al. [51]. On the other hand, Lai et al. [50] presented an increased content of C18:1 in the S. marcescens CH-1 swarmer cells but without a simultaneous decline in the C18:0 level, while
Chow and Schmidt [48] noticed a simultaneous increase in C16:1 and a decrease in C16:0
amounts when C. crescentus cells were swarming. The latter may suggest an enhanced
activity of ∆-9-desaturase creating palmitoleic acid based on palmitic acid. So far, this
enzyme has not been described in the representatives of Caulobacter. y
p
Affinity between lipids may lead to the formation of lipid rafts which stabilize mem-
brane physical properties over varying temperatures and other environmental condi-
tions [28]. 3. Discussion Bacteria modify the acyl chains of their membrane phospholipids in response to chang-
ing environmental conditions. The first of these modifications is cis→trans isomerization. When the phospholipid cis–trans isomerase replaces the cis conformation of the double
bond with the trans conformation, trans FAs gain properties similar to those for saturated
FAs creating membranes with higher transition temperatures [22,46]. The cis to trans iso-
merization of FAs, well described for Pseudomonas putida and the marine psychrophilic
Vibrio, seems to be an adaptation of the lipid composition to higher temperatures [46]. This
adjustment in the membrane composition does not seem to be useful during the formation
of P. mirabilis swarmers. Both short and long forms of the ATCC 29906 and 1984 rods
contain only cis fatty acids. Another modification is cyclopropanation of double bonds. When the cyclopropane fatty acid synthase converts cis double bonds of unsaturated fatty
acyl chains to cyclopropane rings by transfer of a methylene moiety from S-adenosyl-L- Int. J. Mol. Sci. 2023, 24, 16461 11 of 18 11 of 18 methionine, newly formed cyclic FAs stabilize the membranes by increased ordering of
the chains, without the limitation of membrane fluidity. This modification, noticed for E. coli and Salmonella enterica is considered to be crucial for the survival of acid shock [47]. Our results showed decreased levels of cy C17:0 in the swarmer cells of the ATCC 29906
and 1984 strains. Similar results were observed for swarmers of Caulobacter crescentus [48]
and Pseudomonas aeruginosa [49]. Limitation of the cyclopropanation reaction seems likely
to be important for the differentiation process of P. mirabilis elongated cells. The next
modification is changing branched-chain fatty acids from iso to anteiso form, which leads to
altering the position of the methyl group on the acyl-chain. The anteiso fatty acids promote a
higher membrane fluidity than the iso fatty acids, due to a dismissed distance of the methyl
branch from the FA end. Such adjustment in the membrane FA branching, described for
Listeria monocytogenes, is regarded as a membrane response to low temperature and pH
stress [22]. This adaptation of membrane composition does not seem to be useful when
P. mirabilis rods undergo the swarming process. In the present study, it was shown that
both short-form and long-form rods of the ATCC 29906 and 1984 strains were devoid of
anteiso fatty acids, whereas Lai et al. 3. Discussion Bramkamp and Lopez [56] paid attention to
membrane hopanoids in the Gram-negative bacterium Rhodopseudomonas palustris, which
divides asymmetrically into a mother cell and a swarmer cell. These molecules, similar
structurally to sterols, were necessary for proper cell division. Lack of hopanoids led to
the formation of cells connected by their cell wall, forming long filaments. Moreover, it
was noted that this sterole-like molecule could replace cholesterol in membranes, as well
as induce phase separation, which makes it probable that hopanoids are involved in the
assembly of lipid rafts, similarly to cholesterol [56]. Research on the lipid rafts in cells
undergoing the differentiation process is rather a novelty, hence there is little available
information. Mileykovskaya and Dowhan [29] compared wild-type of E. coli E614 and its
filamentous mutant devoid of membrane PEs with an inhibited cell division process. The
authors discovered the cardiolipin-enriched domains in both the filamentous mutant and
the septal and cell poles regions of wild-type E. coli. Interestingly, in the bacterial envelope
there were sites with a higher permeability for used fluorophore, which resulted in different
staining of the filamentous mutant and wild-type cells [29]. We obtained similar results. Confocal microscope photographs of Rhod PE-stained membranes visualized a strong
fluorescence intensity of the whole P. mirabilis swarmer cells in contrast to a weak mem-
brane fluorescence of short-form bacterial rods. A rapid intracellular entry of chromophore
could be explained by the lack of membrane tightness of the elongated cells for Rhod PE
molecules. Gué et al. [18] also observed an increased membrane fluidity in the swarmer
cells of P. mirabilis WT19, compared to the vegetative ones. Ghorbal et al. [57] suggested
that the cellular fatty acid profile is strongly correlated to the swarming phenotype of P. aeruginosa PAO1. Furthermore, Lai et al. [50] hypothesized that the conserved mechanism
of swarming in Gram-negative bacteria involves the control of membrane fluidity. g
g
y
Freeze-fracture electron microscopy and spin-label electron paramagnetic resonance
allowed for demonstrating that the mobility of the spin label of the outer membrane of
P. mirabilis P11 swarmer cells was higher than that of the outer membrane of the non-
swarming short cells, which showed a typical rigid profile [58]. 3. Discussion One of the tools currently applied to study lipid rafts uses fluorescent molecular
probes. The most common are lipophilic fluorescent probes which partition specifically into
liquid ordered or liquid disordered phases [30]. In order to search for lipid rafts in the P. mirabilis rods morphologically adapted for the swarming phenomenon, we used the energy
transfer from the electronic excited donor chromophore (pyrene PE) to the acceptor chro-
mophore (rhod PE). Importantly, previous studies had shown only little effect of the FRET
probes presence on domain formation [52]. Differentiation of the P. mirabilis cells occurring
during the swarming process led to a decrease in F/Fo pointing to the enhancement of the
FRET efficiency in the elongated rods of 1984 and ATCC 29906 strains. Melting of rafts
taking place during the bacterial rods elongation resulted in an increase in the disordered
phase area and spreading of donor and acceptor fluorophores in the membrane area. It Int. J. Mol. Sci. 2023, 24, 16461 12 of 18 12 of 18 corresponded to the homogeneity of lipid bilayers lacking ordered domains. A different
result was obtained for the short-form rods of P. mirabilis. Weak FRET (an efficient donor
quenching) caused by a strong partition of the acceptor chromophore into liquid-disordered
domains corresponded to strong membrane ordering of the P. mirabilis short cells. p
g
g
The presence of lipid rafts was noticed in both Gram-negative and Gram-positive bacte-
ria [28,29]. Formation of ordered-domains in Borrelia burgdorferi B31 turned out to be similar
to the process occurring in eukaryotic cells but with the participation of saturated phos-
phatidylcholines instead of sphingolipids, crucial components of rafts in eukaryotes [53]. On the other hand, phosphatidylethanolamines behaving similarly to unsaturated PLs
were not able to form and stabilize an ordered domain formed spontaneously in the Heli-
cobacter pylori membrane lipid extracts [54]. According to the present knowledge, lipids
with unsaturated acyl chains are characterized by a decreased ability to create ordered
domains relative to lipids containing saturated acyl chains [55]. However, the most notable
difference in the phospholipid profiles of the short- and long-form cells of the P. mirabilis
was a drastic reduction in the PE 33:1 and PG 31:2 total content (~32%) in the first ones. This
may indicate an involvement of PE 33:1 and PG 31:2 in the formation of ordered domains
in the ATCC 29906 and 1984 short-form rods. 4.3. Phase-Contrast Microscopy Imaging 4.3. Phase-Contrast Microscopy Imaging An eclipse TE2000-S Inverted Microscope (Nikon, Tokyo, Japan) equipped with an
air condenser (Nikon LWD, numerical aperture (NA) 0.52) and a 20× phase contrast lens
(Nikon, LWD, Ph1, NA 0.40) was applied for imaging the P. mirabilis rods spreading over
the agar surface. After 18 h of the swarm plate assay (Section 4.2) duration, part of the agar
containing the outermost edge of the swarm colony was cut with a scalpel (Swann-Morton,
Sheffield, England) and transferred onto a microscopic slide (Menzel Gläser, Braunschweig,
Germany). Initially, the sample was illuminated through a 4× objective (NA = 0.10) to
capture a wide field. Subsequently, the objective LWD 20× was used for the presentation
of the P. mirabilis colony movement in 4-min intervals. Phase-contrast microscopy was also applied for imaging the elongated P. mirabilis
rods. After 18 h of the swarm plate assay (Section 4.2) duration, the bacterial cells from the
outermost edge of the swarm colony were imprinted to microscopic slides (Menzel Gläser,
Braunschweig, Germany). Observations were carried out using a 60× Plan Fluor objective
(Nikon, ELWD, Ph2, NA 0.70). 4.1. Bacterial Strains Two P. mirabilis strains capable of swarming motility were investigated in this study. The first one, the clinical strain named 1984, came from the strain’s collection of the Depart-
ment of Biology of Bacteria (University of Lodz, Lodz, Poland). The second, ATCC 29906,
which was a reference strain, was purchased from the American Type Culture Collection
(Manassas, VA, USA). These bacteria had been described in our previous papers [60,61]. 4.4. Preparing Bacteria for Further Research After 18 h of the swarm plate assay (Section 4.2) duration, the P. mirabilis cells from two
zones, i.e., consolidation and the outermost edge of the swarm colony, were collected from
the agar plate surface using sterile swabs (PROFILAB, Warsaw, Poland) and then suspended
in 1 mL of sterile distilled water or PBS solution. Optical densities of the cell suspensions
were adjusted spectrophotometrically to 0.4 (Multiskan EX, LabSystem, Cracow, Poland)
at λ = 550 nm. The short- and long-form cells prepared in this way were used for further
experiments. Each of them was preceded by the verification of the microscopic morphology
of gram-stained bacterial forms. 3. Discussion It suggested physical
reorganization of the outer membrane of swarmer cells associated with an increase in the
flagella number per area unit and LPS rearrangement within the membrane, resulting
in the exposure of phospholipid regions to the external environment and then altered
permeability [17]. Spin labels are usually less rigid in the inner membrane, where the
regions of the lipid bilayer enable a rotation of the lipid polar heads and flexing of the
nonpolar tails [58]. Molecular relaxations in the acyl region regulate diffusion through the
viscosity and elastic properties of the bilayer, such as the bending modulus through the
area compressibility [28]. Auer et al. [59] proved that the lengthening of P. mirabilis HI4320
cells was accompanied by a large increase in flexibility. The cell stiffness (bending rigidity)
was lowered from 1.4 × 10−20 N m−2 to 5.5 × 10−22 N m−2 during rods differentiation. Int. J. Mol. Sci. 2023, 24, 16461 13 of 18 The authors found that enhanced flexibility of P. mirabilis swarmer cells was caused by
modifications in the composition and thickness of the peptidoglycan layer in the walls of
their rods [59]. 4.2. Swarm Plate Assay The P. mirabilis strains were cultured in TSB medium (BioMaxima S.A., Lublin, Poland)
at 37 ◦C for 18 h. Swarming motility was assessed by spotting 5 µL of the late-logarithmic-
phase bacterial culture, adjusted to an optical density of 5 on the McFarland scale, onto
the center of an LB swarm plate containing 1.5% agar (BioMaxima S.A., Lublin, Poland)
followed by incubation at 37 ◦C. The diameter of the swarm colony was measured at
two-hour intervals for 24 h by using a caliper (Schut Geometrical Metrology, Giethoorn,
The Netherlands). The Netherlands). 4.6. Isolation and Identification of Bacterial Fatty Acids The P. mirabilis fatty acids contained in the extract prepared according to the description
in Section 4.5 were released and derivatized to form fatty acid methyl esters (FAMEs) using
the previously described procedure [61]. In short, methanolic HCl (8%) and toluene (Avantor
Performance Materials Poland S.A, Gliwice, Poland) were added to the lipid extracts and then
the solutions were heated at 45 ◦C for at least 18 h (Eppendorf Thermomixer R with Eppendorf
2 ml Thermoblock; Eppendorf, Hamburg, Germany). After derivatization, the samples were
cooled and the FAMEs were extracted with ultrapure hexane (J.T. Baker Chemical Company,
Deventer, the Netherlands) for further GC–MS analyses. )
y
The upper layers of the solutions, containing methylated bacterial fatty acids, were
placed into the inserts of chromatographic vials (Supelco Inc., Bellefonte, PA, USA) and
then analyzed using an Agilent Model 7890 gas chromatograph equipped with a 5975C
mass detector (Santa Clara, CA, USA). Settings of the injection port, temperature gradient of
the capillary column, helium flow, and other method details were provided in our previous
work [61]. The reference standards of bacterial fatty acid methyl esters (Matreya LLC, State
College, PA, USA) were applied for the quantitative method. 4.5. Extraction and Quantification of Bacterial Phospholipids The P. mirabilis lipids were isolated in accordance with our gently modified method
described previously [62]. The bacterial cells obtained according to the description in
Section 4.4 were centrifuged for 15 min at 12,000 rpm (Sigma 1-15 Centrifuge; SIGMA
Laborzentrifugen GmbH, Osterode, Germany). Firstly, 1 mL of the mixture (2:1 v/v)
of chloroform-methanol (Chempur, Piekary Slaskie, Poland) and glass beads (Ø 1 mm)
(Merck, Darmstadt, Germany) were added into an Eppendorf containing the cells washed Int. J. Mol. Sci. 2023, 24, 16461 14 of 18 14 of 18 in distilled water. Rods disintegration and a simultaneous transfer of the lipids to the
organic phase were performed using a ball mill (Retsch MM 400, Haan, Germany) for 5 min
at 30 Hz. Then, 100 µL of 0.85% NaCl was added to the homogenate devoid of glass beads. After quick mixing on the vortex, the sample was centrifuged (Sigma 1-15 Centrifuge;
SIGMA Labor-zentrifugen GmbH, Osterode, Germany) for 5 min at 2000 rpm. A bottom
layer was collected and dried. Lipid extracts were dissolved in ultrapure methanol (J.T. Baker Chemical Company,
Deventer, the Netherlands) and then analyzed using an ExionLC AC UHPLC system
(Sciex, Framingham, MA, USA) coupled with a 4500 QTRAP mass spectrometer (Sciex,
Framingham, MA, USA). Ion source, column specification, gradient and flow of solvents,
and other parameters had been presented in more detail in our earlier paper [61]. To
acquire accurate and precise quantification results, the internal standards PE 14:0/14:0 and
PG 14:0/14:0 (Merck, Darmstadt, Germany) were applied. For phospholipid analysis, an
information-dependent acquisition method (precursor ion or multiple reaction monitoring
enhanced product ion) was also employed. The mass spectra of PGs and PEs were acquired
in the range of m/z 100–1500 and exhibited the ions corresponding to the deprotonated
molecules [M −H]−. Values of the double bond index referring to the degree of unsaturation of phospho-
lipid tails were calculated in accordance with Su et al. [63]. 4.9. Statistical and Other Analyses The presented data are shown as the mean value and standard deviation of three
separate analyses. The significance of results was determined by Fisher’s tests and two-
sample T tests assuming that equal or unequal variances were appropriate. All p-values
are one tailed at a 95% confidence interval. Analyses were performed by using Excel
2016 (Microsoft Corporation, Redmond, WA, USA), the GraphPad Prism 9.1.0 software
(GraphPad, San Diego, CA, USA), and the Analyst v1.6.3 software (Sciex, Framingham,
MA, USA). 4.7. Förster Resonance Energy Transfer Technique Segregation of lipids in the short- and long-form rods of P. mirabilis was detected
using a FRET technique in accordance with the modified method of Huang et al. [54]. Two membrane probes labeled at the polar head group constituting a donor-acceptor pair
were applied. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl)—
pyrene PE was a donor fluorophore, while 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-
N-(lissaminerhodamine B sulfonyl)—rhod PE (Avanti Polar Lipids, Alabaster, AL, USA)
was an acceptor fluorophore. p
p
Bacterial cells suspended in PBS solution (prepared according to the description in
Section 4.4) were portioned by 200 µL into the wells of a black 96-well plate (Thermo
Fisher Scientific, Roskilde, Denmark) obtaining ~6.5 × 106 cells per well, and then labeled
with membrane probes. The F wells contained the FRET donor (to a final concentration of
0.02 µg mL−1) and the FRET acceptor (to a final concentration of 1 µg mL−1). The Fo wells
contained only the donor. Backgrounds for the F wells (containing unlabeled cells and the
acceptor, but no donor) and for the Fo wells (containing only unlabeled cells) were also
prepared. The plate was incubated in the dark at room temperature for 1 h before fluores-
cence measurements. By applying the same conditions of the experiments, paying special Int. J. Mol. Sci. 2023, 24, 16461 15 of 18 15 of 18 attention to the age (18 h) and density of the analyzed bacterial cells (~6.5 × 106 cells), the
concentration of fluorophores, and the labeling time, the impact of the above factors on the
stability of the experimental FRET values, as well as on the repeatability of the obtained results,
was minimalized. Fluorescence intensity was measured in the Laboratory of Microscopic Imaging and
Specialized Biological Techniques, Faculty of Biology and Environmental Protection, Uni-
versity of Lodz, Poland using a Spectramax i3 Multi-Mode Microplate Reader (Syngen
Biotech, Wroclaw, Poland) with the following parameters: λex = 350 nm and λem = 379 nm,
6 flashes on read, a linear shaking before read for 5 s. The F/Fo values were calculated after
the subtraction of backgrounds. The values of fractions of energy transfer were calculated
with the following formula 1-F/Fo. 4.8. Confocal Microscopy Imaging The P. mirabilis rods obtained according to the description in Section 4.4 were trans-
ferred into a 24-well black plate with a transparent glass bottom (Greiner Bio-One GmbH,
Kremsmünster, Austria) and then labeled with Rhod PE (Avanti Polar Lipids, Alabaster, AL,
USA) for 18 h, allowing the cells to adhere to the glass surface. Labeled cells were imaged
using a DMI 6000 CS inverted microscope with the TCS SP8 confocal system, operated by
the LAS 2.0.215022 software (Leica Microsystems, Wetzlar, Germany) in the Laboratory
of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and
Environmental Protection, University of Lodz, Poland. The observations were made using
an HC PLAPO CS2 100×/1.40 oil immersion objective. The 560 nm supercontinuum white
light laser (WLL) (10% intensity) was used to excite the fluorophore-stained membranes. A
fluorescence emission was collected in the range of 570–699 nm by a conventional detector
photomultiplier tube (PMT) or by a hybrid detector (HyD) working in bright R mode. To
enhance the contrast, a green channel was applied. Confocal scans were performed at a
speed of 600 Hz, zoom from 1.8 to 3.61, a pinhole 151.5 µm, and a line average set at 7. Image logical size was 512 × 512. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Yuan, F.; Huang, Z.; Yang, T.; Wang, G.; Li, P.; Yang, B.; Li, J. Pathogenesis of Proteus mirabilis in Catheter-Associated Urinary Tract
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sition and organization in P. mirabilis rods. We have proved that drastic modifications
occurring during the formation of the long-form cells of uropathogens are directly related
to changes in their phospholipid profile, while showing weak relation with the alterations
in their fatty acid composition. We have indicated some phospholipid species as probable
participants in the formation and stabilization of lipid rafts present in the short-form rods
of P. mirabilis. Melting of these domains during the rod elongation has been found to ensure
homogeneity of the lipid bilayers and enhance their permeability for macromolecules. In this study, we have been able to gain important insights into the relevance of the
lipid adjustment, a factor involved in phenotypic modifications induced in P. mirabilis rods
during swarming motility. However, this preliminary report requires expansion in order to
search for other potential lipid virulence factors associated with the elongated morphotype
of swarming rods that potentially contribute to UTIs development. Int. J. Mol. Sci. 2023, 24, 16461 16 of 18 Author Contributions: Conceptualization, P.S.; Methodology, P.S. and P.B.; Formal analysis, A.R.;
Investigation, P.S. and P.B.; Data Curation, P.S.; Writing—Original Draft Preparation, P.S.; Writing—
Review and Editing, P.B. and A.R.; Visualization, P.S.; Project Administration, P.S. and A.R.; Funding
Acquisition, A.R. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. It was supported by the Department of Biology
of Bacteria (University of Lodz, Lodz, Poland). Funding: This research received no external funding. It was supported by the Department of Biology
of Bacteria (University of Lodz, Lodz, Poland). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. References Diffusible Signal Factor-Dependent Quorum Sensing in Pathogenic Bacteria and Its Exploitation for Disease Control. g
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Molecular Structure and Dynamics of Water on Pristine and Strained Phosphorene: Wetting and Diffusion at Nanoscale
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Scientific reports
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Wei Zhang1,2,*, Chao Ye1,*, Linbi Hong3, Zaixing Yang4 & Ruhong Zhou1,3,5 Wei Zhang1,2,*, Chao Ye1,*, Linbi Hong3, Zaixing Yang4 & Ruhong Zhou1,3,5 Phosphorene, a newly fabricated two-dimensional (2D) nanomaterial, has emerged as a promising
material for biomedical applications with great potential. Nonetheless, understanding the wetting
and diffusive properties of bio-fluids on phosphorene which are of fundamental importance to these
applications remains elusive. In this work, using molecular dynamics (MD) simulations, we investigated
the structural and dynamic properties of water on both pristine and strained phosphorene. Our
simulations indicate that the diffusion of water molecules on the phosphorene surface is anisotropic,
with strain-enhanced diffusion clearly present, which arises from strain-induced smoothing of the
energy landscape. The contact angle of water droplet on phosphorene exhibits a non-monotonic
variation with the transverse strain. The structure of water on transverse stretched phosphorene
is demonstrated to be different from that on longitudinal stretched phosphorene. Moreover, the
contact angle of water on strained phosphorene is proportional to the quotient of the longitudinal and
transverse diffusion coefficients of the interfacial water. These findings thereby offer helpful insights
into the mechanism of the wetting and transport of water at nanoscale, and provide a better foundation
for future biomedical applications of phosphorene. As a newly fabricated 2D nanomaterial composed of phosphorus atoms, phosphorene possesses a direct band gap
which makes it a natural semiconducting nanomaterial1,2. Because of its unique electrical, optical and mechani-
cal properties, phosphorene can be used in a wide range of biomedical applications with promising potential3–5. Notably, using a few layers of phosphorene, researchers have successfully fabricated the field-effect transistor6,7,
which is a desirable device for biodetection and biosensing8–10. Simulation and experimental results showed that
phosphorene in water is more stable than that in air11,12. The good stability of phosphorene in water creates
the chances for phosphorene’s applications in biological fields. Compared to graphene, phosphorene is more
biological-friendly due to its less disruption to proteins4. Moreover, as photothermal and photodynamic agents,
black phosphorus nanosheets (multiple layers of phosphorene) have exhibited excellent potential in killing cancer
cells5,12. Naturally, wider applications of this novel nanomaterial do require more extensive studies on its interac-
tions with biomolecules, including the wetting and diffusive properties of bio-fluids on its surface.h fl
The structure and dynamics of interfacial water at various nanomaterial surfaces are of fundamental impor-
tance for developing these nanomaterials’ potential applications in biomedicine and nanofluidics. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Molecular Structure and Dynamics
of Water on Pristine and Strained
Phosphorene: Wetting and
Diffusion at Nanoscale received: 08 April 2016
accepted: 08 November 2016
Published: 06 December 2016 received: 08 April 2016
accepted: 08 November 2016
Published: 06 December 2016 Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 Wei Zhang1,2,*, Chao Ye1,*, Linbi Hong3, Zaixing Yang4 & Ruhong Zhou1,3,5 The structure
of water is perturbed heavily near the surfaces which have the potential to affect water diffusion13,14 and proteins’
adsorption15. During recent years, the wetting and diffusive properties of water in contact with two-dimensional
(2D) nanomaterials, such as graphene16–20, boron-nitride sheets16,21,22, WS2 and MoS2
23,24, have attracted great
attention. These studies have helped the promotion of potential applications of these 2D nanomaterials in both 1Institute of Quantitative Biology and Department of Physics, Zhejiang University, Hangzhou 310027, China. 2Department of Physics, College of Sciences, China University of Mining and Technology, Xuzhou 221116, China. 3Computational Biology Center, IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598, USA. 4Institute of Quantitative Biology and Medicine, SRMP and RAD-X, Collaborative Innovation Center of Radiation
Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China. 5Department of
Chemistry, Columbia University, NY 10027, USA. *These authors contributed equally to this work. Correspondence
and requests for materials should be addressed to W.Z. (email: wzhangph@gmail.com) or R.Z. (email: ruhosngz@
us.ibm.com) Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 1 www.nature.com/scientificreports/ Figure 1. (a) Perspective view of transverse and longitudinal directions upon which strains were imposed. (b) A snapshot of water droplet on phosphorene (ε = 0; pristine) at the end of the MD simulation. (c) The water
contact angle θ as a function of the transverse and longitudinal strain. Figure 1. (a) Perspective view of transverse and longitudinal directions upon which strains were imposed. (b) A snapshot of water droplet on phosphorene (ε = 0; pristine) at the end of the MD simulation. (c) The water
contact angle θ as a function of the transverse and longitudinal strain. iomedicine and nanofluidics. However, the structure and dynamics of water on phosphorene, a new member o
D nanomaterials, are still elusive to the best of our knowledge.l Another promising aspect of phosphorene is its great mechanical flexibility25, due to the hexagonally arranged
phosphorus atoms and the subsequently formed puckered honeycomb structure inside the monolayer of phos-
phorene. Peng’s work demonstrated that phosphorene can withstand a uniaxial tensile strain up to 0.3026. Recent
computational studies based on the density functional theory (DFT) showed that a single-layer phosphorene
could actualize tensile strain (stretching) up to 0.54 while maintaining its original P-P bond and “two-sublayer”
(non-planar) structure27,28. It has also been demonstrated that all the electrical29–32, optical33,34, thermoelectric35–37
and mechanical27,30,38 properties of phosphorene could be modified upon mechanical strain. Methods
l MD simulations were performed to explore the wetting and diffusive properties of water on both pristine and
strained phosphorene. The Gromacs package 4.5.7 42 and OPLS-AA force field43 were used for the simula-
tions. The SPC/E model was used to model water molecules. Phosphorene with a dimension of 156 Å × 17 Å
was chosen for this study. The bond length and bond angle of phosphorene were presented in Fig. S1 in the
Supporting Information (SI). The phosphorus atoms were modeled as uncharged Lennard–Jones particles. The
depth of potential well εPP, cross sections σPP and bond strength constants are set at 0.400 kcal mol−1, 3.33 Å and
297 kcal mol−1 Å2, respectively, following a previous study based on DFT computations and experimental results44. Using the umbrella pulling code of Gromacs, we applied a harmonic potential between the centers of mass
of two groups of P atoms at two side of phosphorene sheet to produce the strained phosphorene, during which
phosphorene was flexible. The strained phosphorene was then fixed throughout the water-phosphorene interac-
tion simulation. Note that the strain being discussed in this study only refers to tensile strain. Phosphorene was
therefore stretched along two typical directions: transverse (perpendicular to the pucker) and longitudinal (par-
allel to the pucker), as shown in Fig. 1(a). Phosphorene with strain ε = 0 corresponds to the pristine phosphorene. Figure S2 in the Supporting Information shows four representative configurations of strained phosphorene. The
initial water droplet which was configured as a cubic consisting of 2828 molecules was set onto phosphorene
under various strain conditions. During the first few nanoseconds of the simulation, the water droplet gradually
converted from a cubic into a hemisphere, as illustrated in Fig. 1(b). We chose 10 morphologies of phosphorene
with different degrees of transverse strain, as well as another 10 phosphorene morphologies with different degrees
of longitudinal strain for our current study of the interaction with water droplet. Each phosphorene-water system
was simulated for 16 ns, and the trajectory of the last 1 ns was extracted for further analysis. Following similar MD simulations were performed to explore the wetting and diffusive properties of water on both pristine and
strained phosphorene. The Gromacs package 4.5.7 42 and OPLS-AA force field43 were used for the simula-
tions. The SPC/E model was used to model water molecules. Phosphorene with a dimension of 156 Å × 17 Å
was chosen for this study. Wei Zhang1,2,*, Chao Ye1,*, Linbi Hong3, Zaixing Yang4 & Ruhong Zhou1,3,5 The puckered struc-
ture of phosphorene brings anisotropy and negative Poisson’s ratio27,35. The strain also gives rise to phosphorene’s
transition between metal and semiconductors32,35,38. Therefore, it is of great interest to learn if there is any change
in both the structure and dynamics of water on phosphorene upon strain. y
p
p
p
In this study, we use molecular dynamics (MD) simulations, which are widely used in the study of the inter-
action of (bio)molecules with nanomaterials39–41, to investigate the contact angle, diffusion coefficient, and dis-
tribution of water on both pristine and strained phosphorene. We found that the diffusion of water molecules
at the surface of phosphorene is anisotropic, and strain-enhanced water diffusion is clearly present. The struc-
ture and wetting of water on transverse stretched phosphorene differs from that on the longitudinal stretched
one. The contact angle of water droplet non-monotonously changes with imposed transverse strain displaying
a parabola-like curve (first increases, then decreases), whereas it increases nearly linearly with imposed longi-
tudinal strain. Also, the distribution of water near the surface of phosphorene exhibits obvious changes when
imposed transverse strain increases, while the distribution of water barely changes with the longitudinal strain. Additionally, we found that the contact angle of water on strained phosphorene is proportional to the quotient
of the longitudinal and transverse diffusion coefficients of the interfacial water. The dispersion energy and free
energy profile of water were further calculated to interpret the above phenomena. Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 Results and Discussionh The Equation 1 can be written as
θ
θ
τ γ
=
+
∞
r
cos
cos
/
LV B, where θ∞ is the contact angle for macroscopic droplet. By changing the number of
water molecules of droplet (the droplet size), one could derive the macroscopic contact angle θ∞ of droplet on
phosphorene with a certain strain57 (see ref. 57).i In addition to the droplet size, the contact angle of a water on nanomaterials changes significantly as a func-
tion of the water-nanomaterial interaction energy55. If one uses a single sheet of LJ atoms in a specific lattice and
tunes the interaction energy with the water molecules one would find the initial increase in contact angle due to
the higher interaction with the substrate.ff Along with the differences in water contact angles, the transverse and longitudinal strains also cause different
deformations of the phosphorene surface. While strain in the transverse direction effectively flattens the puckers
of phosphorene, longitudinal strain, on the other hand, has little effect on the bending structure of the puckers’
ring. This distinct deformation of the phosphorene monolayer leads to different interactions between the water
droplet and phosphorene.h p
p
p
The contact angle θ non-monotonically changes with the transverse strain, which is primarily caused by
non-monotonic variations of the interaction energy Ews. To analyze the influences of the transverse strain on Ews
in detail, we calculated the interaction energy between water droplet and phosphorous atoms in the bottom (Ews
B )
and upper (Ews
T ) surfaces of the puckered monolayer of phosphorene, respectively (see Fig. 2). Here,
=
+
E
E
E
ws
ws
B
ws
T . With the increase of the transverse strain εT, the density of the atoms in the upper surface
decreases, thus Ews
T weakens remarkably (Fig. 2, blue curve), resulting in a less favorable interaction and a higher
water contact angle. As for the atoms in the bottom surface, they move toward the water droplet (closer to water
molecules) which is favorable for the interaction. However, its density decreases at the same time and hence is
unfavorable for the interaction. When εT < ~0.16, the decrease in the density of atoms in the bottom surface
dominates the interaction, and Ews
B weakens slightly (see Fig. 2, red curve). Results and Discussionh The Young’s modulus of phosphorene is sensitive to the direction upon which strains were imposed, due to the
nanomaterial’s anisotropic nature. Figure S4 in Supporting Information illustrates the relationship between the
strain at transverse and longitudinal directions and the pull force exerted along these directions. The slope of
transverse strain as a function of pull force is clearly steeper than that of longitudinal strain, indicating less stiff-
ness in phosphorene when stretched in the transverse direction. These results are consistent with previous calcu-
lations based upon first-principles density functional theory25,27.hf i
The strains imposed upon phosphorene not only affect its electronic and mechanical properties, but also
change the water contact angle on its surface. As shown in Fig. 1(c), the strains exerted in different directions
have distinct effects on the water contact angle θ. An increase of the transverse strain εT causes θ to first increase,
and then to decrease after the peak is reached (parabola-like curve or inverted U-shaped curve). At εT ~ 0.3, we
observed the maximal contact angle θmax. However, for strains imposed upon the longitudinal direction, the
relationship between θ and the longitudinal strain rate εL is clearly monotonic (roughly linear). A comparison
between the contact angles of water droplet on phosphorene under different directional strains also showed an
overall larger contact angles under transverse than longitudinal strain.hi The microscopic contact angle has been given by the modified Young’s equation. It relates the surface tensions
γ of the relevant phase (subscripts S, L, and V for solid, liquid, and vapor phase, respectively), the line tension τ
and the droplet base radius rB (see Fig. S3) with the contact angle θ as55: γ
γ
γ
θ
τ
=
+
+
r
cos
(1)
SL
LV
B
SV (1) This equation implies that the contact angle is related to the size of droplet due to line tension. For the sessile
water drops with linear shape, it was shown that the wetting depends on the contact line56. In this paper, the mol-
ecule number of a water droplet on every strained phosphorene is the same (2828 water molecules), but the base
radius of the hemispherical water droplet changes with the strain, as shown in Fig. S5. This is one of the reasons
why the contact angle of water droplet changes with the strain (Fig. 1c). www.nature.com/scientificreports/ protocols used in our previous studies45–51, the particle-mesh Ewald method52 with a grid spacing of 1.2 Å was
applied to simulate the long-range electrostatic interactions, and a typical cutoff of 10 Å was applied for the van
der Waals interactions. We increased the z-dimension of the box (a box height of 3 times the slab height) to pro-
duce a pseudo-2D Ewald summation. Yeh and Berkowitz’s scheme for 2D Ewald summaries is another general
method for systems with slab geometry53. All simulations were performed in an NVT ensemble at a constant
temperature of 298 K by using v-rescale thermostat54. p
y
g
The water contact angle θ was measured by fitting the time-averaged liquid-vapor interface55. The liquid-vapor
interface is defined as the contour with half of the bulk density, while the number density of water droplet was cal-
culated by the time-averaged spatial mesh with a grid spacing of 0.5 Å at a fixed azimuthal angle. The liquid-vapor
interface was then fitted into an arc and θ was calculated as the angle of contingence at the liquid-solid interface
(refer to Figure S3 in the Supporting Information). Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 Methods
l The bond length and bond angle of phosphorene were presented in Fig. S1 in the
Supporting Information (SI). The phosphorus atoms were modeled as uncharged Lennard–Jones particles. The
depth of potential well εPP, cross sections σPP and bond strength constants are set at 0.400 kcal mol−1, 3.33 Å and
297 kcal mol−1 Å2, respectively, following a previous study based on DFT computations and experimental results44. Using the umbrella pulling code of Gromacs, we applied a harmonic potential between the centers of mass
of two groups of P atoms at two side of phosphorene sheet to produce the strained phosphorene, during which
phosphorene was flexible. The strained phosphorene was then fixed throughout the water-phosphorene interac-
tion simulation. Note that the strain being discussed in this study only refers to tensile strain. Phosphorene was
therefore stretched along two typical directions: transverse (perpendicular to the pucker) and longitudinal (par-
allel to the pucker), as shown in Fig. 1(a). Phosphorene with strain ε = 0 corresponds to the pristine phosphorene. Figure S2 in the Supporting Information shows four representative configurations of strained phosphorene. The
initial water droplet which was configured as a cubic consisting of 2828 molecules was set onto phosphorene
under various strain conditions. During the first few nanoseconds of the simulation, the water droplet gradually
converted from a cubic into a hemisphere, as illustrated in Fig. 1(b). We chose 10 morphologies of phosphorene
with different degrees of transverse strain, as well as another 10 phosphorene morphologies with different degrees
of longitudinal strain for our current study of the interaction with water droplet. Each phosphorene-water system
was simulated for 16 ns, and the trajectory of the last 1 ns was extracted for further analysis. Following similar Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 2 www.nature.com/scientificreports/ Results and Discussionh When εT > ~0.16, however, the
approach toward the water droplet of the atoms in the bottom surface dominates the interaction, enhancing Ews
B
dramatically (Fig. 2, red curve). The combined effect on the interaction from both the upper and bottom surfaces
atoms is thus as following, with the increase of εT the interaction energy Ews increases firstly and then decreases
after εT reaches ~0.3, displaying a parabola-like curve as shown in the inset of Fig. 2. On the other hand, as men-
tioned above, the longitudinal strain has little effect on the bending structure of the pucker ring, thus it displays a
monotonic behavior in both the interaction energy with water and the water contact angle (Fig. 1(c)).f gy
g
g
In addition to water contact angle, the strain may also affect the structure of water near phosphorene
order to understand the structure of the interfacial water molecules, we measured the density distribu Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 3 www.nature.com/scientificreports/ Figure 2. The interaction energy between the water droplet and phosphorous atoms in the bottom (Ews
B red
circles) and upper (Ews
T blue pentagons) surfaces of the puckered phosphorene as a function of the strain
along the transverse direction. The inset shows the combined interaction energy
=
+
E
E
E
ws
ws
B
ws
T . Figure 2. The interaction energy between the water droplet and phosphorous atoms in the bottom (Ews
B red
circles) and upper (Ews
T blue pentagons) surfaces of the puckered phosphorene as a function of the strain
along the transverse direction. The inset shows the combined interaction energy
=
+
E
E
E
ws
ws
B
ws
T . circles) and upper (Ews blue pentagons) surfaces of the puckered phosphorene as a function of the strain
along the transverse direction. The inset shows the combined interaction energy
=
+
E
E
E
ws
ws
B
ws
T . Figure 3. (a) A snapshot of the simulation system for studying the density distribution and diffusion of water
molecules. (b) and (c) The density distribution function of oxygen atoms g(z) as a function of the distance z
under different magnitudes of strain. Figure 3. (a) A snapshot of the simulation system for studying the density distribution and diffusion of water
molecules. Results and Discussionh (b) and (c) The density distribution function of oxygen atoms g(z) as a function of the distance z
under different magnitudes of strain. function (DDF) of the oxygen atoms along the direction normal to the surface (z axis). DDF g(z) is defined as in
Equation (2): ρ
ρ
=
. g z
z
( )
( )/
(2) (2) ρ
ρ
=
. g z
z
( )
( )/ where ρ(z) is the density of oxygen atoms within a thin slice of height z parallel to the surface (the thickness of the
slice is set at 0.2 Å), and ρ is the mean density of oxygen atoms in the bulk. Note that the zero point of z corre-
sponds to the geometric center of phosphorene in the z-axis direction. A new and much larger thick 2D slab sys-
tem consisting of 25,662 water molecules, as shown in Fig. 3(a), was used to study the density distribution and
diffusion of the water molecules. f
Figure 3(b) and (c) show the g(z) of the oxygen atoms near phosphorene under various transverse and lon-
gitudinal strains. Due to the dispersive interaction between the phosphorous atoms and water molecules, the
structure of water near phosphorene is considerably perturbed from its bulk structure, which displays a double
peak character. The double peaks of g(z) in both Fig. 3(b) and (c) indicate the two-layer structure of water in the
vicinity of phosphorene, with the first peak corresponding to the first layer of water. The density of the oxygen
atoms in the first water layer can be 2.8 times larger than that in the bulk. While z > ~1.0 nm, the density fluctu-
ation disappears and the bulk density is recovered. Meanwhile, the difference between Fig. 3(b) and (c) is worth
noting. In Fig. 3(b), as the transverse strain increases, both the critical position zc where oxygen atoms appear and
the maximal DDF gmax decrease, but the half-width of the first peak W increases (see Table 1). As for the longitu-
dinal strain, it showed almost no effect on g(z), as shown in Fig. 3(c).hf f
g
g
The above results demonstrated that the effects of transverse and longitudinal strains on the static structure
of interfacial water are distinct, which can be partly attributed to the anisotropic mechanical properties of phos-
phorene. Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 Results and Discussionh In the following discussion, we present the effects of the strain on the dynamic properties of interfacial Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 4 www.nature.com/scientificreports/ εT
zc (nm)
gmax
W (nm)
0
0.36
2.85
0.08
0.03
0.35
2.68
0.10
0.07
0.34
2.64
0.10
0.16
0.32
2.38
0.10
0.24
0.30
2.26
0.12
0.28
0.30
2.21
0.12
0.31
0.28
2.24
0.12
0.36
0.25
2.14
0.14
0.43
0.24
2.08
0.15
0.52
0.22
1.97
0.18
Table 1. The critical position zC, the height gmax and half-width W of the first peak of the density
distribution function (DDF) of oxygen atoms corresponding to various values of the transverse strain εT. εT
zc (nm)
gmax
W (nm)
0
0.36
2.85
0.08
0.03
0.35
2.68
0.10
0.07
0.34
2.64
0.10
0.16
0.32
2.38
0.10
0.24
0.30
2.26
0.12
0.28
0.30
2.21
0.12
0.31
0.28
2.24
0.12
0.36
0.25
2.14
0.14
0.43
0.24
2.08
0.15
0.52
0.22
1.97
0.18
Table 1. The critical position zC, the height gmax and half-width W of the first peak of the density
distribution function (DDF) of oxygen atoms corresponding to various values of the transverse strain εT. Table 1. The critical position zC, the height gmax and half-width W of the first peak of the density
distribution function (DDF) of oxygen atoms corresponding to various values of the transverse strai Table 1. The critical position zC, the height gmax and half-width W of the first peak of the density
distribution function (DDF) of oxygen atoms corresponding to various values of the transverse strain εT. Figure 4. The diffusion coefficient D of water molecules in the first layer as a function of (a) the transverse and
(b) longitudinal strain. Figure 4. The diffusion coefficient D of water molecules in the first layer as a function of (a) the transverse a
(b) longitudinal strain. water. To illustrate the dynamical properties clearly, we denote zm and zv as the z position of the first peak and
valley of DDF, respectively.f In order to examine the dynamic properties of liquid on phosphorene, we begin with the self-diffusion of
water molecules in the first layer. Results and Discussionh The mean squared displacement (MSD) was measured according to the follow-
ing equation:
τ
τ
=
∑
Θ
⋅
+
−
Θ
⋅∏
Θ
∑
Θ
⋅
Θ
⋅∏
Θ
τ
τ
=
=
+
′
=
=
+
′
′
′
r
r
r
t
t
t
t
( )
(0)
(
)
( )
(t)
( )
(0)
(t)
( )
,
(3)
i
N
i
i
i
i
t
t
t
i
t
i
N
i
i
t
t
t
i
t
2
1
2
1 (3) where ri(t) is the position of oxygen atoms of the i th water molecule at time t; τ is the lag time and N is the num-
ber of molecules included in this calculation. The angle brackets <…>t indicate the average over the time t. The
function of Θ (t)
i
is 1 if the water molecule i belongs to the first layer at time t and is otherwise 0. MSD in Eq. 3 was
evaluated only for the survived water molecule in the first layer (the position of the oxygen atom of water in the
region zc < z < zv). Water molecule in the first layer calculated in original time would jump out from this layer, and
the continuous survival probability (CSP)58 decreases with the duration time (Fig. S6 in SI). The life-time of water
in the first layer decreases with the transverse strain in the range (0.00–0.36). Because in this range the interaction
energies between water molecules and strained phosphorene decreases with the strain.h g
p
p
The transverse and longitudinal mean squared displacement (MSDT = 〈Δx2(τ)〉 and MSDL = 〈Δy2(τ)〉 respectively)
increases linearly with the time interval τ, as shown in Fig. S7. The transverse and longitudinal self-diffusion
coefficients, DT and DL, are derived from the following equations: 〈Δx2(τ)〉 = 2DTτ and 〈Δy2(τ)〉 = 2DTτ.ffii fi
T
L
g q
〈
〉
T
〈
y
〉
T
Figure 4 shows the transverse and longitudinal diffusion coefficients of water molecules in the first layer with
various strain conditions. It is clear from the separation of the curves as shown in Fig. 4, that the diffusion of water
molecules on pristine phosphorene is anisotropic, mainly due to the puckering surface morphology of phos-
phorene. Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 Results and Discussionh Compared to motions in the transverse direction, a higher DL coefficient indicates that it is much easier
for water molecules to move along the longitudinal direction. The transverse diffusion coefficient DT decreases
monotonically with increased transverse strain εT. The longitudinal diffusion coefficient DL increases while εT is
smaller than 0.16, and decreases after εT rises above 0.16, as shown in Fig. 4(a) and its inset (again, parabola-like
curve or inverted U-shaped curve). The effects of longitudinal strain on the diffusion of interfacial water differ
from that of transverse strain. Here, as the longitudinal strain εL increases, DL also increases but DT decreases. As
shown in Fig. 4(b), DL increases from 2.37 to 2.61 (10−5 cm2/s) in the measured range of εL. In contrast, the change Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 5 www.nature.com/scientificreports/ Figure 5. Free energy profile of water within the first layer ΔF(x, y) scaled by kBT for three values of εT. Here, the scaling parameter L = 0.05 nm. Figure 5. Free energy profile of water within the first layer ΔF(x, y) scaled by kBT for three values of εT. Here, the scaling parameter L = 0.05 nm. of DT is not as significant. Clearly, the effects of εT and εL on the diffusion of interfacial water are complicated, and
all of an anisotropic manner.fi of DT is not as significant. Clearly, the effects of εT and εL on the diffusion of interfacial water are complicated, and
all of an anisotropic manner. p
To rationalize the diffusion behaviors of the interfacial water, we computed the free energy profile ΔF(x, y)
of water within the first layer (zc < z < zv) (with a bin width of 5 nm in x and y-directions at the center of phos-
phorene sheet) with the equation: ∆
= −
F x y
k TlnP x y
( ,
)
( ,
)
(4)
B
o ∆
= −
F x y
k TlnP x y
( ,
)
( ,
)
B
o (4) Here, Po(x, y) is the spatial probability distribution function of the oxygen atoms of water within the first layer
at coordinate (x, y). This approach has been previously applied to examine the friction of water on graphene and
boron nitride22, as well as the free energy landscape of protein folding59–61. In Fig. Results and Discussionh 5, we present ΔF(x, y) as scaled
by k T
B under different transverse strains. The free energy profile ΔF(x, y) obviously exhibits a “grooved
morphology”, mimicking the phosphorene surface, which is a clear indication of anisotropy. The minimal free
energy regions appear in the grooves of phosphorene. The variation of the free energy profile with the strain arises
from the change of the structure of phosphorene. The hopping mechanism of interfacial water would depend
upon free energy barrier. A smooth free energy profile is beneficial to the diffusion of the interfacial water. The
smoother the free energy profile is (corresponding to smaller energy barrier), the larger the diffusion coefficient
of interfacial water becomes.i For the pristine phosphorene (εT = 0 and εL = 0), the free energy profile ΔF(x, y) shows clear “zigzag” patterns
in the grooved region (see Fig. 5(a)), similar to the so-called “swallow gird”. The maximal energy barrier for
water molecules to translate along the longitudinal direction is about 1.5 kBT, while it is ~1.7 kBT to move in the
transverse direction. Thus, it is more difficult for interfacial water molecules to move in the transverse than in the
longitudinal direction. As a result, the diffusion coefficient of interfacial water along the longitudinal direction is
larger than that along the transverse direction. Rationalizing the effects of the transverse strain εT on the transverse diffusion coefficient DT. The strain εT acts to broaden the interval of free energy ribbons (low-energy region along the groove) which
corresponds to the groove width of phosphorene. The broadening of free energy ribbon interval constrains the
diffusion of water molecules along the transverse direction, since it introduces more difficulty in water molecules’
crossing of broadened energy barrier. Meanwhile, the strain εT increases the mean energy barrier for water mol-
ecules to move in the transverse direction, thus hindering the motion of water molecules along the transverse
direction. Consequently, the transverse diffusion coefficient DT decreases as εT increases, as shown in Fig. 4(a). Rationalizing the effects of the transverse strain εT on the longitudinal diffusion coefficient DT Under the combined influences of these two factors (the increased height and decreased width of
energy barrier), as εL increases, the transverse diffusion coefficient DT decreases slightly.h ffi
Though the longitudinal strain εL obviously smoothes the free energy ribbon and increases the longitudinal
diffusion coefficient of interfacial water, it has almost no effects on the density distribution function (DDF) g(z)
(see Fig. 3(c)). The DDF g(z), however, obviously changes with the transverse strain εT (Fig. 3(b)), which is caused
by the flattening of the puckered surface of phosphorene. The flattening of the surface of phosphorene leads to the
decrease of the critical position zc. The increase of the half-width W and the decrease of the height gmax are related
to the energy profile of single water molecule on phosphorene surface as a function of distance from surface (see
Fig. S10). As shown in Fig. S10, energy profile curve are similar to LJ potential and has a minimum value. With
the increase of the transverse strain, the minimum energy value and the half-width of the energy valley increase. Consequently, DDF g(z) is related not only to the dispersion energy between water and phosphorene, but also to
the distribution of water molecules on phosphorene’s surface. p
p
Interestingly, the quotient DL/DT, instead of DL or DT alone, exhibits a roughly linear relationship with the
contact angle θ of water droplet, as shown in Fig. 6. The variation of the width and depth of the groove of phos-
phorene caused by the strain directly affects the interaction energy Ews and the free energy profile ΔF(x, y). The
contact angle θ and the diffusion coefficient of water molecules in the first layer, on the other hand, are mainly
determined by the interaction energy Ews and free energy profile ΔF(x, y). The free energy profile ΔF(x, y) jointly
affects the transverse and longitudinal diffusion of water molecules. In this sense, the contact angle θ should be
related to the diffusion of water molecules along transverse and longitudinal directions. However at present, we
have not yet found a quantitative interpretation regarding the relationship between the ratio DL/DT and water
contact angle θ, which needs further investigation and experimental validation.f g
g
p
Changing the pattern of the puckering surface of phosphorene by the strain could effectively enhance or
attenuate the diffusion of interfacial water molecules, which might provide insight for controlling/designing the
motion of interfacial molecules. Rationalizing the effects of the transverse strain εT on the longitudinal diffusion coefficient DT By controlling the strain, one could construct continuous diffusion (or wetting)
gradient, which is of interest in artificial microscopic walk62,63. The anisotropic diffusion of water molecules near
phosphorene surface may also affect phosphorene’s motion in complex biological systems, which is of importance
for phosphorene’s potential biomedical applications, such as localized bioprobes and drug delivery. Rationalizing the effects of the transverse strain εT on the longitudinal diffusion coefficient DT Rationalizing the effects of the transverse strain εT on the longitudinal diffusion coefficient DT. With the increase of strain εT, the “zigzag” pattern gradually disappears in general and the free energy ribbons
becomes smooth in the longitudinal direction (the low-energy region inter-connects, see Fig. 5(b) and (c)), which
is favorable for water molecules to diffuse in the longitudinal direction. Thus, the longitudinal diffusion coeffi-
cient DL increases with the increase of εT, up to 0.16, as shown in the inset of Fig. 4(a). With further increase of εT
to be larger than 0.16, the influence of phosphorus atoms at the bottom layer of the groove begins to dominate as
mentioned above, which affects water’s diffusion as well. As εT increases above 0.16, DL decreases, because of the
increased attraction from phosphorus atoms at the bottom of the groove (see Fig. 2). Rationalizing the effects of the longitudinal strain εT on the longitudinal and transverse diffusion
coefficients. When phosphorene is under longitudinal strain, the variations of the free energy profile ΔF(x, y)
is different from those under transverse strain (see Fig. S8 in Supporting Information). The longitudinal strain
εL obviously attenuates the “zigzag” pattern of the free energy ribbon, which accounts for the enhancement of DL
as εL increases. However, unlike the effect of εT, the longitudinal strain εL only narrows the groove width, while
shows little effect on its depth. In other words, it increases the mean height of energy barrier (see Fig. S9) but
decreases its width. The increased energy barrier constrains the diffusion of water molecules along the transverse
direction. On the other hand, the decrease of the width of energy barrier is in favor of water diffusion, thus an Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 6 www.nature.com/scientificreports/ Figure 6. The quotient of DL/DT as a function of the contact angle θ of water droplets. The fitting line has the
form: DL/DT = θ ∙ 0.016(0)−1 − 0.06. Figure 6. The quotient of DL/DT as a function of the contact angle θ of water droplets. The fitting line has the
form: DL/DT = θ ∙ 0.016(0)−1 − 0.06. increased DT. Under the combined influences of these two factors (the increased height and decreased width of
energy barrier), as εL increases, the transverse diffusion coefficient DT decreases slightly.h increased DT. Conclusions In this study, we investigated the wetting and diffusive properties of water near both pristine and strained phos-
phorene with MD simulations. It was found that the pristine phosphorene is of weak hydrophobicity, with a water
contact angle of ~72°. As for the interactions between water droplet and phosphorene under different strains
(stretchings), we discovered that the water contact angle θ displays a parabola-like curve with the transverse
strain (first increases, then decreases, as the transverse strain reaches a critical threshold of 0.3). However, for
the longitudinal strain, the contact angle θ increases monotonically as the strain increases. The changes in water
contact angle are mainly determined by the interaction energy between the water droplet and phosphorene. The
structure of the interfacial water dramatically changes with the transverse strain εT, but the longitudinal strain εL
has almost no effect on water’s structure.f f
While the diffusion of water molecules near pristine phosphorene surface is anisotropic, the longitudinal dif-
fusion coefficient DL is larger than that (DT) in the transverse direction. As the transverse strain εT increases, DT
decreases monotonically, while DL exhibits a parabola-like curve (or inverted U-shaped curve). The longitudinal
strain εL, on the other hand, causes DL to increase, and DT to decrease monotonically. We also calculated the free
energy profile ΔF(x, y) to determine the main cause of variations in the diffusion of water molecules near phos-
phorene. It was shown that the smoothing of the energy landscape enhances DL, and the increased energy barrier
causes DT to decrease. Last but not least, we found that interestingly the quotient DL/DT is positively correlated
with the contact angle θ. Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 7 www.nature.com/scientificreports/ As a novel 2D nanomaterial, phosphorene has the potential to be widely used in biomedical applications in
the near future. Therefore it is necessary to investigate their interactions with various bio-fluids, including the
wetting and diffusive properties of water on both pristine and strained phosphorene. Our study should help with
understanding and manipulating the wetting and diffusive properties of liquid on phosphorene, which is critical
for phosphorene’s application in the fields of biomedicine and nanofluidics. References & Dong, M. Two-dimensional material confined water. Accounts of chemical research 48, 119–127
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(2011). Scientific Reports | 6:38327 | DOI: 10.1038/srep38327 8 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Zhang, W. et al. Molecular Structure and Dynamics of Water on Pristine and Strained
Phosphorene: Wetting and Diffusion at Nanoscale. Sci. Rep. 6, 38327; doi: 10.1038/srep38327 (2016). How to cite this article: Zhang, W. et al. Molecular Structure and Dynamics of Water on Pristine and Strained
hosphorene: Wetting and Diffusion at Nanoscale. Sci. Rep. 6, 38327; doi: 10.1038/srep38327 (2016). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Classification of Wheezing Children in Rural Bangladesh by Intensity of <i>Ascaris</i> Infection, Total and Specific IgE Levels, History of Pneumonia, and Other Risk Factors
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Journal of immunology research
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cc-by
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Haruko Takeuchi
,1 Md Alfazal Khan,2 Khalequz Zaman,3 Sayaka Takanashi,4
S. M. Tafsir Hasan
,2 Mohammad Yunus,5 and Tsutomu Iwata6 1Department of Community and Global Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan n and Clinical Services Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 S
n Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh 3Maternal and Child Health Division, icddr,b, 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
4Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan 5Emeritus Scientist, Maternal and Child Health Division, International Centre for Diarrhoeal Disease Research, B
68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
6 5Emeritus Scientist, Maternal and Child Health Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b),
68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
6 6The Graduate School of Humanities and Life Sciences, Tokyo Kasei University, Tokyo, Japan 6The Graduate School of Humanities and Life Sciences, Tokyo Kasei University, Tokyo, Japan Correspondence should be addressed to Haruko Takeuchi; htakeuchi-tky@umin.net Correspondence should be addressed to Haruko Takeuchi; htakeuchi-tky@umin.net Received 5 August 2019; Accepted 16 November 2019; Published 5 December 2019 Received 5 August 2019; Accepted 16 November 2019; Published 5 December 2019 Guest Editor: Barbara C. Figueiredo Guest Editor: Barbara C. Figueiredo Copyright © 2019 Haruko Takeuchi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited. Ascaris lumbricoides is the most common soil-transmitted helminth and infects 447 million people in impoverished areas
worldwide. It causes serious morbidity including wheezing and influences various aspects of human immunity, such as type 2
innate lymphoid cells, regulatory T cell function, and acquired immunity. Thus, it is crucial to elucidate its influence on human
immunity. We aimed to classify wheezing children based on their Ascaris infection intensity and other risk factors using
hierarchical cluster analysis to determine the mechanisms of and the degree to which Ascaris contributes to childhood wheezing
in rural Bangladesh. We analyzed relevant data collected in 2001. The participants included 219 5-year-old wheezing children
who were randomly selected from 1705 children living in the Matlab Health and Demographic Surveillance area of the
International Centre for Diarrhoeal Disease Research, Bangladesh. Hierarchical cluster analysis was conducted using variables of
history of pneumonia, total and specific immunoglobulin E levels, Ascaris infection intensity, and parental asthma. Three
distinct wheezing groups were identified. Hindawi
Journal of Immunology Research
Volume 2019, Article ID 4236825, 8 pages
https://doi.org/10.1155/2019/4236825 Hindawi
Journal of Immunology Research
Volume 2019, Article ID 4236825, 8 pages
https://doi.org/10.1155/2019/4236825 1. Introduction mentation of a national deworming program, indicating that
the decrease in the prevalence of Ascaris infection did not
increase wheezing [15]. Ascaris lumbricoides is the most common soil-transmitted
helminth (STH), and Ascaris infection is one of 13 neglected
tropical diseases of great concern. The STH affects approx-
imately 1.5 billion people worldwide, and Ascaris infects
447 million people in impoverished areas of Africa, Asia,
and Central and South America [1, 2]. The people at risk
are preschool children and school-age children [1]. The
WHO has implemented a program since 2001 for people
at risk in endemic areas in order to eliminate STH infec-
tions to reduce intensity of infection and to protect infected
individuals from morbidity related to the worms harbored
[1]. Although the eradication program of helminthic infec-
tions has been on the way, an unacceptably large number of
individuals continue to suffer from them despite the pro-
gram [2]. The morbidity related to the worms harbored
includes abdominal pain, general malaise and weakness,
intestinal obstruction, and impaired cognitive and physical
development. In addition to these symptoms, Ascaris causes
wheezing; it migrates through the lungs during maturation,
where it induces the type 2 inflammatory response, called
Löffler’s syndrome [3]. It appears likely that Ascaris infections are associated
with increased wheezing. A systematic review and meta-
analysis of 22 studies found an association between Ascaris
infection and wheezing [16]. Another systematic review con-
ducted in Latin America reported an association of a higher
risk of asthma or wheezing with an Ascaris infestation [17]. However, this relationship remains controversial because
the results of multiple epidemiological studies both support
and refute the protective effects of helminths on asthma
and allergies [13–18]. In 2001, we also reported that anti-Ascaris IgE was an
increasing risk factor for childhood wheezing in rural areas
of Bangladesh [19], and in 2005, we found that anti-Ascaris
IgE was an increasing risk factor for childhood BHR in the
same rural areas [20]. In these studies, Ascaris infection itself
was not a risk factor for wheezing. The antiparasite role of
IgE antibody against helminths is thought to be a normal
component of the protective response of the host during
infection, and they are not usually associated with allergic
symptoms. However, allergic manifestations have been
described in some helminth infections such as Ascariasis
and Anisakiasis [21]. Haruko Takeuchi
,1 Md Alfazal Khan,2 Khalequz Zaman,3 Sayaka Takanashi,4
S. M. Tafsir Hasan
,2 Mohammad Yunus,5 and Tsutomu Iwata6 Children in Cluster 1 (n = 50) had the highest titers of the total, anti-Ascaris, anti-
Dermatophagoides pteronyssinus, and anticockroach IgEs and experienced the fewest episodes of pneumonia. Cluster 2 (n = 114),
the largest group, experienced few episodes of pneumonia and had the lowest titers of the total, anti-Ascaris, anti-Dp, and
anticockroach IgEs. Cluster 3 (n = 32) consisted of participants with the most episodes of pneumonia and lower titers of the
total and specific IgEs. The extremely high prevalence of Ascaris infection found in Clusters 1-3 was 78%, 77%, and 72%,
respectively. Childhood wheezing in rural Bangladesh could be divided into three groups, with 26% of wheezing attributable
to anti-Ascaris IgE and 16% to history of pneumonia during early childhood, and 58% might have been due to Ascaris
infection without elevated anti-Ascaris IgE. Journal of Immunology Research 2 1. Introduction y
A potential explanation for the role of Ascaris infection
in wheezing might be pulmonary inflammation of type 2
immunity induced by type 2 innate lymphoid cells (ILC2s). Animal worms, such as Nippostrongylus, known as the rat
hookworm, which have a larval stage in the lungs, have been
linked to lung damage, type 2 immune responses, and long-
term changes in lung function and structure in nonhuman
hosts, which are consistent with allergic airway disease [4]. Migration of Nippostrongylus larvae through the lungs
causes damage to the epithelium, promoting the release of
damage-associated molecular patterns from epithelial cells
in the airway [4–6]. The release of interleukin-33 (IL-33)
and IL-25 promotes the activation of ILC2s, leading to an
increase in the release of the type 2 cytokines, IL-4, IL-5,
and IL-13 [4, 6], which have been found to be part of a path-
way in both the innate and adaptive responses to lung larval
migration in mice [5, 6]. Furthermore, Ascaris larval migra-
tion causes significant pulmonary damage, including bron-
chial hyperreactivity (BHR) and type 2 inflammatory lung
pathology resembling an extreme form of allergic airway dis-
ease in mice [7]. Ascaris influences on various aspects of human immu-
nity, such as type 2 innate lymphoid cells (ILC2s), Treg func-
tion, and acquired immunity; hence, childhood wheezing in
rural Bangladesh might be attributable to Ascaris infection
through a complex interplay between innate, regulatory,
and acquired immunity. The mechanism by which Ascaris
is involved in the development of wheezing and asthma
symptoms has caught attention given the serious morbidity
caused by this helminth. Therefore, the study’s purpose was
to classify wheezing children, who participated in the 2001
study, based on the intensity of their Ascaris infection, total
and specific IgEs including anti-Ascaris IgE, parental asthma,
and other risk factors to determine the mechanism by which
Ascaris causes childhood wheezing in this rural area of Ban-
gladesh and the degree to which it contributes to the develop-
ment of wheezing. 2. Methods Table 2 shows the total and specific IgE levels, prev-
alence and intensity of Ascaris infection, and pneumonia his-
tory. The first group consisted of 50 (26%) children who had
the highest titers of the total, anti-Ascaris IgEs, anti-Dp, and
anticockroach IgEs and the lowest frequency of pneumonia
episodes. The second group consisted of 114 (58%) children
who had a moderate level of pneumonia history and the low-
est titers of the total and anti-Ascaris IgEs. The third group
consisted of 32 (16%) children who had the highest fre-
quency of pneumonia episodes and low IgEs. The dataset of the 2001 study included information about
wheezing, family history of allergies, socioeconomic status,
environmental factors, helminth infections, serum total and
antigen-specific IgE levels, and the frequency of pneumonia
episodes during the earliest years of childhood. We included
the following variables in the present analysis: frequency of
pneumonia episodes when the children were 0, 1, and 2 years
of age; total, anti-Ascaris, anti-Dermatophagoides pteronyssi-
nus (Dp), and anticockroach IgE levels; history of parental
asthma; and helminth infections. SPSS 22 (IBM Japan, Tokyo, Japan) was used to perform
the cluster analysis with Ward’s minimum-variance hierar-
chical clustering method. The variables were standardized
to equalize the standard deviation of the scales. To compare
differences
among
the
clusters,
analysis
of
variance
(ANOVA) was used for parametric tests of the continuous
variables and the Chi-square test was used to analyze the cat-
egorical variables. The significance level for all statistical
analyses was set at P < 0:05. 3.2.1. Cluster 1. Twenty-six percent of the participants
(n = 50) were grouped into Cluster 1. This cluster was charac-
terized by significantly high serum levels of the total and anti-
Ascaris IgE levels, and high anti-Dp and anticockroach IgE
levels, and a significantly lower number of children with a
history of pneumonia when they were 0, 1, and 2 years of
age. A significantly lower number of the mothers in Cluster
1 were educated at the primary level, and their household
income (monthly) was lower than that of the households in
the other groups, although the difference was not significant. We found no significant differences from other groups, with
respect to sex, physical measurements, parental history of
asthma, or type of cooking fuel. Cluster 1 had the highest
prevalence and intensity of Ascaris infection (78%) although
the differences between the three groups were not significant. 2. Methods On the other hand, the sharp rise in the worldwide prev-
alence of bronchial asthma since the 1970s, with children liv-
ing in industrial and urban areas experiencing higher asthma
rates than those in rural area [8–12], has led to the hypothesis
that helminthic infections might provide protection against
asthma by suppressing the host’s immune response. Helmin-
thic infections activate regulatory T cells and induce the pro-
duction of IL-10, thereby playing a protective role against
asthma and allergies. Studies have shown that IL-10 induced
in chronic schistosomiasis suppresses atopy in African chil-
dren [13], and infection with Schistosoma mansoni has been
associated with a reduced course of asthma [14]. However,
we found concurrent decreases in the prevalence of Ascaris
infection and wheezing from no less than 72% in 2001, to
18% in 2016, and from 16% to 9%, respectively, after imple- The present study reanalyzed the data collected in 2001. The
procedures used for the data collection used are described
elsewhere [19]. In short, the study population consisted of
1705 5-year-old children randomly selected from Matlab, a
riverine rural area located 55 km southeast of Dhaka, the cap-
ital of Bangladesh. Children (n = 256) who had experienced
wheezing during the previous 12 months were identified
using a questionnaire adapted from the standardized ques-
tionnaire of the International Study of Asthma and Allergies
in Childhood (ISAAC) [22]. We retrieved information about
the children’s history of pneumonia from the routine data-
collection system of the International Centre for Diarrhoeal
Disease Research, Bangladesh (icddr,b) in the Matlab Health
and Demographic Surveillance System (HDSS) area [23]. We
also collected blood and stool samples to measure serum total Journal of Immunology Research 3 (Figure 1). Tables 1 and 2 show the characteristics of the cur-
rent- and never-wheezing participants and the three clusters. and specific IgEs and to detect helminth infections among
the 219 wheezing children whose guardians gave us writ-
ten informed consent. At that time, the risk factors that
were assessed for childhood wheezing included family his-
tory of allergies, serum total and anti-Ascaris IgE levels,
and frequency of pneumonia episodes at 0 years, 1 year,
and 2 years of age. 3.2. Cluster Analysis. We identified three clusters through the
analysis (Figure 2). Table 1 shows the physical status, family
history, and sociodemographic characteristics of the three
groups. 2. Methods The Ethics Committee of Tokyo Kasei University
approved the study’s protocol (Sayama H27-09), and the
Ethics Committee of The University of Tokyo approved the
protocol (11956). The dataset of the study conducted in
2001 was used in the current study, and the protocol (2000-
038) was approved by the Ethical Review Committee of the
icddr,b. The 2001 study involved human participants; there-
fore, it followed the ethical principles of the Declaration of
Helsinki. Written informed consent was obtained from the
legal guardians of all the participants. 3.2.2. Cluster 2. Cluster 2 was the largest group (n = 114;
58%) of participants. It consisted of children with a moderate
frequency of pneumonia episodes and the lowest serum levels
of the total, anti-Ascaris, anti-Dp, and anticockroach IgE,
which were comparable to the levels of the never-wheezing
children. Their mothers had significantly more education, a
relatively high household income (monthly), and a higher
prevalence of asthma than the mothers did in Cluster 3,
although the differences were not significant. The prevalence
(77%) and intensity (37.7%) of Ascaris infection were as high
as Cluster 1. 3. Results 3.1. Characteristics of the Participants. The initial study’s
dataset contained 1705 children who were selected using
random-cluster sampling and 1580 of them agreed to par-
ticipate in the first questionnaire survey. Two hundred
fifty-six (16.2%) children were found to have wheezing
during the previous 12 months (current wheezing), and
219 participated in the subsequent nested case-control
study and submitted blood and stool samples. The infor-
mation collected from these 219 current wheezing children
was used for the cluster analysis. Two hundred fifty-six of
the 1324 children with no current wheezing had been ran-
domly selected as the control group and 183 of them
agreed to participate in the nested case-control study. The children without current wheezing were divided into
two groups. One of the groups consisted of 122 children
who had never experienced wheezing (never wheezing) and
the other group included 61 children who had experienced
wheezing (ever wheezing), but not within the previous 12
months. Data from the 122 children who had never experi-
enced wheezing were used as the comparison group 3.2.3. Cluster 3. Cluster 3, which was the smallest cluster
(n = 32; 16%), was characterized by their frequency of pneu-
monia episodes when they were 0, 1, and 2 years old. They
experienced a significantly higher frequency of pneumonia
episodes than the children in the other groups did. They
had more Trichuris infections, and their mothers had a lower
rate of asthma than the mothers did in the other two groups,
although the differences were not significant. The prevalence
(71.9%) and intensity (21.9%) of Ascaris infection were lower
in Cluster 3 than in the other two groups and were almost
comparable to the never-wheezing group (71.6% and
29.4%, respectively). 3.2.3. Cluster 3. Cluster 3, which was the smallest cluster
(n = 32; 16%), was characterized by their frequency of pneu-
monia episodes when they were 0, 1, and 2 years old. They
experienced a significantly higher frequency of pneumonia
episodes than the children in the other groups did. They
had more Trichuris infections, and their mothers had a lower
rate of asthma than the mothers did in the other two groups,
although the differences were not significant. The prevalence
(71.9%) and intensity (21.9%) of Ascaris infection were lower
in Cluster 3 than in the other two groups and were almost
comparable to the never-wheezing group (71.6% and
29.4%, respectively). 3. Results Journal of Immunology Research Current wheezing
256
No current-wheezing
1324
No current-wheezing
with consent
183
Current wheezing
with consent
219
All five-year-old children
in the icddr,b HDSS service area
(67 villages)
Participants with consent
1580
Random cluster sampling
All
ISAAC questionnaire
1705
(51 villages)
Data of these children were analyzed
Random sampling
Ever wheezing
61
Never wheezing
122
Figure 1: Flowchart of the sampling procedure for the dataset. All five-year-old children
in the icddr,b HDSS service area
(67 villages) Random cluster sampling 1705
(51 villages) All Random sampling Current wheezing
with consent Figure 1: Flowchart of the sampling procedure for the dataset. 4. Discussion
The major finding of this analysis was that three distinct clus-
ters of wheezing children in rural Bangladesh were identified,
with children having a high titer of anti-Ascaris IgE compris-
ing Cluster 1. Participants in this group (n = 50) had higher
titers of the total IgE, anti-Ascaris IgE, anti-Dp IgE, and
anticockroach IgE levels and experienced fewer episodes of
pneumonia. Children in Cluster 2 (n = 114) had a low fre-
quency of pneumonia episodes and lower titers of the total,
anti-Ascaris, anti-Dp, and anticockroach IgE. Cluster 3
(n = 32) consisted of children with a higher frequency of
pneumonia episodes and lower titers of the total and specific
IgEs. The prevalence of Ascaris infection was high in all
Table 1: Comparisons of physical status, family history, and sociodemographic characteristics of the three groups. Total
Cluster 1
Cluster 2
Cluster 3
Never-wheezing
n
219
50
114
32
P=
122
Sex, female (%)
108 (49)
22 (44)
63 (55)
12 (38)
0.138
66 (49)
Physical measurements (n)
194
49
113
32
122
Height (cm)
103.0
102.4
103.6
102.3
0.213
103.7
Weight (kg)
14.7
14.5
14.8
14.5
0.492
14.8
DPT3 vaccine coverage (%)
185 (94.4)
45 (90.0)
108 (94.7)
32 (100)
0.154
Measles vaccine coverage (%)
188 (95.9)
47 (94.0)
109 (95.6)
32 (100)
0.395
Family history
Mother’s asthma (%)
42 (19)
11 (22.0)
26 (22.8)
2 (6.3)
0.106
12 (9.8)
Father’s asthma (%)
25 (12)
4 (8.2)
16 (14.0)
4 (12.5)
0.578
2 (1.7)
Dry leaves as fuel
183 (86)
42 (89)
93 (83)
28 (88)
0.548
91 (75)
Mother’s education (none) (%)
93 (43)
29 (58)
41 (36)
17 (53)
0.018
48 (39)
Monthly income (BTk)
3943
2896
4164
3712
0.054
4755
DPT3: diphtheria, pertussis, tetanus vaccine; BTk: Bangladesh Taka. 3. Results Table 1: Comparisons of physical status, family history, and sociodemographic characteristics of the three groups. Total
Cluster 1
Cluster 2
Cluster 3
Never-wheezing
n
219
50
114
32
P=
122
Sex, female (%)
108 (49)
22 (44)
63 (55)
12 (38)
0.138
66 (49)
Physical measurements (n)
194
49
113
32
122
Height (cm)
103.0
102.4
103.6
102.3
0.213
103.7
Weight (kg)
14.7
14.5
14.8
14.5
0.492
14.8
DPT3 vaccine coverage (%)
185 (94.4)
45 (90.0)
108 (94.7)
32 (100)
0.154
Measles vaccine coverage (%)
188 (95.9)
47 (94.0)
109 (95.6)
32 (100)
0.395
Family history
Mother’s asthma (%)
42 (19)
11 (22.0)
26 (22.8)
2 (6.3)
0.106
12 (9.8)
Father’s asthma (%)
25 (12)
4 (8.2)
16 (14.0)
4 (12.5)
0.578
2 (1.7)
Dry leaves as fuel
183 (86)
42 (89)
93 (83)
28 (88)
0.548
91 (75)
Mother’s education (none) (%)
93 (43)
29 (58)
41 (36)
17 (53)
0.018
48 (39)
Monthly income (BTk)
3943
2896
4164
3712
0.054
4755
DPT3: diphtheria, pertussis, tetanus vaccine; BTk: Bangladesh Taka. 1: Comparisons of physical status, family history, and sociodemographic characteristics of the three groups. Table 1: Comparisons of physical status, family history, and sociodemographic characteristi 4. Discussion This polygonal line graph shows the standardized value of the
variables regarding the number of the history of pneumonia at 0,
1, and 2 years of age, Ascaris infection intensity, Trichuris
infection intensity, parental asthma, and total and specific IgE levels. Table 2: Comparisons of serum IgE levels, helminth infections, and pneumonia history among the three groups. Total
Cluster 1
Cluster 2
Cluster 3
Never-wheezing
n (%)
196 (100)
50 (25.5)
114 (58.2)
32 (16.3)
P=
122
Total IgE (IU/ml)
13598
3705
3959
<0.001
3686
Specific IgE (UA/ml)
Anti-Ascaris IgE
30.8
62.5
20.3
24.8
<0.001
14.9
Anti-Dp IgE
4.1
7.8
1.8
2.7
<0.001
1.8
Anticockroach IgE
4.2
8.1
2.3
4.0
<0.001
2.8
Helminth infection
199
Ascaris egg (+) (%)
152 (76.4)
39 (78.0)
88 (77.2)
23 (71.9)
0.789
78 (71.6)
(+++) (%)
71 (35.7)
21 (42.0)
43 (37.7)
7 (21.9)
0.158
32 (29.4)
Trichuris (+) (%)
100 (50.3)
22 (44.0)
56 (49.1)
20 (62.5)
0.252
66 (60.6)
Pneumonia history (+) n (%)
At 0 years
56 (25.6)
6 (12.0)
31 (27.2)
12 (37.5)
0.024
16 (13.1)
1 year
44 (20.1)
2 (4.0)
21 (18.4)
18 (56.3)
<0.001
4 (3.3)
2 years
38 (16.4)
2 (4.0)
0 (0.0)
32 (100)
<0.001
2 (2.0)
IgE: immunoglobulin E; Dp: Dermatophagoides pteronyssinus. 2: Comparisons of serum IgE levels, helminth infections, and pneumonia history among the three groups. 2.50000
Ward method
1
2
3
Mean Z
1.50000
1.00000
0.50000
–0.50000
Z (ar0)
Z (ar1)
Z (ar2)
Z (Ascaris intensity)
Z (Trichius intensity)
Z (anti-Ascaris IgE)
Z (anti-Dp IgE)
Z (anti-cockroach IgE)
Z (Total IgE RIST)
Z (Parental asthma)
0.00000
2.00000
Figure 2: Graph of the three clusters. Three clusters were identified. This polygonal line graph shows the standardized value of the
variables regarding the number of the history of pneumonia at 0,
1, and 2 years of age, Ascaris infection intensity, Trichuris
infection intensity, parental asthma, and total and specific IgE levels. study, which was conducted in the same region of Bangla-
desh in 2008, when the infection prevalence was 17.4%. That study reported that anti-Ascaris IgE was associated
with an increased risk of ever having asthma among 5-
year-old children [24]. Studies conducted in Latin America
have also reported similar results [25–27]. The fact that
children in Cluster 1 had higher titers of anti-Dp and
anticockroach IgE may be explained by a predisposition
to atopy among the children in this group. 4. Discussion anticockroach IgE levels and experienced fewer episodes of
pneumonia. Children in Cluster 2 (n = 114) had a low fre-
quency of pneumonia episodes and lower titers of the total,
anti-Ascaris, anti-Dp, and anticockroach IgE. Cluster 3
(n = 32) consisted of children with a higher frequency of
pneumonia episodes and lower titers of the total and specific
IgEs. The prevalence of Ascaris infection was high in all anticockroach IgE levels and experienced fewer episodes of
pneumonia. Children in Cluster 2 (n = 114) had a low fre-
quency of pneumonia episodes and lower titers of the total,
anti-Ascaris, anti-Dp, and anticockroach IgE. Cluster 3
(n = 32) consisted of children with a higher frequency of
pneumonia episodes and lower titers of the total and specific
IgEs. The prevalence of Ascaris infection was high in all The major finding of this analysis was that three distinct clus-
ters of wheezing children in rural Bangladesh were identified,
with children having a high titer of anti-Ascaris IgE compris-
ing Cluster 1. Participants in this group (n = 50) had higher
titers of the total IgE, anti-Ascaris IgE, anti-Dp IgE, and Journal of Immunology Research 5 clusters (78%, 77.2%, and 72%), and it was higher in Clusters
1 and 2 than in Cluster 3, although the differences were
not significant. Th
hild
i Cl
t
1 h d hi h
tit
f th t t l
d
study, which was conducted in the same region of Bangla-
desh in 2008, when the infection prevalence was 17.4%. That study reported that anti-Ascaris IgE was associated
with an increased risk of ever having asthma among 5-
year-old children [24]. Studies conducted in Latin America
have also reported similar results [25–27]. The fact that
children in Cluster 1 had higher titers of anti-Dp and
anticockroach IgE may be explained by a predisposition
to atopy among the children in this group. In other words,
the children in Cluster 1 are likely to produce high titers
of anti-Ascaris IgE when they were infected with Ascaris
because they were atopic. This group may resemble to
multisensitized atopic wheezing cluster in other studies
[28]. However, neither the family history of asthma nor
allergies were obvious in Cluster 1. Another explanation for the elevated levels of anti-Asca-
ris and anti-Dp IgE is the cross-reactivity between the Ascaris
and the house-dust mite antigens. 4. Discussion The Ascaris antigen’s
cross-reactivity with that of the house-dust mite through
tropomyosin might stimulate the production of elevated
anti-Dp and anticockroach IgE [29]. Therefore, if Dermato-
phagoides antigen is abundant in the environment and is
inhaled, it might join with anti-Ascaris IgE on the mast cell
surface of the airway and result in wheezing [30, 31]. It is
understandable that the group with a high level of anti-Asca-
ris IgE comprises one cluster, as anti-Ascaris IgE was an inde-
pendent risk factor for wheezing [19]. In the study in 2001,
whose participants are the target population of the present
study, the odds ratios of anti-Ascaris IgE levels for current
wheezing increased and P values decreased as the children
expressed severer symptoms. This association was not found
in total, anti-DP, or anticockroach IgEs. Th
hildr n in Cl
t r 2
p ri n
d r l ti
l f
pi
Table 2: Comparisons of serum IgE levels, helminth infections, and pneumonia history among the three groups. Total
Cluster 1
Cluster 2
Cluster 3
Never-wheezing
n (%)
196 (100)
50 (25.5)
114 (58.2)
32 (16.3)
P=
122
Total IgE (IU/ml)
13598
3705
3959
<0.001
3686
Specific IgE (UA/ml)
Anti-Ascaris IgE
30.8
62.5
20.3
24.8
<0.001
14.9
Anti-Dp IgE
4.1
7.8
1.8
2.7
<0.001
1.8
Anticockroach IgE
4.2
8.1
2.3
4.0
<0.001
2.8
Helminth infection
199
Ascaris egg (+) (%)
152 (76.4)
39 (78.0)
88 (77.2)
23 (71.9)
0.789
78 (71.6)
(+++) (%)
71 (35.7)
21 (42.0)
43 (37.7)
7 (21.9)
0.158
32 (29.4)
Trichuris (+) (%)
100 (50.3)
22 (44.0)
56 (49.1)
20 (62.5)
0.252
66 (60.6)
Pneumonia history (+) n (%)
At 0 years
56 (25.6)
6 (12.0)
31 (27.2)
12 (37.5)
0.024
16 (13.1)
1 year
44 (20.1)
2 (4.0)
21 (18.4)
18 (56.3)
<0.001
4 (3.3)
2 years
38 (16.4)
2 (4.0)
0 (0.0)
32 (100)
<0.001
2 (2.0)
IgE: immunoglobulin E; Dp: Dermatophagoides pteronyssinus. 2.50000
Ward method
1
2
3
Mean Z
1.50000
1.00000
0.50000
–0.50000
Z (ar0)
Z (ar1)
Z (ar2)
Z (Ascaris intensity)
Z (Trichius intensity)
Z (anti-Ascaris IgE)
Z (anti-Dp IgE)
Z (anti-cockroach IgE)
Z (Total IgE RIST)
Z (Parental asthma)
0.00000
2.00000
Figure 2: Graph of the three clusters. Three clusters were identified. 4. Discussion p
g
Acute lower respiratory infections (ALRI) have been
major causes of morbidity and mortality in Bangladesh; how-
ever, improvements in the management of childhood ill-
nesses have successfully decreased deaths caused by ALRI
among young children [38]. Thus, it is understandable that
these children had a higher risk of developing asthma in sub-
sequent years. The symptoms of children in Cluster 3 were
compatible with these observations, indicating the need for
attention to wheezing post-ALRI in order to stem the
increase in asthma in rural Bangladeshi children. This group
might be comparable to the nonatopic postviral bronchial
hyperresponsiveness group of Tucson Study [39]. We found 3 clusters as predictive index for asthma in
infants and preschoolers in rural Bangladesh. Anti-Ascaris
IgE was an independent risk factor for wheezing against the
fact that anti-DP IgE was not a risk factor for wheezing. However, children with high anti-Ascaris IgE and anti-Dp
IgE seem to comprise 1 group, which indicates that the chil-
dren with high anti-Ascaris IgE might emerge as children
who have high anti-Dp IgE with the development of the soci-
ety in the future. Since the children in this group might
develop persistent wheezing in the future through early sen-
sitization by any antigen, early sensitization with Ascaris
antigen by Ascaris infection should absolutely be prevented. Therefore, we think children with high anti-Ascaris IgE
might need to be followed up carefully, before the develop-
ment of the future atopic type to curb the increase of persis-
tent asthma. In animal models, worms have been linked to type 2
immune responses through ILC2s in the lungs, including air-
way hyperresponsiveness which resembles an extreme form
of allergic airway disease [4–7]. Although the function of
human ILC2s in Ascaris infection should be investigated epi-
demiologically and experimentally in future studies, it has
been reported that Ascaris induces an inflammatory response
in the lungs independent of its effect on IgE production, which
may explain some of the contradictory findings of studies
examining the association between geohelminth infection,
atopy, and asthma [18]. As anti-Ascaris IgE increases only in
individuals with current or past Ascaris infections, the notion
that childhood wheezing in rural Bangladesh might be attrib-
utable to Ascaris infection is reasonable. 4. Discussion This finding was supported by a subsequent Journal of Immunology Research 6 this group or with Ascaris infection intensity as measured by
Ascaris egg count in the stool. Therefore, we analyzed Ascaris
infection intensity and family history asthma and allergies by
the demographic and health-related characteristics of the
three groups: sex; history of diarrhea; physical status; number
of family members; number of older or younger children;
number of rooms in the house; duration of exclusive breast
feeding; coverage for the diphtheria, pertussis, and tetanus
vaccine and the measles and bacillus Calmette-Guerin vac-
cines; eczema; allergic rhinitis; household smoking; water
supply; and parental education. However, no specific charac-
teristics of the children were found to be significant in the
analysis, except for a higher level of maternal education. Then, what factor contributed to wheezing in Cluster 2? This
group and Cluster 1 had a higher prevalence of Ascaris infec-
tion than did Cluster 3, although the difference was not sig-
nificant. The main difference between Clusters 1 and 2 was
the serum levels of IgE, indicating that children in Cluster 1
had the capacity to produce high titers of IgE than Cluster
2. In 2015, we conducted an epidemiological study, which
found concurrent decreases in the prevalence of wheezing
and Ascaris infection among 5-year-old children in rural
Bangladesh [15]. The study also showed that wheezing chil-
dren had a significantly higher rate of Ascaris infection com-
pared to never-wheezing children, although Ascaris infection
was not a risk factor for wheezing. However, it was evident
that wheezing and the prevalence of Ascaris infection
decreased simultaneously. ducted in Dhaka, Bangladesh [33]. Respiratory syncytial
virus, which is known to cause recurrent wheezing and
BHR in later life [34, 35], was the most common virus
detected in children less than 2 years old who were hospital-
ized due to severe lower respiratory tract infections. Rhinovi-
rus, a causative agent of the common cold, is also related to
exacerbations of asthma attacks in 80% of children and might
have been present in 50% of adults [36, 37]. Although we did
not examine the etiologic agents of pneumonia in our study,
we speculate that the majority of episodes might have been
due to these pathogens. 4. Discussion In other words,
the children in Cluster 1 are likely to produce high titers
of anti-Ascaris IgE when they were infected with Ascaris
because they were atopic. This group may resemble to
multisensitized atopic wheezing cluster in other studies
[28]. However, neither the family history of asthma nor
allergies were obvious in Cluster 1. g
Another explanation for the elevated levels of anti-Asca-
ris and anti-Dp IgE is the cross-reactivity between the Ascaris
and the house-dust mite antigens. The Ascaris antigen’s
cross-reactivity with that of the house-dust mite through
tropomyosin might stimulate the production of elevated
anti-Dp and anticockroach IgE [29]. Therefore, if Dermato-
phagoides antigen is abundant in the environment and is
inhaled, it might join with anti-Ascaris IgE on the mast cell
surface of the airway and result in wheezing [30, 31]. It is
understandable that the group with a high level of anti-Asca-
ris IgE comprises one cluster, as anti-Ascaris IgE was an inde-
pendent risk factor for wheezing [19]. In the study in 2001,
whose participants are the target population of the present
study, the odds ratios of anti-Ascaris IgE levels for current
wheezing increased and P values decreased as the children
expressed severer symptoms. This association was not found
in total, anti-DP, or anticockroach IgEs. Figure 2: Graph of the three clusters. Three clusters were identified. This polygonal line graph shows the standardized value of the
variables regarding the number of the history of pneumonia at 0,
1, and 2 years of age, Ascaris infection intensity, Trichuris
infection intensity, parental asthma, and total and specific IgE levels. clusters (78%, 77.2%, and 72%), and it was higher in Clusters
1 and 2 than in Cluster 3, although the differences were
not significant. The children in Cluster 2 experienced relatively few epi-
sodes of pneumonia and had the lowest titers of the total
and anti-Ascaris IgE and the lowest anti-Dp IgE level. Before
the analysis, we expected to find an association of family his-
tory of asthma and allergies with a high titer of anti-Dp IgE in The children in Cluster 1 had higher titers of the total and
anti-Ascaris IgE and slightly elevated anti-Dp and anticock-
roach IgE levels. We reported that in 2005, elevated serum
anti-Ascaris IgE was associated with BHR in children in rural
Bangladesh [20]. 4. Discussion These findings indi-
cate that the high prevalence of Ascaris infection in Clusters
1 (78%) and 2 (77.2%) might be a contributing factor to the
wheezing of the children in these groups. References [16] J. Leonardi-Bee, D. Pritchard, J. Britton, and the Parasites in
Asthma Collaboration, “Asthma and Current Intestinal Para-
site Infection,” American Journal of Respiratory and Critical
Care Medicine, vol. 174, no. 5, pp. 514–523, 2006. [1] “Soil-transmitted helminth infections,” July 2019, https://www
.who.int/news-room/fact-sheets/detail/soil-transmitted-
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prevalence of Ascaris infection might have been a contrib-
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childhood wheezing merits further investigation. Cluster 3 (n = 32) consisted of children with a higher fre-
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and specific IgEs. In the present study, information about
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Prof. Masamine Jimba, the professor and the chair of the
Department of Community and Global Health of The
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Prevalence of SARS-CoV-2 antibodies and associated factors in the adult population of Belgium: a general population cohort study between March 2021 and April 2022
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Prevalence of SARS-CoV-2 antibodies and
associated factors in the adult population of
Belgium: a general population cohort study between
March 2021 and April 2022
Johan Van der Heyden ( johan.vanderheyden@sciensano.be )
Epidemiology and public health, Sciensano
Victoria Leclercq
Epidemiology and public health, Sciensano
Els Duysburgh
Epidemiology and public health, Sciensano
Laura Cornelissen
Epidemiology and public health, Sciensano
Isabelle Desombere
Immune response, Sciensano
Inge Roukaerts
Viral diseases, Sciensano
Lydia Gisle
Epidemiology and public health, Sciensano
Research Article
Keywords: Seroprevalence, SARS-CoV-2 antibodies, COVID-19, cohort study, saliva, trends, Belgium,
population-based
Posted Date: April 18th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2802349/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read
Full License
Page 1/25
Abstract
Background
This cohort study assessed seroprevalence trends of SARS-CoV-2 antibodies in the general Belgian
population between March 2021 and April 2022, and explored factors associated with seropositivity among
the vaccinated and unvaccinated population. Seroreversion and its potential determinants were also
examined.
Methods
A random sample of the adult population in Belgium was invited to provide a saliva sample and to
complete a survey questionnaire. Participants were followed up twice for a new saliva sample and updated
information. Antibodies were assessed with a semi-quantitative measure of anti-RBD (Receptor Binding
Domain) IgG ELISA. Seven time periods were defined for estimating SARS-CoV-2 antibody prevalence using
post stratification weights to match the population distribution. Seroreversion was defined as passing from
a positive to a negative antibody test from one data collection point to the next. Potential determinants of
seropositivity were assessed through hierarchical multiple logistic regressions.
Results
In total 6,178 valid observations were obtained from 2,768 individuals. SARS-CoV-2 antibody prevalence
increased from 25.1% in the beginning of the study period to 92.3% in the end. Among the vaccinated
population, factors significantly associated with a higher seropositivity were being younger, having a
bachelor diploma, living with others, having had a vaccine in the last 3 months and having received a
nucleic-acid vaccine or a combination. Lower seropositivity rates were observed among vaccinated people
with a neurological disease and transplant patients. Factors significantly associated with a higher
seropositivity rate among the unvaccinated population were having non-O blood type and being nonsmoker.
Among fully vaccinated people the seroreversion rate was much lower (0.3%) among those who had
received their latest vaccine in the last 3 months compared to those who had received their latest vaccine
more than 3 months ago (2.7%).
Conclusions
The rapid increase in antibody seropositivity in the general adult population in Belgium during the study
period was driven by the vaccination campaign which ran at full speed during this period. Factors
associated with higher and lower seropositivity were identified among the vaccinated and unvaccinated
people.
Background
Page 2/25
The first case of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection in Belgium was
reported on February 4th 2020 (1). The rapid increase of people testing positive thereafter marked the first
epidemic wave, which started on March 1st 2020 (2). A national COVID-19 (Coronavirus Disease 2019)
surveillance system was set up by Sciensano, the Belgian institute of health, at an early stage of the
epidemic, mainly focussing on COVID-related cases, hospitalisations, deaths and later, on vaccination
coverage (2). In parallel, several serological studies were launched according to the WHO recommendations
(3). Former research had indeed shown that specific serum antibodies to SARS-CoV-2 increased 2–3 weeks
following the primary infection and remained detectable for 3 to 6 months after (4, 5). Serology tests can
thus be used to evaluate the number of people, including asymptomatic, that get infected with the virus and
to estimate the cumulative prevalence of infection and disease transmission over time (6).
Seroepidemiological studies can also provide an important empirical input for mathematical models in the
analysis and prediction of the pandemic (7). During the vaccination campaign, serological surveillance
remains relevant for assessing the prevalence of SARS-CoV-2 infection among the unvaccinated people
and for comparing the immune response status between naïve and previously infected individuals among
the vaccinated (8). The SARS-CoV-2 seroprevalence studies in Belgium were conducted in several settings,
at first on residual blood samples (9) but also on specific subpopulation sera, e.g. from blood donors (2),
healthcare workers in hospitals (10), primary healthcare providers (11), school children (12) and nursing
home residents and staff (13, 14). This paper reports findings of a seroprevalence study conducted in a
general population sample of adults randomly selected from the Belgian national register. Similar
serological studies in community settings were initiated in other European countries (15). Ours took place in
the period running from March 2021 to April 2022, with three data collection points within the same study
sample.
Although serum-based methods are the gold standard to assess the presence of SARS-CoV-2 antibodies,
research using blood samples is difficult to implement in a geographically scattered random selection of
the general population. The major barriers encountered in this context are low participation acceptance as
well as high economic, logistical and time constraints related to drawing blood samples and delivering
them to a lab in optimal conditions. This led us to consider an alternative method for community-based
surveillance, i.e., using salivary antibody tests. Two independent studies from the US and from Belgium had
shown this to be a non-invasive scalable substitute to serology tests (16, 17). Another study confirmed that
both serum and salivary IgG antibodies to SARS-CoV-2 persisted in the majority of COVID-19 patients for at
least 3 months after symptom onset (18).
During the course of our study, Belgium faced four COVID-19 waves and a steep increase in the COVID-19
vaccination rate (2). The study objectives were adapted to the rapidly progressing dynamics of the
epidemic and considered the impact of the vaccination campaign on antibody prevalence. Three research
questions are tackled in the framework of this paper:
How did the prevalence of SARS-CoV-2 antibodies evolve during the 13-months study period in the
general adult population, and independently among the vaccinated and the unvaccinated people?
What sociodemographic and health-related characteristics associate with seropositivity, in both the
vaccinated and the unvaccinated people?
Page 3/25
To what extent did people with a positive test result serorevert to a negative test result in a following
data collection point, and what factors were associated with seroreversion?
Methods
Study design and study population
The study design has been thoroughly described in former publications (19, 20). In brief, this is a
prospective cohort study in a random sample of adults selected from the Belgian National Register of
residents. A stratified clustered sampling method was used, with private households as primary sampling
unit. Within a selected household, all participants aged 18 years and older were eligible for participation. A
total of 1,339 people belonging to 634 households were invited to participate in a pilot phase of the study.
We used the household composition of the 2018 national Health Interview Survey (21) participants to
calculate, per stratum, the number of households needed in order to reach the predefined representative
number of respondents per gender, age group and region of residence in this pilot phase. Next, sample size
calculations indicated that 1,200 individuals in each of the 3 Belgian regions (thus a total of 3,600) would
be large enough to obtain regional estimates on tSARS-CoV-2 seroprevalence with sufficiently high
precision (19). Based on these predictions and on the participation scheme obtained during the pilot phase,
another 12,862 individuals in 7,598 households were selected to supplement the baseline data collection
point. For practical reasons, invitations to participate in this cohort were sent out in 3 distinct time batches
within a period of 4 weeks. Because the fieldwork procedures were not modified between the pilot and
supplementary baseline data collection phases, the observations gathered in these two phases were
merged into a total “wave 1” dataset. This allowed to increase the power of the analysis and to assess
finetuned trends in antibody seroprevalence over an expanded timeline during the first study period. Finally,
the wave 1 participants who agreed to follow-up were reinvited for the wave 2 and wave 3 data collection
points. People were thus contacted 3 times for the study: between 25/03/2021 and 15/06/2021 for wave 1,
between 23/09/2021 and 28/10/2021 for wave 2 and between 25/01/2022 and 08/02/2022 for wave 3.
Participants got access to their test results through the project website or by phone, using a code.
Data collection
Saliva samples were obtained from the participants through self-collection. People selected for the study
received an invitation letter, two consent forms, an Oracol® tube (Malvern Medical Developments Ltd) for
collecting saliva, a user’s guide (and online video) on how to proceed and how to obtain their test result, a
survey questionnaire (paper or online) and a prepaid return envelope. All invitations were sent through a
third trusted party (Statbel, the national office of statistics) detaining participants’ names and addresses for
follow-up. Data were collected from 29th March 2021 until 25th April 2022.
Assessment of outcome variables
The saliva samples were returned by post and analysed in Sciensano’s laboratories. Semi-quantitative
measurements of anti-receptor-binding domains IgG (anti-RBD IgG) were performed using the WANTAI
SARS-CoV-2 IgG ELISA (Quantitative Wantai Bio-Pharm, cat n° WS-1396) customized for saliva (in house
Page 4/25
protocol). The cut-off value for anti-RBD IgG positivity in saliva was previously established using PCRconfirmed samples from adults for whom both serum and saliva were available. Saliva from positive PCR
cases were tested using the in house ELISA-protocol. Assay performance at each individual cut-off was
evaluated using ROC (Receiver Operating Characteristic) analyses and a specificity-optimized cut-off value
for anti-RBD IgG positivity in saliva was determined. This cut-off was used to create a binary variable
reflecting the presence of SARS-CoV-2 antibodies in saliva with a specificity of 96.7% and a sensitivity of
95.1%. Another outcome variable in our study is that of seroreversion, which was defined as passing from a
positive to a negative antibody test result from one data collection point to the next.
Assessment of potential determinants
The potential determinants of antibody seroposivity and/or seroreversion in this study comprised baseline
measures of sociodemographic and occupational information (age, sex, region of residence, living
situation, working in the health care sector), health and biological characteristics (self-rated health, activity
limitations, blood type), important health risk factors (obesity and smoking) and chronic disease status.
The latter expressed having at least one chronic disease or condition from a list of 12 that were defined by
the Superior Health Council as priority criteria for COVID-19 vaccination in Belgium (22). Information
regarding COVID-19 illness and vaccination was gathered at each data collection point. Information on age,
sex and region of residence was obtained directly from the national register. All other potential
determinants were assessed through the self-administered questionnaires. Supplementary file 1 provides
detailed information on all covariates included in the analyses, including the vaccination status and the
presence of chronic diseases and conditions.
Statistical analyses
First, we performed the trend analysis of community-wide SARS-CoV-2 seroprevalence using post
stratification survey weights. These were calculated with reference to both the Belgian population structure
on January 1, 2021 established by Statbel and the official national vaccination records. Weighted
seroprevalence estimates with 95% confidence intervals were calculated for seven time points during the
full study period. Details on the weight calculations are provided in Supplementary file 2.
Subsequently, we assessed potential factors associated with the presence of SARS-CoV-2 antibodies in
saliva samples. To take into account within-subject observations across the three data collection points,
hierarchical multiple logistic regressions with three levels (observation, individual, household) were
modelled, including the week number when the saliva was collected as covariate in the model. These
analyses were executed with the PROC GLIMMIX procedure in SAS® (23) and were conducted separately
for fully vaccinated and unvaccinated people. Observations of the partially vaccinated were not involved in
these analyses. A two-step approach was applied: first, the association of each independent variable with
the SARS-CoV-2 antibody outcome was assessed separately. Next, the variables found to be significantly
associated (p < 0.05) with having SARS-CoV-2 antibodies in the univariate analyses were modelled in a
multivariable logistic regression with age and gender. If associations remained significant (p < 0.05), the
interaction of the variable with time (week number) was also tested. This allowed assessing whether the
associations changed over time.
Page 5/25
Lastly, antibody seroreversion was examined among individuals with at least two consecutive observation
points. Possible factors associated with seroreversion were additionally explored. A p-value < 0.05 was
considered statistically significant for all analyses.
Results
Study population
Figure 1 indicates the participation flow across the different study waves. In total, 2,768 individuals
participated in wave 1, among whom 1,389 in all 3 waves. Merging the data from the three waves added up
to 6,178 valid observations: 4,205 observations in individuals who were fully vaccinated at the time saliva
was collected, 852 observations in those who were partially vaccinated and 1,121 observations in the
unvaccinated. Supplementary file 3 provides information on the number of saliva samples collected per
week during the study period, the evolution of the number of COVID-19 cases during this period (showing
four epidemic waves), the evolution of the national vaccination coverage, and the geographical spread of
the study participants by municipality.
Table 1 provides a comparison of the distribution of the participants by gender, age group and region of
residence with the equivalent distribution in the total Belgian population. As shown, participation drop-out
between wave 1 and wave 3 was considerable, especially in the two youngest age groups (18–29 and 30–
49). However, considering the characteristics of those who participated in the 3 waves, all population
groups remained well represented.
Page 6/25
Table 1
Distribution of participants by gender, age group and region in relation to this distribution in the Belgian
population, 2021
Gender
Age
group
Region
SalivaHIS sample
Participants wave 1
SalivaHIS sample
Number
%
Number
%
%
Men
1,248
45.1
646
46.5
48.8
Women
1,520
54.9
743
53.5
51.1
18–29
years
423
15.3
133
9.6
17.9
30–49
years
862
31.1
355
25.6
32.6
50–69
years
825
29.8
459
33.1
32.1
70 +
years
658
23.8
442
31.8
17.3
Flanders
1,160
41.9
610
43.9
58.2
Brussels
819
29.6
404
29.1
10.3
Wallonia
789
28.5
375
27.0
31.5
2,768
100.0
1,389
100.0
100.0
Total
Participants wave 1,
2 and 3
Belgian population
2021 (18+)*
* Source: Statbel
Prevalence trend of SARS-CoV-2 antibodies
The trend in prevalence of antibodies to SARS-CoV-2 observed in the general adult population and seperatly
among the fully vaccinated and unvaccinated individuals is presented in Table 2. In the general population,
prevalence of antibody increased from 25.1% in the first half of April 2021 to 92.3% in March 2022.
Although the prevalence of SARS-CoV-2 antibodies among the fully vaccinated was high throughout the
study period, it still rose as time progressed. During the pilot phase in March 2021, a relatively low antibody
prevalence was found among the fully vaccinated (81.3%), but this result is based on the rather small
number (n = 18) of fully vaccinated in our sample at that time, mostly consisting of older people, with a
shorter time lag between the latest vaccine and saliva collection than during the main baseline study. More
precisely, between May 2021 and April 2022, the prevalence of SARSCoV-2 antibodies among the fully
vaccinated people increased from 92.5–99.9%.
Page 7/25
Table 2
Prevalence trend of SARS-CoV-2 antibodies in the population aged 18 years and older in Belgium, global
results and results by vaccination status, March 2021 until April 2022 §
Total population°
Fully vaccinated and
last vaccination in
past 3 months
Fully vaccinated
and last
vaccination ≥ 3
months ago
Unvaccinated
Period
n/N*
%**
(95%
CI)
n/N*
%**
(95% CI)
n/N*
n/N*
%**
(95%
CI)
29/3–
11/4/21
72/284
25.1
(18.7–
31.6)
14/18
81.3
(64.9–
97.7)
0/0
38/223
16.6
(10.1–
23.2)
609/1111
47.8
(44.1–
51.5)
298/324
92.5
(89.095.9)
25/27
91.3%
(80.2–
100.0)
70/410
17.5
(13.3–
21.6)
550/780
69.0
(63.3–
74.6)
335/350
96.3
(94.098.7)
38/38
100.0 (-)
34/175
21.9
(10.1–
33.7)
764/841
84.5
(79.3–
89.6)
219/227
98.0
(96.4–
99.7)
517/559
93.6
(91.4–
95.8)
7/33
16.3
(2.1–
30.6)
663/713
89.1
(84.9–
93.2)
127/134
97.8
(95.1–
100.0)
509/535
96.0
(94.2–
97.8)
7/24
24.8
(6.1–
43.6)
1049/1076
91.3
(87.1–
95.5)
766/770
99.8
(99.6–
100.0)
235/237
99.0
(97.6–
100.0)
10/30
17.8
(0.4–
35.3)
229/242
92.3
(86.8–
97.9)
133/134
99.9
(99.6–
100.0)
87/88
99.3
(97.8–
100.0)
3/13
11.7
(2.3–
21.1)
W1
17/5–
13/6/21
W1
14/6–
11/7/21
W1
27/9–
24/10/21
W2
25/10–
21/11/21
W2
24/1–
20/2/22
W3
21/2–
20/3/22
W3
§
%**
(95% CI)
Observations from samples that were taken outside the 7 defined time periods are not considered here
° Including partially vaccinated people
* Number seropositive /total
** Weighted percentage
W1 wave1
W2 wave 2
W3 wave 3
Page 8/25
Within each of the 7 time periods considered for the trend analysis, antibody prevalence was higher in fully
vaccinated people having recieved their last shot in the past 3 months than in those having received it more
than 3 months ago, but the difference between these two groups was only statistically significant (p =
0.005) within one time period (September 27 to October 24). Regarding the unvaccinated people, there was
a clear increase in the prevalence of SARS-CoV-2 antibodies during the first data collection point of the
study, rising from 16.6% at the time of the pilot study to 21.9% by the end of wave 1. During wave 2 and 3,
the number of unvaccinated people was too low to estimate sufficiently precise prevalence rates by time
period.
Characteristics associated with SARS-CoV-2 seropositivity
The potential determinants of seropositivity were explored separately for observations of the fully
vaccinated and unvaccinated individuals (Tables 3 and 4). The only factor significantly associated with
higher seropositivity in both the fully vaccinated and unvaccinated was a history of COVID-19 infection.
Among the fully vaccinated population, additional factors significantly associated with a higher
seropositivity were being younger (age group 18–39 years versus 65 + years), having a bachelor diploma
(versus those with only secondary education and lower), living with others (versus living alone), having no
chronic disease (versus at least one chronic disease), having had a vaccine in the last 3 months (versus
vaccine more than 3 months ago) and having received the nucleic-acid vaccine or a combination (versus a
viral-vectored vaccine only). Factors significantly associated with a higher seropositivity rate among the
unvaccinated population were having non-O blood type (versus O blood type) and being non-smoker
(versus smokers).
Page 9/25
Table 3
Results of simple and multiple logistic regression analyses on antibody seropositivity for the fully
vaccinated population including the observations from the three waves1
Crude OR
Adjusted OR
(95% CI)
(95% CI)
Age group
p-value time
interaction
0.1707
18–39 years
Ref
Ref
40–64 years
0.43 (0.23–0.82)*
0.66 (0.33–1.34)
65 + years
0.24 (0.13–0.43)*
0.49 (0.25–0.96)*
Man
Ref
Ref
Woman
1.65 (1.17–2.33)*
1.49 (0.99–2.23)
Gender
Region
Flanders
Ref
Brussels
1.03 (0.68–1.57)
Wallonia
1.04 (0.67–1.60)
Education
0.8584
Secondary or lower
Ref
Ref
Bachelor
2.11 (1.32–3.38)*
2.44 (1.40–4.22)*
Master or higher
1.54 (1.02–2.33)*
1.43 (0.89–2.30)
Living situation
Ref
Ref
Alone
1.70 (1.14–2.55)*
1.76 (1.10–2.83)*
With others
Health care worker
Yes
1.83 (0.87–3.86)
No
Ref
1
Vaccination status assessed at the moment the saliva test was done
2Global
Activity Limitation Indicator
3potentially
a booster vaccination
Page 10/25
0.1329
Crude OR
Adjusted OR
(95% CI)
(95% CI)
At least one chronic disease
0.0692
Yes
0.47 (0.33–0.68)*
0.59 (0.37–0.94)*
No
Ref
Ref
Good to very good
Ref
Ref
Fair, bad to very bad
1.78 (1.19–2.66)*
0.96 (0.54–1.71)
Limited
0.48 (0.32–0.71)*
0.69 (0.39–1.23)
Not limited
Ref
Ref
Self-perceived health
Long term limitation (GALI2)
Blood type
O blood type
Ref
Non-O blood type
1.02 (0.66–1.58)
1Vaccination
2Global
3
p-value time
interaction
status assessed at the moment the saliva test was done
Activity Limitation Indicator
potentially a booster vaccination
Page 11/25
Crude OR
Adjusted OR
(95% CI)
(95% CI)
p-value time
interaction
Obesity (BMI ≥ 30 kg/m²)
Yes
0.67 (0.44–1.03)
No
Ref
Smoking
Yes
0.65 (0.40–1.07)
No
Ref
COVID-19 infection
Yes
No
Time since last vaccination
dose3
< 3 months
0.0927
10.14 (3.19–
32.24)*
12.55 (2.96–
53.16)*
Ref
Ref
0.6514
1.38 (099-1.92)
1.76 (1.09–2.85)*
Ref
Ref
≥ 3 months ago
0.1249
Type of vaccine received
Nucleic-acid or combination
8.44 (5.90-12.07)*
7.70 (5.07–11.69)*
Only viral-vectored
Ref
Ref
1
Vaccination status assessed at the moment the saliva test was done
2Global
3
Activity Limitation Indicator
potentially a booster vaccination
Page 12/25
Table 4
Results of simple and multiple logistic regression analyses on antibody seropositivity for the
unvaccinated population including the observations from the three waves1,
Crude OR
Adjusted OR
(95% CI)
(95% CI)
18–39 years
Ref
Ref
40–64 years
1.06 (0.71–1.58)
0.81 (0.45–1.48)
65 + years
0.86 (0.45–1.64)
0.55 (0.18–1.70)
Man
Ref
Ref
Woman
1.34 (0.94–1.90)
0.98 (0.56–1.69)
Age group
Gender
Region
Flanders
Ref
Brussels
1.44 (0.92–2.26)
Wallonia
1.25 (0.79–1.97)
Education
Secondary or lower
Ref
Bachelor
1.13 (0.73–1.77)
Master or higher
0.96 (0.62–1.48)
Living situation
Alone
Ref
With others
1.25 (0.71–2.20)
Health care worker
Yes
0.97 (0.41–2.28)
No
Ref
1Vaccination
status assessed at the moment the saliva test was done
2
Global Activity Limitation Indicator
3
potentially a booster vaccination
Page 13/25
p-value time interaction
Crude OR
Adjusted OR
(95% CI)
(95% CI)
p-value time interaction
At least one chronic disease
Yes
1.14 (0.69–1.88)
No
Ref
Self-perceived health
Good to very good
Ref
Fair, bad to very bad
1.43 (0.16–12.82)
Long term limitation (GALI2)
Limited
0.63 (0.31–1.28)
Not limited
Ref
Blood type
0.9803
O blood type
Ref
Ref
Non-O blood type
1.67 (1.05–2.67)*
1.74 (1.01–3.01)*
1
Vaccination status assessed at the moment the saliva test was done
2
Global Activity Limitation Indicator
3potentially
a booster vaccination
Page 14/25
Crude OR
Adjusted OR
(95% CI)
(95% CI)
p-value time interaction
Obesity (BMI ≥ 30 kg/m²)
Yes
1.12 (0.65–1.92)
No
Ref
Smoking
0.4346
Yes
0.43 (0.25–0.74)*
0.33 (0.13–0.82)*
No
Ref
Ref
0.0825
Last COVID-19 infection3
< 3 months ago
14.23 (8.10–25.0)*
16.59 (7.10-38.77)*
≥ 3 months ago
8.99 (4.83–16.71)*
7.61 (2.97–19.52)*
No COVID-19 infection
Ref
Ref
1Vaccination
2Global
3
status assessed at the moment the saliva test was done
Activity Limitation Indicator
potentially a booster vaccination
The association between the chronic disease indicator and seropositivity among the fully vaccinated
people was further explored in a specific analysis. For that, the chronic disease indicator was replaced by
the 12 diseases and conditions separately. Detailed results are presented in Supplementary file 4. The main
finding is that, after adjustment for the potential confounders, the fully vaccinated people with a
neurological disease and those with a transplant were significantly less inclined to present a seropositive
test (respectively [ORa 0.33; 95% CI 0.13–0.84] and [ORa 0.01; 95% CI < 0.001–0.07]) compared to people
without those problems.
Seroreversion and associated characteristics
Seroreversion, defined here as passing from a positive to a negative antibody test from one data collection
point to the next, was assessed between the first and second data collection points (waves 1 and 2) and
between the second and third data collection points (wave 2 and 3). Out of 909 people with a positive
antibody test in wave 1, 32 (3.5%) seroreversed in wave 2. Out of 1,065 seropositive people in wave 2, only
8 (0.8%) seroreversed in wave 3. So in total, seroreversion occurred in 40 cases during the study period.
Seroreversion was significantly associated with the vaccination time and status: seroreversion rate was
lowest (0.3%) among the fully vaccinated people having received their latest vaccine in the last 3 months
Page 15/25
compared to those who received their latest vaccine more than 3 months ago (2.7%). In contrast, the
percentage of seroreversion reached 36.0% in people who were not or partially vaccinated.
Discussion
Time trends
We aimed to determine the prevalence and evolution of SARS-CoV-2 antibodies among the general
population aged 18 years and older during the 13-months study period. The observed increase in SARSCoV-2 antibody prevalence, from 25.1% in April 2021 to 92.3% in March 2022, was essentially the
consequence of the vaccination campaign which was running at full scale during this period. We compared
our results with those from the Belgian blood donors (2) between April and December 2021, a period during
which data were available for both studies, and found that antibody prevalence rates were consistently
lower in our study. Though antibody testing in serum is more common, the salivary test we used was
validated against COVID-19 PCR and paired serum/saliva samples with 95.1% sensitivity. The difference
obtained in these comparative results highlights the value of general population studies to complement the
scope of national serological surveillance.
Importantly, this study allowed assessing the trend in prevalence of antiSARSCoV-2 antibodies in both
vaccinated and unvaccinated people from the general population. Although the prevalence of antibodies in
vaccinated people was high throughout the study period, it increased slightly as time progressed. One
ought to consider the seroprevalence of 81.3% observed during the pilot phase with caution due to the very
small number of observations involved. However, from the next wave 1 (baseline) period onwards, the
number of vaccinated participants was high enough to give reasonably precise estimates. Here it appeared
that the prevalence of SARS-CoV-2 antibodies in fully vaccinated people increased from 92.5–99.9%
between May 2021 and March 2022. One explanation for the lower values could be that in the beginning of
the study, the vaccine campaign predominantly focused on older people and people with chronic
morbidities, who may have a lower immune response to vaccination. Factors that may explain the increase
in immune response over time, is the possible multiple exposure to the antigen as time progressed (through
COVID-19 infection or vaccine, including booster). Among the unvaccinated people, an increase in the
prevalence of SARS-CoV-2 antibodies was observed during the first data collection period (wave 1). In wave
2 and wave 3 antibody prevalence was more variable across time and lower than expected, but again, the
low number (N = 100) of unvaccinated people in wave 2 and wave 3 call for caution in interpreting the
results. A hypothesis for the low antibody prevalence among unvaccinated people could be that these
people have been particularly careful in preventing infection by adhering more strickly to the sanitary
measures put in place (e.g. lock-down, tele-working, mask wearing). This is supported by the fact that the
proportion of people who reported a history of a COVID-19 infection was lower than expected from the
official COVID-19 infection statistics.
Characteristics associated with SARS-CoV-2 seropositivity
Page 16/25
Among fully vaccinated people seropositivity was significantly lower among people with a chronic disease.
Further exploration identified two specific groups in which this was particularly the case: people with a
neurological disease and people with a transplant. This result needs further investigation, as our study only
relies on self-reported diseases, with a limited number of affected individuals and a quite heterogeneous
variety of neurological diseases reported. However, some literature findings do support our results. For
instance in patients with multiple sclerosis, a neurological affection, disease-modifying therapies were seen
to reduce humoral immunity after SARS-CoV-2 vaccination (24). Furthermore, despite the small number of
observations for transplant individuals in our study, the seropositivity rate in this group (58.3%) was
remarkably similar to the SARS-CoV-2 anti-Spike seroprevalence of 52.4% found in a study among renal
transplant patients (25, 26).
Social factors were also influencial. First, a higher seropositivity was observed among vaccinated people
with a bachelor diploma compared to those with only secondary education and lower. A possible
hypothesis is that higher socio-economic status is associated with a better health status and behaviors,
hence a stronger immune system. However, higher seropositivity rates were not found among people with a
master degree and above. Second, the higher seropositivity among vaccinated people living with others
(compared to those living alone) may be because those people are more likely to be (even unknowingly)
exposed to the virus. Studies have shown that e.g. living with children does increase your risk of SARS-CoV2 infection (28, 29).
Finally, although both types of vaccines (nucleic-acid and viral-vectored) have been shown to be effective
and are associated with a higher presence of antibodies, some studies showed a higher seroprevalence
among people who received a nucleic-acid vaccine compared to those with a viral-vectored vaccine (27).
This was also observed in our study. This vaccine response difference could be explained by the fact that
people in the group who received a viral-vector vaccine in our study did not receive a booster shot, as the
booster dose was a nucleic-acid vaccine. Nonetheless, if we only consider the results of the first wave of
this study, that is, when people had not yet received a booster, a higher seropositivity is also observed
among those having received a nucleic-acid vaccine compared to those having received a viral-vectored
vaccine (19).
Among the unvaccinated people, a higher seropositivity was observed for those with a non-O blood type
than for those with an O blood type. A systematic review and meta-analysis indicated that blood group A
may be especially a risk factor for COVID-19, whereas the blood group O appears to be somewhat
protective (30). To what extent and how this relates with our findings remains unclear.
Seroreversion
Seroreversion occurred in only 40 study participants. This low number may be related to epidemic and virus
dynamics. Between wave 2 and 3 Delta and Omicron variants emerged which resulted in many reinfections,
hence less seroreversion.
Clearly, the time since the latest vaccination was an important predictor of seroreversion, but the results
mainly showed a much higher risk of seroreversion among the partially or unvaccinated people compared
Page 17/25
to people who were fully vaccinated. An explanation for this observation is that the antibodies developed
following vaccination or following a mix of vaccination and COVID-19 infection were more robust and wane
less rapidly than those developed after natural infection only (31, 32).
Our results are based on a binary categorization of the serological outcome variable (seropositive-vs.seronegative) instead of a continuous measure of antibody tests results which is more precise.
Seroreversion in this study thus mainly indicates the waining of antibodies below a certain threshold.
Limitations and strengths of the study
Our study has some important limitations regarding serological surveillance. First, for logistical and
practical reasons, it was opted to detect antibodies in saliva. Although it was considered to be a valid
alternative to serum-based methods, the latter are the preferred reference for seroprevalence studies.
Despite this, the performance characteristics of the in house SARS-CoV-2 RBD IgG ELISA test on saliva were
good with a high sensitivity and specificity. This RBD-test that was used to provide quick results, but
unfortunately no distinction could be made between antibodies generated as result of natural infection and
of vaccination. Furthermore, the antibody outcome reported in this study is a dichotomous indicator. This
had an impact on the level of analyses, but also on the interest of the study to participants. Indeed, even
though semi-quantitative results were available, the threshold of protection was not yet known and so they
were not communicated to the participants. This definitely had an impact on the low response rate and the
substantial drop of the participation rate in wave 2 and wave 3. Feedback on the test result to the
participants was initially an important incentive to participate in the study, but as information on their level
of protection was not provided, their motivation for participation dropped substantially, especially after the
majority had been vaccinated and the epidemic was on decline. Finally, the saliva collection was executed
by the participants themselves, without supervision. The method to collect saliva (Oracol®) is designed for
self-use and much effort had been put in giving clear instructions in a leaflet and a video. Nevertheless, it
appeared that 17% of the initial swap samples did not contain enough saliva to be analysed. In the next
waves, the importance to correctly follow the instructions for the saliva collection was emphasized. The
percentage of invalid samples dropped thereon, although it was still 9% for the wave 3 saliva collection
point.
The study also bears important strengths. It is a population-based probability sample including residents
from 317 of the 581 Belgian municipalities. Even though non-response and drop-out were substantial and
biases are inevitable, the use of post-stratification weights with both the national register and the
exhaustive Belgian vaccination record database as auxiliary data sets, ensured that results were as
representative as possible of the Belgian population aged 18 years and above.
Furthermore, the questionnaires that accompanied the three data collection waves allowed to gather
extensive information from the participants in many different domains: socio-demographic information,
health related factors, health behaviors, COVID-19 infection, vaccination status, etc. Additionally, over 90%
of the study participants agreed that their saliva samples could be stored in a biobank and that their results
could be linked with administrative databases for futher research. Finally, 85% of the wave 3 participants
Page 18/25
agreed to being recontacted in the context of supplemental public health studies. These outcomes provide
promising opportunities for future research.
Conclusions
This study provided useful information for monitoring the COVID-19 pandemic in the general adult
population in Belgium. It allowed to identify, among vaccinated and unvaccinated people, factors that were
associated with a lower humoral immune response and provided some insights on waning of SARS-CoV-2
antibodies among vaccinated people in the general population.
Because COVID-19 continues to be a public health concern, monitoring antibodies to SARS-CoV-2 in the
general population remains useful, as in other countries (33, 34), but improvements in the study set-up are
necessary:
The methods used to test seropositivity should be able to distinguish between antibodies generated as
a result of vaccination and antibodies generated as a result of infection.
Future monitoring procedures could consider postal collection of blood samples by means of a finger
prick for example. However, this may have an impact on participation bias and rate, since a blood
collection is more invasive than a saliva collection.
Growing evidence is available on the antibody levels associated with protection against SARS-CoV-2
(35). A surveillance system should be able to assess the level of protection of the general population,
in specific population groups and for different variants.
From a cost-effectiveness point of view, it should be investigated if such a surveillance could be
integrated in a more global serosurveillance system in which antibodies against other pathogens are
also included, as is for instance the case in the Netherlands in which the monitoring of the SARS-CoV-2
seroprevalence has been integrated in the national seroepidemiological (PIENTER) studies (36, 37).
Abbreviations
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
COVID-19 Coronavirus Disease 2019
IgG Imunnoglobuline G
ELISA Enzyme-linked immunoassay
RBD Receptor-binding domain
PCR Polymerase chain reaction
ROC Receiving operating characteristic
SAS Statistical analysis software
Page 19/25
PIENTER Peiling Immunisatie Effect Nederland Ter Evaluatie van het Rijksvaccinatieprogramma
Declarations
Ethics approval and consent to participate
The study has been approved by the ethics committee of the University of Ghent and the University hospital
of Ghent on 16th February 2021 (reference B6702021000193). Written informed consent was obtained from
all participants before enrollment in the study. To guarantee confidentiality, study laboratory results and
questionnaires were pseudonymized using unique study codes.
Consent for publication
Not applicable.
Availability of data and materials
Data are available on reasonable request. The statistical codes that support the findings of this study are
available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
This study was funded by Sciensano, the Belgian institute of public health, Brussels, Belgium. Sciensano
was involved in all stages of the study, from conception and implementation to analysis and reporting.
Authors' contributions
JV, VL and LG were responsible for designing the objectives and approach of the study. JV and VL
conducted the statistical analyses. JV, VL and LG were involved in the original draft preparation. All authors
critically revised the manuscript, contributed to the contents and read and approved the final manuscript.
Acknowledgements
We would like to thank Veronik Hutse, who was involved in setting up the study, Nayema Van den Houte,
who was part of the research team that implemented the study, Cyril Barbezange for scientific guidance,
and lab technicians Vera Verburgh and Marthe Depessemier. Furthermore we thank the administrative team
of the department Epidemiology and public health of Sciensano and Statbel for logistical support during
the fieldwork. Finally we would like to express our gratitude to all participants of the study.
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Page 20/25
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Figures
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Figure 1
Participation flow among Belgian adults
* Participants who had indicated that they did not want to take part in the next wave were not invited for
follow-up waves
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The ESO Diffuse Interstellar Bands Large Exploration Survey (EDIBLES)
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1. Introduction et al. 2001; Schmidt et al. 2014) and C+
60 have been identified
as pure polyatomic carbon species in the diffuse ISM and this
leaves a large gap to be filled in our current understanding of
the carbon chemical network in diffuse clouds. It is possible
that the detection of C+
60 hints at a long predicted important role
of polycyclic aromatic hydrocarbons (PAHs) in the ISM (Van
der Zwet & Allamandola 1985; Léger & d’Hendecourt 1985;
Salama et al. 1996). Recent laboratory (Zhen et al. 2014) and
modelling (Berné et al. 2015) works support the proposal by
Berné & Tielens (2012) that fullerenes may form upon photo-
dissociation of large PAH precursors. The unknown identity of the carriers of all but two diffuse in-
terstellar bands (DIBs) constitutes the longest standing spectro-
scopic enigma of modern astronomy (Sarre 2006). Two features
at 5797 and 5780 Å, which are now known to be interstellar in
origin, were first noted by Heger (1922) and studied in relation to
interstellar gas and dust by Merrill & Wilson (1938). At present,
over 400 of these interstellar absorption features are known (for
a handful of sightlines), superimposed on an otherwise nearly
smooth interstellar extinction curve (Herbig 1995; Galazutdinov
et al. 2000; Hobbs et al. 2008). Only recently has the attribu-
tion of a pair of near-infrared DIBs (Foing & Ehrenfreund 1994)
to C+
60 been confirmed with laboratory gas phase experiments
(Campbell et al. 2015; Kuhn et al. 2016) along with the tentative
astronomical detection of three more predicted bands (Walker
et al. 2015, 2016), though this needs further verification and in-
vestigation (Galazutdinov et al. 2017; Cordiner et al. 2017). This
is an exciting result because C60 (Cami et al. 2010; Sellgren et al. 2010), and C+
60 (Berné et al. 2013) have also recently been de-
tected in space through their mid-infrared emission spectra. This
identification may be a chemical clue towards identifying fur-
ther DIB carriers; so far only C3 (Haffner & Meyer 1995; Maier Observational surveys (e.g. Herbig 1993; Friedman et al. 2011; Kos & Zwitter 2013) have shown that the strength of the
strongest ∼20 of the DIBs correlates roughly linearly with the
amount of dust and gas, measured by the reddening E(B −V) or
the column density of atomic hydrogen N(H I), respectively. This
indicates a thorough mixing of the DIB carriers with interstellar
matter (Cox 2011). The ESO Diffuse Interstellar Bands Large Exploration Survey
EDIBLES I. Project description, survey sample and quality assessment
Nick L. J. Cox1, 2, Jan Cami3, 4, Amin Farhang5, Jonathan Smoker6, Ana Monreal-Ibero7, 8, 9, Rosine Lallement7,
Peter J. Sarre10, Charlotte C. M. Marshall10, Keith T. Smith11, 12, Christopher J. Evans13, Pierre Royer14,
Harold Linnartz15, Martin A. Cordiner16, 17, Christine Joblin1, 2, Jacco Th. van Loon18, Bernard H. Foing19,
Neil H. Bhatt3, Emeric Bron20, Meriem Elyajouri7, Alex de Koter21, 14, Pascale Ehrenfreund22, Atefeh Javadi5,
Lex Kaper21, Habib G. Khosroshadi5, Mike Laverick14, Franck Le Petit23, Giacomo Mulas24, Evelyne Roueff23,
Farid Salama25, and Marco Spaans26 (Affiliations can be found after the references)
Received March 2017; Accepted ABSTRACT The carriers of the diffuse interstellar bands (DIBs) are largely unidentified molecules ubiquitously present in the interstellar medium
(ISM). After decades of study, two strong and possibly three weak near-infrared DIBs have recently been attributed to the C+
60 fullerene
based on observational and laboratory measurements. There is great promise for the identification of the over 400 other known DIBs,
as this result could provide chemical hints towards other possible carriers. In an effort to systematically study the properties of the DIB carriers, we have initiated a new large-scale observational survey: the
ESO Diffuse Interstellar Bands Large Exploration Survey (EDIBLES). The main objective is to build on and extend existing DIB
surveys to make a major step forward in characterising the physical and chemical conditions for a statistically significant sample of
interstellar lines-of-sight, with the goal to reverse-engineer key molecular properties of the DIB carriers. EDIBLES is a filler Large Programme using the Ultraviolet and Visual Echelle Spectrograph at the Very Large Telescope at Paranal,
Chile. It is designed to provide an observationally unbiased view of the presence and behaviour of the DIBs towards early-spectral-
type stars whose lines-of-sight probe the diffuse-to-translucent ISM. Such a complete dataset will provide a deep census of the atomic
and molecular content, physical conditions, chemical abundances and elemental depletion levels for each sightline. Achieving these
goals requires a homogeneous set of high-quality data in terms of resolution (R ∼70 000 – 100 000), sensitivity (S/N up to 1000
per resolution element), and spectral coverage (305–1042 nm), as well as a large sample size (100+ sightlines). In this first paper the
goals, objectives and methodology of the EDIBLES programme are described and an initial assessment of the data is provided. Key words. ISM: lines and bands – ISM: clouds – ISM: molecules – ISM: dust – local interstellar matter – Stars: early-type Key words. ISM: lines and bands – ISM: clouds – ISM: molecules – ISM: dust – local interstellar matter – Stars: c⃝ESO 2017 c⃝ESO 2017 Astronomy & Astrophysics manuscript no. ediblesI_v2
June 29, 2017 The ESO Diffuse Interstellar Bands Large Exploration Survey
EDIBLES I. Project description, survey sample and quality assessment
Nick L. J. Cox1, 2, Jan Cami3, 4, Amin Farhang5, Jonathan Smoker6, Ana Monreal-Ibero7, 8, 9, Rosine Lallement7,
Peter J. Sarre10, Charlotte C. M. Marshall10, Keith T. Smith11, 12, Christopher J. Evans13, Pierre Royer14,
Harold Linnartz15, Martin A. Cordiner16, 17, Christine Joblin1, 2, Jacco Th. van Loon18, Bernard H. Foing19,
Neil H. Bhatt3, Emeric Bron20, Meriem Elyajouri7, Alex de Koter21, 14, Pascale Ehrenfreund22, Atefeh Javadi5,
Lex Kaper21, Habib G. Khosroshadi5, Mike Laverick14, Franck Le Petit23, Giacomo Mulas24, Evelyne Roueff23,
Farid Salama25, and Marco Spaans26 1. Introduction Setting
Arm
Slit width
Resolving
Spectral range
(nm)
(′′)
power
(nm)
346
blue
0.4
71 000
304.2 – 387.2
564
red-L
0.3
107 000
461.6 – 560.8
red-U
566.9 – 665.3
437
blue
0.4
71 000
375.2 – 498.8
860
red-L
0.3
107 000
670.4 – 853.9
red-U
866.0 – 1042.0 Studies of selected bands in a dozen sightlines have revealed
a complex substructure in the narrowest bands (Sarre et al. 1995;
Ehrenfreund & Foing 1996; Galazutdinov et al. 2008) which
show small variations with local temperature (Cami et al. 2004;
Ka´zmierczak et al. 2010a), typical for a molecular carrier. Sub-
structure has also been identified in weak DIBs, but line-
of-sight variations are less well studied (Galazutdinov et al. 2005). On the other hand, broader DIBs do not contain sub-
structure (Snow 2002; Galazutdinov et al. 2008), which may
be due to lifetime broadening of the absorption band (Lin-
nartz et al. 2010). Considerations of the available elemental
abundances and plausible oscillator strengths lead to the conclu-
sion that abundant large organic molecules are suitable candi-
dates (Léger & d’Hendecourt 1985; Huang & Oka 2015). The
combination of observational studies, theoretical models, and
laboratory astrophysics indicates that candidate carriers should
primarily be sought among a large number of possible carbon-
based organic molecules [see Sarre (2006) for a review, and
Cami & Cox (2014) for an overview of recent progress]. the astronomical data is found. With the notable exception of
the above mentioned work on C+
60 previous studies have thus far
failed, such as attempts to link PAH cations (Bréchignac & Pino
1999; Salama et al. 2011, 1999; Romanini et al. 1999), neutral
PAHs (Salama et al. 2011; Gredel et al. 2011), carbon chains
(Motylewski et al. 2000; Maier et al. 2004), or H2 (Sorokin &
Glownia 1999; Ubachs 2014) to the DIBs. The search for a lab-
oratory match can be optimised if the most likely candidates can
be pre-selected out of the vast collection of possible species, and
if the relevant conditions can be accurately constrained. Hence
it is necessary to unravel the physical and chemical properties of
the DIB carriers through analysis and modelling of observations. This includes deriving environmental conditions that affect their
strength/profile shapes, as well as understanding the molecular
physics and spectroscopy of candidate carriers. 1. Introduction However, a large real scatter is observed in
these relations with gas and dust, and the relative strengths of
several bands are known to have an environmental dependence
(Cox & Spaans 2006); some bands vary as a function of radiation
strength between different lines-of-sight (Krełowski & Walker Article number, page 1 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Table 1. UVES instrument setups used. The two spectrograph arms are
used to collect data for a pair of wavelength regions simultaneously. A
second setup allows the gaps to be covered with another pair of settings. For each setting we give the slit width, nominal resolving power, and
nominal wavelengths covered. Red-L and Red-U refer to the spectra
recorded with the Red Lower EEV CCD and the Red Upper MIT CCD
(c.f. Sect. 5). Table 1. UVES instrument setups used. The two spectrograph arms are
used to collect data for a pair of wavelength regions simultaneously. A
second setup allows the gaps to be covered with another pair of settings. For each setting we give the slit width, nominal resolving power, and
nominal wavelengths covered. Red-L and Red-U refer to the spectra
recorded with the Red Lower EEV CCD and the Red Upper MIT CCD
(c.f. Sect. 5). 1987; Cami et al. 1997; Vos et al. 2011; Friedman et al. 2011). The scatter is also partly due to multiple cloud structures along
sightlines. The relationship between several DIBs and, for ex-
ample, C2 and CN, has been investigated (Thorburn et al. 2003; Weselak et al. 2008), but generally the link with di-
atomic species is not well understood. This relation between
DIBs and reddening has not been investigated for the remaining
>380 bands. Whether or not there is a direct physical connection
between DIB carriers and dust grains, e.g. in terms of depletion
onto grains or as carrier formation sites, remains to be seen –
so far no polarisation signal has been detected for the twenty
strongest DIBs (Cox et al. 2011). Article number, page 2 of 30page.30 1. Introduction Ongoing and future large spectroscopic surveys offer the
possibility to study (mostly the strongest) DIBs in large areas of
the sky. For example, Lan et al. (2014) and Baron et al. (2015)
constructed DIB strength maps from SDSS spectra, Kos et al. (2014) produced pseudo-3D maps for the 8621 Å DIB using the
RAVE survey, Zasowski et al. (2015) and Elyajouri et al. (2016)
used the APOGEE near-infrared survey to study the distribution
of the 15267 Å near-infrared DIB. The spatial distribution and
properties of DIBs can also be studied in smaller fields-of-view
(van Loon et al. 2009; Raimond et al. 2012; Puspitarini et al. 2015) or closer regions, such as the Local Bubble (Farhang et al. 2015; Bailey et al. 2016). This paper presents the observational overview of the ESO
Diffuse Interstellar Bands Large Exploration Survey (EDIBLES)
and how we intend to use the obtained spectra in our long-term
goal of reverse-engineering the molecular characteristics of DIB
carriers. In Sect. 2 we describe the scientific goals and immediate
objectives of EDIBLES. Sect. 3 describes the methodology and
survey design. The survey target selection is discussed in Sect. 4
and the data processing steps are described in Sect. 5. Sect. 6
discusses several confounding factors such as telluric and stellar
spectral lines. In Sect. 7 we present a preview of the EDIBLES
data and illustrate their scope and quality. A brief summary is
given in Sect. 8. In the last decade it has also been firmly established that
many band carriers are universal; DIBs have been detected and
surveyed in the Magellanic Clouds (Cox et al. 2006, 2007; Welty
et al. 2006; van Loon et al. 2013; Bailey et al. 2015), in M31 and
M33 (Cordiner et al. 2008, 2011), and in individual sightlines in
more distant galaxies (Junkkarinen et al. 2004; Sollerman et al. 2005; Lawton et al. 2008; Cox & Patat 2008, 2014; Monreal-
Ibero et al. 2015). DIB carriers therefore constitute an important
reservoir of (organic) material throughout the Universe. 3. Methodology & survey design 7. Variations in interstellar species due to the small-scale struc-
ture of the diffuse ISM (Cordiner et al. 2013; Smith et al. 2013). EDIBLES
provides
the
community
with
optical
(∼305–
1042 nm) spectra at high spectral resolution (R ∼70 000 in the
blue arm and 100 000 in the red arm) and high signal-to-noise
(S/N; median value ∼500–1000), for a statistically significant
sample of interstellar sightlines. Many of the >100 sightlines in-
cluded in the survey already have auxiliary available ultraviolet,
infrared and/or polarisation data on the dust and gas components. To achieve our objectives efficiently we use the Ultra-
violet Visual Echelle Spectrograph (UVES; Dekker et al. 2000;
Smoker et al. 2009) mounted on the 8-metre second Unit Tele-
scope (UT2) of the ESO (European Southern Observatory) Very
Large Telescope at the Paranal Observatory. The relative bright-
ness of nearby early-type stars allows the observation strategy to
take advantage of poor observing conditions and twilight hours
that would otherwise be under-utilised. The programme is run-
ning as a Large ‘Filler’ Programme (ESO ID 194.C-0833, PI. N.L.J. Cox), which has been allocated 280 hours of observing
time. About 8 500 science exposures with a total exposure time
of 229 hours (with blue and red arm exposures taken simulta-
neously) have been collected between September 2014 and May
2017. The program is expected to be completed by late 2017. Studies of DIBs typically report data such as equivalent
width, central depth, profile shape and substructure identifica-
tion. These cannot easily be compared between surveys due
to differences in the instrumentation, data quality and analy-
sis procedures – e.g. continuum normalisation and measurement
of spectroscopic lines. Archival material comprises a heteroge-
neous sample of spectra with varying S/N, resolving power, and
spectral coverage. To achieve the goals and objectives described
above requires a large and homogeneous survey of UV/visible
spectroscopic tracers across a broad spectral range, covering
a broad variety of interstellar environments. From this self-
consistent set of observations we can extract: UVES has two arms, red and blue, which can be used simul-
taneously by inserting a dichroic mirror (Dekker et al. 2000). To obtain coverage of the entire spectral range accessible with
UVES, we use two instrumental settings per target: setting #1:
346+564 and setting #2: 437+860, where the pairs of num-
bers refer to the central wavelengths in nanometres of the two
arms. 2. Scientific goals & immediate objectives The primary science goal of EDIBLES is to reverse-engineer
molecular characteristics of DIB carriers, through studying the
behaviour of DIBs in relation to the physical and chemical pa-
rameters of their environment. This approach differs from earlier
work in which attempts to identify DIBs were based mainly on
direct comparisons of astronomical and laboratory or theoretical
spectra. A large systematic high-fidelity survey of the diffuse-to-
translucent ISM is necessary to realise this approach. Identifying the DIB carriers and understanding their prop-
erties must come from high-quality data in the nearby Galactic
interstellar medium (ISM). Identification of the carrier species
will directly impact our understanding of interstellar chemistry,
and can help reconstruct 3D line-of-sight properties if related to
specific environments. It is clear that the ultimate confirmation
must come from a direct comparison between astronomical, the-
oretical, and laboratory spectra over a broad wavelength range. A commonly applied and straightforward approach is to acquire
laboratory spectra of possible candidate carriers taken under as-
trophysical relevant conditions until an unambiguous match with The aim is to assemble a sample of interstellar spectra with
sufficiently high spectral resolution and signal-to-noise ratio to
allow detailed analysis of numerous DIBs and known atomic
and molecular absorption lines in the same lines-of-sight. At the
same time, our sample is designed to sample a wide range of
interstellar conditions, in terms of reddening, molecular content
and radiation field, within a practical observing time. With EDIBLES we plan to compile the global properties of
a large ensemble of both weak and strong DIBs, and variations N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey therein, as a function of depletion (patterns) and local physical
conditions. The new dataset should allow us to: (a) determine
the relation between weak and strong DIBs by identifying corre-
lations and sequences; (b) identify (sets of) DIBs that correlate
with different physical conditions in the ISM, and assess whether
the DIBs can be used to determine those conditions as a remote
diagnostic tool; (c) study the physico-chemical parameters that
influence the DIB properties, by using state-of-the-art chemical
modelling, combined with extensive auxiliary line-of-sight data
(e.g. 2. Scientific goals & immediate objectives Updated measurements of peak positions of weak (per unit
reddening) diffuse bands along single cloud sightlines. 4. Measurements and cross-correlation of over 50 weak
and strong bands along the most reddened sightlines
(E(B −V) > 0.4 mag). Correlations between strong and
weak bands might reveal additional information on
groups (or families) of DIBs, but it should be noted that
a strong correlation between DIBs is not a necessarily a
guarantee that they have a common carrier (McCall et al. 2010; Krełowski et al. 2016). 4. Measurements and cross-correlation of over 50 weak
and strong bands along the most reddened sightlines
(E(B −V) > 0.4 mag). Correlations between strong and
weak bands might reveal additional information on
groups (or families) of DIBs, but it should be noted that
a strong correlation between DIBs is not a necessarily a
guarantee that they have a common carrier (McCall et al. 2010; Krełowski et al. 2016). 5. Stacking analyses to search for molecules and/or DIBs which
are too weak to be seen in individual spectra. 6. Firm detection limits or abundance constraints on specific
molecular carriers for which laboratory spectra are obtained. 2. Scientific goals & immediate objectives on dust); and (d) constrain the chemical composition of the
DIB carriers by studying their relation to interstellar elemen-
tal abundances (depletion levels) and dust grain properties and
composition derived from, for example, the UV-visual extinc-
tion (including the conspicuous 2175 Å UV bump) and optical
polarisation curves. energy dissipation models (c.f. Flower & Pineau des Forêts
2015; Godard et al. 2014; Bron 2014). For the photo-chemistry and derivation of particle density,
radiation fields, turbulent energy dissipation, the key tran-
sitions are those of CN λλ3874, 7906, CH λλ3879, 4300,
and CH+ λλ3958, 4232 Å, together with H2 (from archival
UV spectroscopy). For example, rotational temperatures can
be derived from bands of C2 (λ8756) and C3 (λ4053), and
cosmic ray ionisation rates can be derived from OH+ abun-
dances (λλ3300–3600 Å). CH measurements can be used
to estimate the H2 column density (Danks et al. 1984; We-
selak et al. 2004). 2. 2. Accurate measurements of DIB profiles (asymmetries,
wings, substructure) and variations therein. Substructure can
be related to molecular properties / sizes of carrier species
(Kerr et al. 1996; Ehrenfreund & Foing 1996; Huang & Oka
2015) with variations due to changes in the rotational tem-
perature (Cami et al. 2004; Ka´zmierczak et al. 2010b) or the
presence of hot bands (Marshall et al. 2015). A number of studies have attempted to investigate links be-
tween the physical and chemical conditions of the ISM and the
properties of the DIBs. However, most studies focus only on a
few strong bands in a moderate-to-large number (≈100) of sight-
lines (Friedman et al. 2011; Vos et al. 2011; Zasowski et al. 2015), or on many DIBs in just a few sightlines (Cami et al. 1997; Tuairisg et al. 2000; Hobbs et al. 2008, 2009). Hence,
more recent progress in the field has been limited to the study
of only a handful of the strongest DIBs due to high demands on
the signal-to noise ratio (S/N), spectral resolution, the removal
of stellar and telluric lines, and the lack of large, uniform data
sets. EDIBLES is designed to fill this gap, making just such a
large, uniform data set available and thus enabling a large and
systematic study of the physical and chemical parameters that
are expected to directly influence the formation efficiency and
spectroscopic response of DIB carriers. 3. 3. Methodology & survey design Together this provides near-continuous wavelength cover-
age from ∼305 to 1042 nm. The blue and red arm slit widths are
0.4′′ and 0.3′′, respectively, yielding nominal resolving powers
R = λ/∆λ of ∼71 000 and ∼107 000. Table 1 presents a sum-
mary of the instrumental setups. The EDIBLES data presented
here were collected over a period of two years, and it is there-
fore important to realize that the UVES resolution is not fully 1. Accurate column density measurements (or upper limits) for
the most important atomic and molecular species, across a
wide spectral range. These can be used to assess the veloc-
ity structure of the line-of-sight (in particular to determine
radial velocity differences between species; e.g. Bondar
et al. 2007), derive depletion levels of metals, infer and com-
pute physical conditions (within the limitations imposed
by the current knowledge on interstellar processes), using
diffuse cloud PDR models (Le Petit et al. 2006) or turbulent Article number, page 3 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 -150°-120° -90° -60° -30°
0°
30°
60°
90° 120° 150°
-75°
-60°
-45°
-30°
-15°
0°
15°
30°
45°
60°
75°
Galactic latitude (deg)
EDIBLES targets
0.15
0.30
0.45
0.60
0.75
0.90
1.05
E(B-V)
Fig. 1. Galactic distribution of EDIBLES targets. The symbol size reflects the value of RV, while the interior colour represents the line-of-sight
reddening, E(B −V). Symbols with green edges represent the observed targets, while blue edges correspond to the targets to be observed by the
end of the programme. -150°-120° -90° -60° -30°
0°
30°
60°
90° 120° 150°
-75°
-60°
-45°
-30°
-15°
0°
15°
30°
45°
60°
75°
Galactic latitude (deg)
EDIBLES targets
0.15
0.30
0.45
0.60
0.75
0.90
1.05
E(B-V) EDIBLES targets Fig. 1. Galactic distribution of EDIBLES targets. The symbol size reflects the value of RV, while the interior colour represents the line-of-sight
reddening, E(B −V). Symbols with green edges represent the observed targets, while blue edges correspond to the targets to be observed by the
end of the programme. Galactic long
stable with time1, but the values listed in Table 1 were typically
realised in the actual spectra. tude (deg)
→translucent clouds, (b) study the effect of shock- and photo-
processing, (c) probe the behaviour of DIBs with respect to grain
properties, and (d) identify unusual DIB environments. 3. Methodology & survey design The “filler”-type observation strategy means that observa-
tions are often executed in non-optimal (and unpredictable) con-
ditions e.g. in terms of seeing, cloud coverage, sky emission/air
glow, lunar phase, and water vapour content. This needs to be
taken into account in the implementation of the observations. Despite these limitations, S/N ratios of 200–300 per exposure
can be reached in short exposure times for the bright (2 < V <
6 mag) targets, and up to 20 exposures (to avoid saturation of
individual frames) are obtained for each to build up higher S/N. Observations are divided into observing blocks (OBs) for a spe-
cific instrument setting and target. OB execution times range
from ∼20 minutes for the bright (V < 6 mag) stars up to ∼45
minutes for the fainter (6 < V < 9) stars. During target selection the following factors were taken into
account: – Given that fH2depends non-linearly on AV, due to the transi-
tion from atomic to molecular hydrogen driven by H2 self-
shielding, we require numerous sightlines probing AV ∼
1 −3 mag and below, in small increments ∆AV. – The dust grain properties and attenuation of UV photons (im-
portant for photo-chemistry) are constrained by the extinc-
tion curve, i.e. the AV and RV parametrisation (Valencic et al. 2004; Fitzpatrick & Massa 2007) or from fitting with a well-
defined dust-PAH extinction model (Mulas et al. 2013). Additional flat-field calibration exposures were taken during
the day-time (when possible, subject to operational constraints)
to reduce residual fringing that would persist with the standard
UVES calibration plan and to increase the overall S/N ratio. De-
tails of the flat-field corrections are given in Sect. 5. – Preference
is
given
to
sightlines
with
auxiliary
atomic/molecular data, such as H I and H2 measure-
ments (Jenkins 2009; Gudennavar et al. 2012), optical
polarisation data (Whittet et al. 1992; Weitenbeck 2008), or
Mg/Fe abundances (Voshchinnikov et al. 2012). Where two targets with similar interstellar conditions are
available, we preferentially selected targets which are brighter
and/or of earlier spectral type. 1 http://www.eso.org/observing/dfo/quality/
UVES/reports/HEALTH/trend\_report\_ECH\
_RESOLUTION\_DHC\_HC.html 4. Target survey sample selection and
characteristics The target list is given in the Appendix, Table A.1 (their
Galactic distribution is shown in Fig. 1). Columns (1) to (3) pro-
vide basic information on the target id (HD number) and co-
ordinates (RA/Dec). Columns (4) and (5) list the spectral type
and corresponding literature reference. The interstellar line-of-
sight dust extinction properties, E(B −V), RV, AV, are given
in columns (6) to (9). Columns (10) and (11) list the atomic
and molecular hydrogen abundances with the molecular fraction
fH2listed in column (12). We constructed a statistically representative survey sample that
probes a wide range of interstellar environment parameters
including reddening E(B −V), visual extinction AV, total-to-
selective extinction ratio RV, and molecular hydrogen fraction
fH2. This is essential to (a) trace depletion patterns from diffuse Article number, page 4 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
0.0
0.2
0.4
0.6
0.8
1.0
1.2
E(B-V) (mag)
0
2
4
6
8
10
12
14
16
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 94
0
1
2
3
4
5
AV (mag)
0
2
4
6
8
10
12
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 80
2
3
4
5
6
RV
0
2
4
6
8
10
12
14
16
18
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 80
0.0
0.2
0.4
0.6
0.8
1.0
f(H2)
0
2
4
6
8
10
12
14
16
18
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 58
Fig. 2. Number of selected targets as function of reddening E(B −V), extinction AV, the ratio of total-to-selective extinction RV (= AV/E(B −V)),
nd the fraction of molecular hydrogen fH2for the target sample. The number of observed targets with reported values for each quantity are given
t the top of each panel. The dark blue and light blue distributions correspond to the samples of observed and observed + foreseen targets. The
vertical red lines indicate the value of the 5, 10, 25, 50, 75, 90, and 95 percentiles of each sample. 4. Target survey sample selection and
characteristics 0.0
1.0
2.0
3.0
4.0
5.0
AV
18.5
19.0
19.5
20.0
20.5
21.0
21.5
22.0
log N(X)
H(=H I+ 2H2)
H I
H2 Article number, page 5 of 30page.30 4. Target survey sample selection and
characteristics The labels are located such that they trace the
umulative target distribution. 0
1
2
3
4
5
AV (mag)
0
2
4
6
8
10
12
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 80 0.0
0.2
0.4
0.6
0.8
1.0
1.2
E(B-V) (mag)
0
2
4
6
8
10
12
14
16
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 94 0.0
0.2
0.4
0.6
0.8
1.0
f(H2)
0
2
4
6
8
10
12
14
16
18
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 58 2
3
4
5
6
RV
0
2
4
6
8
10
12
14
16
18
Targets per bin
[5]
[10]
[25]
[50]
[75]
[90]
[95]
Total number of targets = 80 Fig. 2. Number of selected targets as function of reddening E(B −V), extinction AV, the ratio of total-to-selective extinction RV (= AV/E(B −V)),
and the fraction of molecular hydrogen fH2for the target sample. The number of observed targets with reported values for each quantity are given
at the top of each panel. The dark blue and light blue distributions correspond to the samples of observed and observed + foreseen targets. The
vertical red lines indicate the value of the 5, 10, 25, 50, 75, 90, and 95 percentiles of each sample. The labels are located such that they trace the
cumulative target distribution. 0.0
1.0
2.0
3.0
4.0
5.0
AV
18.5
19.0
19.5
20.0
20.5
21.0
21.5
22.0
log N(X)
H(=H I+ 2H2)
H I
H2
Fig. 3. Relation between visual extinction, AV, and neutral hydrogen
column density N(H I), molecular hydrogen column density N(H2), and
total hydrogen column density N(Htot), computed as N(H I)+2N(H2). Note that some EDIBLES lines-of-sight are not included since no direct
H I or H2 measurements are available. 0.0–0.8). The histograms in Fig. 2 illustrate the range of param-
eters included in the survey sample. In Fig. 3 we show com-
parisons between visual extinction, AV, and the measured neu-
tral hydrogen column density N(H I), molecular hydrogen col-
umn density N(H2), and total hydrogen column density N(Htot)
(=N(H I)+2N(H2)). As noted above H2 can be estimated using
the CH transitions (Danks et al. 1984; Weselak et al. 2004). 5. Data processing The S/N ratios plotted for each instrument setting are
average values of S/N measured in five different continuum regions in
the respective setting. Fig. 4. Comparison of the S/N of HD 23180 with changing number
of flat frames. The S/N ratios plotted for each instrument setting are
average values of S/N measured in five different continuum regions in
the respective setting. Fig. 5. (a) Part of the extracted spectrum of HD 23180 taken using the
Red Lower EEV CCD (Red-L). (b) Ditto but for Red Upper MIT CCD
(Red-U). The latter is a thick chip so fringing is much reduced compared
with the EEV detector. The vertical scale is the same in both cases. Within a sequence of observations (20–40 minutes including
overheads) the change in barycentric velocity correction is small
(< 0.1 km s−1 per hour) so the spectra can be averaged without
compromising the velocity precision. However, exposures taken
on different nights were not averaged. This is to preserve multi-
epoch information – specifically for spectroscopic binaries and
the search for time-variable interstellar absorption – and to
avoid addition of misaligned interstellar features due to varia-
tions in the barycentric velocity of the frame of the observer. Order definition
In order to find the physical position of
echelle orders in the X and Y directions of spectral frames for a
given instrument setting, esorex uses a physical model based
on the instrument configuration, ambient pressure, the humidity,
slit width, central wavelength, camera temperature and CCD ro-
tation angle. The physical model then predicts the X and Y pixel
position corresponding to the nominal orders and stores the cal-
culations into the guess line and order tables. These tables are
then normally used as the initial values for identifying the spe-
cific positions of the orders. The data reduction was performed by two semi-independent
teams, one using version 5.7.0 of the UVES pipeline (Ballester
et al. 2000), esorex (version 3.12; ESO CPL Development
Team 2015) integrated in a python pipeline (hereafter Reduction
’A’), and the other using the 4.4.8 version of the UVES pipeline
(Reduction ’B’). p
To accurately detect the order positions, esorex defines a
search box on the detected lines in the arc lamp frames and tries
to match the predicted position of the physical model lines. 5. Data processing The
following procedure robustly detects the order positions: Each set of around 20 science frames was processed with the
same set of calibration frames. For the format check, order defi-
nition and wavelength calibrations these were the nearest in time,
with the master bias and master flats using frames taken typically
over several days or weeks. In general, both data reductions used
similar parameters for the different pipeline recipes, but with a
few differences. Reduction ‘A’ adopts optimal merging, applies
the blaze correction and uses about 100–130 flats for each arm,
while reduction ‘B’ uses optimal merging and takes the near-
est 140 flat field frames. 1. measure the raw X and Y pixel positions of the thorium-
argon lines on an arc frame exposure by defining a 60 × 60
pix2 square search box, 2. compute the difference of predicted and detected order posi-
tions, 3. decrease the size of the search box to 40 × 40 pix2, iterate
the X and Y shifts to search for residuals less than ±1 pixel
and to reduce the root-mean-square (RMS) values, In the following we provide a detailed description of the dif-
ferent data processing steps and discuss their impact on the qual-
ity of the reduced spectra. The differences between reductions
‘A’ and ‘B’ are discussed in relation to the blaze correction and
order merging step. q
(
)
4. fit a 2D Gaussian function to XY pixel positions within a ‘fit
box’ centred at the predicted line positions, p
p
5. For reduction ‘B’ additional iterations of the format
check are done using different values of the CCD rota-
tion offset, selecting the one with the maximum number
of lines found, 6. and, finally, perform a 2D second-order polynomial fit in
XY to the fitted line positions. Bias
To subtract the CCD bias level in science frames, we
created a master bias frame by median-stacking a set of 50
(reduction ‘A’) or 25 (reduction ‘B’) bias exposures, using the
uves_cal_mbias recipe. To handle the bad columns in the
REDL CCD, by default the recipe interpolates across bad pix-
els but does not apply this interpolation in science frames. This
inconsistency creates boxy emission-like artifacts in the reduced
spectra. To avoid this from occurring, high-quality bias frames
are carefully pre-selected and the data are processed without in-
terpolating over bad pixels. Article number, page 6 of 30page.30 5. Data processing Acquiring high-S/N spectra with UVES is challenging in the
context of EDIBLES for a number of reasons. For example,
small errors in the wavelength calibration at the edges of indi-
vidual adjacent orders can cause the appearance of ripples in the
continuum in high-S/N exposures. Moreover, the unpredictable
seeing and other observing conditions inherent in a filler pro-
gramme mean that individual exposure times cannot be opti-
mised for the actual sky conditions. Fig. 3. Relation between visual extinction, AV, and neutral hydrogen
column density N(H I), molecular hydrogen column density N(H2), and
total hydrogen column density N(Htot), computed as N(H I)+2N(H2). Note that some EDIBLES lines-of-sight are not included since no direct
H I or H2 measurements are available. The overall quality of all 8 500 science and ∼1600 flat expo-
sures was checked visually. This visual inspection led us to dis-
card about 150 exposures that appeared corrupted. This could,
for example, be due to sudden changes in weather conditions
or premature termination of exposures. In addition, we care-
fully inspected for the presence of incorrect thorium-argon lamp,
format-check, and flat field exposures that could result in faulty
order tracing or wavelength calibration solutions (see also be-
low). The total number of selected targets amounts to 114 (of
which 96 have been observed at least once as of May 2017) and
comprises mostly bright O and B stars (V ≈2–7 mag with a
small fraction 7 < V < 9 mag). The sample probes a wide range
of interstellar dust extinction properties (E(B −V)∼0–2 mag;
RV ∼2–6; AV ∼0.1–4.5 mag) and molecular content ( fH2∼ Article number, page 5 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Fig. 5. (a) Part of the extracted spectrum of HD 23180 taken using the
Red Lower EEV CCD (Red-L). (b) Ditto but for Red Upper MIT CCD
(Red-U). The latter is a thick chip so fringing is much reduced compared
with the EEV detector. The vertical scale is the same in both cases. Fig. 4. Comparison of the S/N of HD 23180 with changing number
of flat frames. The S/N ratios plotted for each instrument setting are
average values of S/N measured in five different continuum regions in
the respective setting. Fig. 4. Comparison of the S/N of HD 23180 with changing number
of flat frames. 5. Data processing Arm
uncertainty (Å)
uncertainty (m s−1)
346-nm
6.4 × 10−4
52
437-nm
7.8 × 10−4
54
564-nm L
4.5 × 10−4
26
564-nm U
7.0 × 10−4
34
860-nm L
8.3 × 10−4
33
860-nm U
1.1 × 10−3
36 S/N~1000
S/N~555 the necessity for accurate flat-fielding to reach the required S/N
ratio, we reduced 11 science frames of HD 23180 using a dif-
ferent number of flat field frames to build the master flat frame. As shown in Fig. 4, increasing the number of flat fields helps
to increase the final S/N. More than 100 flat field frames do not
cause the S/N to increase further, indicating that flat fielding is
no longer the limiting factor on the S/N. In the case of the 860-
nm setting the S/N drops between 100 and 130 applied flats due
to the presence of several bad-quality flat frame exposures. For
the final reduction these were rejected and we selected as many
as possible, usually about 100, good quality flat frames. By us-
ing the additional flat fields, we were able to improve the final
S/N by factors of two to five, depending on the wavelength. Note that the intensity (photon counts) of normal flats are
very low in the UV (<320 nm). Therefore, to improve the qual-
ity of the first-orders of the 346-nm arm spectra, a final master
flat is constructed by combining a set of normal flats with flats
obtained by exposing with a deuterium calibration lamp. The
merging was done at orders 145 and 146 around 321 nm with
the master deuterium-lamp flat being used bluewards of this and
the normal flat redwards. This is to avoid as much as possible
spurious absorption-line features in the final science spectrum
caused by emission features in the deuterium lamp redwards of
321 nm. Fig. 7. Comparison between the ADP and EDIBLES processing of
HD184915 spectra (0.02 Å binsize). The S/N measurements, taken
at 4912–4913 Å and 6129–6130 Å for the Red-L and Red-U spectra,
are given in the top and bottom panel. Resampling to 0.04 Å binsize
(i.e. corresponding to the spectral resolution) further increases the
S/N by factor
√
2. near the gap between the upper and lower CCDs. The fringing
is much worse in the lower thinned EEV chip than in the upper
MIT thick chip (Smoker et al. 2009). 5. Data processing The highlighted values in the above steps are our tuned parame-
ters in the uves_cal_predict recipe. Flat fielding
The flat-fielding is applied, to the 2D frame, be-
fore the order extraction (see below). This is known as the pixel
flat-fielding method. The standard UVES pipeline uses only five
flat field frames, which limits the maximum S/N. To demonstrate Article number, page 6 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey order-merging jump
DIB
Stellar HeI
Fig. 6. Close-up view of a region including two overlapping orders
in the 564-nm setting for HD 23180 for both reduction ‘A’ (bottom
red trace) and reduction ‘B’ (top black trace). The small jump in the
continuum at approximately 5855 Å seen in reduction B (top black
trace) is due to imperfect merging of two echelle orders. The ap-
parent difference in S/N is due to alternative choices of wavelength
sampling. order-merging jump
DIB
Stellar HeI Table 3. Median S/N per “resolution-element” (0.04 Å spectral bin)
for each UVES setting/arm configuration for all available EDIBLES
spectra. Setting
Arm
λ-range (nm)
Median S/N
Sample
(0.04 Å bin)
size
346
blue
339.3 – 339.4
780a
204
437
blue
398.8 – 398.9
1070a
187
564
red-L
511.0 – 511.1
1090b
205
red-U
613.1 – 613.2
1020b
205
860
red-L
675.35 – 6754.55
670b
184
red-U
869.9 – 870.0
880b
183
(a) Reduction ‘A’ (b) Reduction ‘B’ Fig. 6. Close-up view of a region including two overlapping orders
in the 564-nm setting for HD 23180 for both reduction ‘A’ (bottom
red trace) and reduction ‘B’ (top black trace). The small jump in the
continuum at approximately 5855 Å seen in reduction B (top black
trace) is due to imperfect merging of two echelle orders. The ap-
parent difference in S/N is due to alternative choices of wavelength
sampling. Reduction A
Reduction B
S/N~830
S/N~580
S/N~1000
S/N~555
Fig. 7. Comparison between the ADP and EDIBLES processing of
HD184915 spectra (0.02 Å binsize). The S/N measurements, taken
at 4912–4913 Å and 6129–6130 Å for the Red-L and Red-U spectra,
are given in the top and bottom panel. Resampling to 0.04 Å binsize
(i.e. corresponding to the spectral resolution) further increases the
S/N by factor
√
2. S/N~830
S/N~580 S/N~830
S/N~580
S/N~1000
S/N~555 Table 2. Wavelength calibration uncertainties for the thorium-
argon frames corresponding each of the observed UVES settings
for HD 23180. 5. Data processing Note also the inter-order
gaps, several are indicated with arrows, in the 860-nm Red-U spectrum above ∼9600 Å as well as several conspicuous regions containing
bands of closely-spaced telluric absorption lines (indicated with red horizontal bars) mostly in the Red-L and Red-U 860-nm spectra
(bottom two panels). Two order-merging jumps are indicated in the fourth panel. A more detailed version of this figure is shown in the
appendix (Fig. B.1) where specific interstellar species are labeled and a synthetic DIB spectrum is shown for comparison. Fig. 8. EDIBLES UVES spectra of HD 170740 (B2 V) for each setting from top to bottom: 346B, 437B, 564L, 564U, 860L, 860U. This overview
figure is a demonstration of the data quality. The main gaps in wavelength coverage are between 5610–5670 Å and 8530–8680 Å which
correspond to the physical separation of the Red-L and Red-U detectors in both the 564 and 860-nm settings. Note also the inter-order
gaps, several are indicated with arrows, in the 860-nm Red-U spectrum above ∼9600 Å as well as several conspicuous regions containing
bands of closely-spaced telluric absorption lines (indicated with red horizontal bars) mostly in the Red-L and Red-U 860-nm spectra
(bottom two panels). Two order-merging jumps are indicated in the fourth panel. A more detailed version of this figure is shown in the
appendix (Fig. B.1) where specific interstellar species are labeled and a synthetic DIB spectrum is shown for comparison. Wavelength calibration
UVES wavelength calibration errors
are typically in the range of 0.1–0.5 km s−1 (Whitmore et al. 2010). To achieve an accurate wavelength calibration, the dis-
persion relation is obtained by extracting the thorium-argon arc
lamp frames using the same weights as those used for the sci-
ence objects. Therefore, first we reduced the science frames with
an optimal extraction method to generate a pixel weight map. Then by applying this weight map to the wavelength calibra-
tion, an accurate wavelength calibration with a statistical error less than 5 × 10−4 Å typically in all central wavelengths (for
more details see Table 2) and a systematic uncertainty less than
1.7×10−4 Å is achieved. To optimize the number of lines used in
the wavelength calibration solution we tested a tolerance value
≃0.07 pixels to reject the line identification with wavelength
residuals worse than the tolerance. 5. Data processing Note also the inter-or
ps, several are indicated with arrows, in the 860-nm Red-U spectrum above ∼9600 Å as well as several conspicuous regions contain
nds of closely-spaced telluric absorption lines (indicated with red horizontal bars) mostly in the Red-L and Red-U 860-nm spec
ottom two panels). Two order-merging jumps are indicated in the fourth panel. A more detailed version of this figure is shown in
pendix (Fig. B.1) where specific interstellar species are labeled and a synthetic DIB spectrum is shown for comparison. A&A proofs: manuscript no. ediblesI_v2
3200
3300
3400
3500
3600
3700
Wavelength (˚A)
0.6
0.7
0.8
0.9
1.0
Normalised intensity
3800
3900
4000
4100
4200
4300
4400
4500
4600
4700
4800
4900
Wavelength (˚A)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
4700
4800
4900
5000
5100
5200
5300
5400
5500
5600
Wavelength (˚A)
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
5700
5800
5900
6000
6100
6200
6300
6400
6500
6600
Wavelength (˚A)
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
6700
6800
6900
7000
7100
7200
7300
7400
7500
7600
7700
7800
7900
8000
8100
8200
8300
8400
8500
Wavelength (˚A)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
03
0.4
0.5
0.6
0.7
0.8
0.9
1.0
ormalised intensity
inter-order gaps
order-merging jumps A&A proofs: manuscript no. ediblesI_v2 3200
3300
3400
3500
3600
3700
Wavelength (˚A)
0.6
0.7
0.8
0.9
1.0
Normalised intensity
3800
3900
4000
4100
4200
4300
4400
4500
4600
4700
4800
4900
Wavelength (˚A)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity 8700
8800
8900
9000
9100
9200
9300
9400
9500
9600
9700
9800
9900
10000 10100
Wavelength (˚A)
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
inter-order ga Fig. 8. EDIBLES UVES spectra of HD 170740 (B2 V) for each setting from top to bottom: 346B, 437B, 564L, Fig. 8. EDIBLES UVES spectra of HD 170740 (B2 V) for each setting from top to bottom: 346B, 437B, 564L, 564U, 860L, 860U. This overview
figure is a demonstration of the data quality. The main gaps in wavelength coverage are between 5610–5670 Å and 8530–8680 Å which
correspond to the physical separation of the Red-L and Red-U detectors in both the 564 and 860-nm settings. 5. Data processing Order extraction
Various possible extraction methods were
tested and we find that the average extraction method with per-
forming a flat fielding before the extraction (see above) leads
to a higher S/N with respect to other methods (such as opti-
mal extraction), which is generally the case for high S/N spec-
troscopy. This method also reduces the fringing seen in the red
wavelengths ≥700 nm, although does not entirely remove it. Fig. 5 shows that displays parts of an extracted science spectrum Cosmic ray rejection and hot pixels
To remove the cosmic
rays and possible hot pixels we applied a sigma-clipping method
to each extracted spectrum. The default sigma-clipping threshold
value in esorex is κ = 10, but to identify all induced hot pixels
we set this κ = 6 to be sure that all cosmic/hot pixels are removed
and no real data are clipped. Article number, page 7 of 30page.30 A&A proofs: manuscript no. ediblesI_v2
3200
3300
3400
3500
3600
3700
Wavelength (˚A)
0.6
0.7
0.8
0.9
1.0
Normalised intensity
3800
3900
4000
4100
4200
4300
4400
4500
4600
4700
4800
4900
Wavelength (˚A)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
4700
4800
4900
5000
5100
5200
5300
5400
5500
5600
Wavelength (˚A)
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
5700
5800
5900
6000
6100
6200
6300
6400
6500
6600
Wavelength (˚A)
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
6700
6800
6900
7000
7100
7200
7300
7400
7500
7600
7700
7800
7900
8000
8100
8200
8300
8400
8500
Wavelength (˚A)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
8700
8800
8900
9000
9100
9200
9300
9400
9500
9600
9700
9800
9900
10000 10100 10200 10300
Wavelength (˚A)
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Normalised intensity
inter-order gaps
order-merging jumps
g. 8. EDIBLES UVES spectra of HD 170740 (B2 V) for each setting from top to bottom: 346B, 437B, 564L, 564U, 860L, 860U. This overv
ure is a demonstration of the data quality. The main gaps in wavelength coverage are between 5610–5670 Å and 8530–8680 Å wh
rrespond to the physical separation of the Red-L and Red-U detectors in both the 564 and 860-nm settings. Article number, page 8 of 30page.30 6.1. Earth transmission spectrum A large number of weak and strong telluric oxygen and water
absorption bands arise from molecules present in the Earth at-
mosphere covering nearly the full DIB range. In addition, for
the near-UV spectral domain (300–350 nm) a correction for at-
mospheric ozone needs to be applied. These telluric lines can be removed or modelled by using
bright, early-type type star spectra recorded in the same con-
ditions as the targets, or by using synthetic atmospheric trans-
mittance spectra. The former method requires observing (unred-
dened) standard stars at roughly similar airmass, shortly before
or after the primary science target observations. This procedure
is not possible within the filler observation strategy, and as the
EDIBLES targets are themselves bright this would double the
required observing time. The latter method has the advantage
of saving observing time, avoiding features associated with the
standard star and benefits from the increase in quality and avail-
ability of the molecular databases. The first tests of atmospheric
spectra correction were performed based on the following ap-
proach: For reduction ‘B’, no blaze correction was performed. Opti-
mal merging provided the best results in the overlapping regions
for both reductions ‘A’ and ‘B’. Fig. 6 shows a comparison of
reductions ‘A’ and ‘B’ for HD 23180 in a spectral region with
two overlapping orders. Reduction ‘B’ reveals a mismatch
of the order-overlapping regions, resulting in a jump in the
spectra at 5855 Å not present in the reduction ‘A’ spectrum. First, the telluric transmittance is optimally adapted to each
target. Paranal observing conditions are downloaded from the
TAPAS facility3 (Bertaux et al. 2014). The TAPAS transmittance
spectra are based on the latest HITRAN molecular database
(Rothman et al. 2013), radiative transfer computations (Line-By-
Line Radiative Transfer Model; Clough & Iacono 1995), and are
computed for atmospheric temperature, pressure and composi-
tion interpolated by the ETHER data centre4 based on a combi-
nation meteorological field observations and other information. Quality control The S/N of the spectra as a function of wave-
length was estimated by fitting a first order polynomial to re-
gions of the spectra in bins of 1 Å and measuring the residual in
each 0.02 Å wavelength bin. Table 3 lists the median S/N (per
2-pixel “resolution-element”) for each setting, together with
the respective continuum wavelength regions. In Fig. 6.1. Earth transmission spectrum 7 we
compare for HD 184915 the spectra obtained with the dedi-
cated EDIBLES processing presented here and the default
archive data products (ADP) provided by ESO. 2 The in-
crease in S/N (labelled in the figure) is almost a factor two
for both the Red-L and Red-U spectra (cf. Table 1). For each
target the different settings/arms generally have different S/N
ratios. This is primarily due to (1) the choice of targets which
are often brighter in V band compared to B band (i.e. redden-
ing), and (2) the lower efficiency of UVES in the very blue and
very red parts of the accessible wavelength range. In terms of
S/N reduction ‘A’ performs slightly better than reduction
’B’ for the 346-nm setting, both perform similar for the 437-
nm and 860-nm settings, while reduction ‘B’ performs better
for the 564-nm setting. The main difference between the two
reductions is in terms of the order-merging jumps. In ad-
dition, there are small, though noticeable differences in the
‘noise’ between both reductions. The reduction scheme ‘A’ g
Then, in regions of moderate absorption (i.e. less than 70%
at line centers before instrumental broadening), a simple method
called rope-length minimization is used (Raimond et al. 2012). Briefly, the algorithm searches for the minimal length of the
spectrum that is obtained after division of the data by the trans-
mittance model. To do so the column of the absorbing species,
the Doppler shift and the width of the instrumental function by
which the transmittance model is convolved are all tuned. The
method uses the fact that when the telluric lines are not well
reproduced, strong maxima and minima remain after the data-
model division and the spectrum length increases. On the con-
trary, if the modelled lines follow very well the observed ones,
the corrected spectrum is smooth. An example of correction is
shown in Fig. 9 for the 6284 Å DIB. Here the rope-length method
has been first applied to the O2 lines, then in a subsequent step
the O2-corrected spectrum is corrected for the weak H2O lines
that are also present in this spectral region. 2 These spectra are generated by ESO’s Quality Control Group for
all UVES point-source observations. The pipeline processing is done
automatically through a dedicated workflow (with no fine-tuning of
pipeline parameters specific to the needs of our programme). 3 http://ether.ipsl.jussieu.fr/tapas/
4 www.pole-ether.fr 5. Data processing For the final iterations of
the fit of the wavelength calibration solutions we set the sigma-
clipping to κ = 3. Further improvements to the wavelength
calibration are being studied for the public release of the N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey is adopted as the primary scheme for results shown in this
work. Both reductions are therefore retained for reference and
as a control for spurious features. data. For example, there is a temperature and density depen-
dent shift (which can be as much as 1 pixel and different for
the different wavelength regions) in the position of the thorium-
argon lines (UVES User Manual). This can be potentially cor-
rected by taking into account the difference between the ob-
servation and calibration temperatures and pressures. Also, we
foresee improvements to the final wavelength calibration in
the 860 nm Red-U arm, where there are few thorium-argon
lines, by cross-referencing with a model telluric transmission
spectrum. data. For example, there is a temperature and density depen-
dent shift (which can be as much as 1 pixel and different for
the different wavelength regions) in the position of the thorium-
argon lines (UVES User Manual). This can be potentially cor-
rected by taking into account the difference between the ob-
servation and calibration temperatures and pressures. Also, we
foresee improvements to the final wavelength calibration in
the 860 nm Red-U arm, where there are few thorium-argon
lines, by cross-referencing with a model telluric transmission
spectrum. As an example, the final continuum normalised EDIBLES
UVES spectrum of HD 170740 (from reduction ‘A’) is shown
in full in Fig. 8, where each panel corresponds to one of the
instrument settings (Table 1). A closer view of this spectrum in
shown in Appendix B. All processed EDIBLES spectra will be
released as new “Phase 3” data to the ESO archive later in
the project. 2 These spectra are generated by ESO’s Quality Control Group for
all UVES point-source observations. The pipeline processing is done
automatically through a dedicated workflow (with no fine-tuning of
pipeline parameters specific to the needs of our programme). The
1d-extracted spectra are ingested into the ESO archive as so-called
“Phase 3” data products. 6. Telluric and stellar features Blaze function and order merging Previous analyses of data
obtained with UVES demonstrate that the shape and position of
the blaze function is the primary source of problems in the or-
der overlap regions. The continuum changes vary smoothly over
subsequent orders, which may be related to the fact that the blaze
profiles produced by UVES are not the same as the theoretical
predictions. Accordingly, the blaze function at the overlapping
regions is not sufficiently well characterised, therefore by av-
eraging the overlap regions some artificial features appear and
cause the signal level to fall off at the end of the orders. Irregular
variations in the continua at the edges of orders are likely due to
a mismatch between the paths of the light from the star and the
flat-field lamp (Nissen 2008) and result in discontinuities where
orders have been merged. 6.2. Stellar spectra We intend to investigate the C+
60 bands in more detail
later in the EDIBLES project, but this will depend upon the
accuracy and success of the telluric line modelling for each
line-of-sight (as there are numerous saturated atmospheric
water absorption lines in this wavelength region) as well as
the stellar atmosphere modelling required to account for, for
example, contribution of Mg II (as discussed in Galazutdinov
et al. 2017). In addition to the goals discussed above, the EDIBLES observa-
tions provide high-quality spectra of the target stars themselves. These span the full spectral range of early-type stars, from early
O-type dwarfs through to late B-type supergiants (plus a cou-
ple of later/cooler stars). Published spectral classifications of the
sample are summarised in Tables A.1 and A.2. All of the O-type EDIBLES targets have been observed as
part of the Galactic O-Star Spectroscopic Survey (GOSSS), a
comprehensive survey of bright Galactic O stars at a resolv-
ing power of R ∼2500 (Sota et al. 2011, 2014). The detailed O-
star classifications quoted in Tables A.1 and A.2 are the GOSSS
types – a thorough description of the classification criteria was
given by Sota et al. (2011), including an overview of the various
classification qualifiers used to convey additional information on
the spectra (their Table 3). In contrast, most of the B-type stars in the EDIBLES sam-
ple have not been subject to such morphological rigour with
high-quality digital spectroscopy. Work is underway within the
GOSSS to better define spectral standards and the classification
framework for early B-type stars (Villaseñor et al. in prep.), with
a few stars overlapping with the EDIBLES sample. Nonetheless,
the high-quality, high-resolution spectra from EDIBLES will be
useful to refine the classification framework for B-type stars, par-
ticularly compared to similar efforts in the Magellanic Clouds
(e.g. Evans et al. 2015). In the short-term we will inspect the EDIBLES data in
the context of classification, to update/refine spectral types as
required – whether arising from the added information of
the high-resolution UVES data (cf. lower-resolution spec-
troscopy from the GOSSS, for example), or simply from in-
trinsic spectral variability, which is seen in many early-type
stars. This will ensure the best parameters are adopted in es-
timating stellar colours, thence the line-of-sight extinctions. We will also look for evidence of spectroscopic binaries in
targets with multiple observations (and/or relevant archival
data, see, e.g., Sect. 7.4). 6.2. Stellar spectra of the instrumental function becomes crucial. The simple rope-
length method alone is no longer appropriate. We have tested an
iterative method that is a combination of the rope-length method
and a classical fitting. In a first step we excluded all spectral
intervals around the centres of the deep lines and performed a
segmented rope-length optimization, i.e. the algorithm searches
for the model parameters that minimize the sum of the lengths of
the individual spectral segments. We then divided the data by the
corresponding adjusted model and performed a running average
of the divided spectrum to obtain an approximate stellar contin-
uum. We then fitted the data to the convolved product of this
continuum and a telluric transmittance. The instrumental func-
tion is now modelled as the sum of a Gaussian and a Lorentzian,
which allows weak extended wings to be taken into account. The
instrumental profile is the same for all lines within the corrected
interval. The parameters defining these two components as well
as the column of the absorbing species are free to vary during the
adjustment. The data were then divided by this updated model. This process can be iterated and stopped when there is no longer
any decrease of the "rope-length". An example of such a two-
step correction is shown in Fig. 9 for the spectral region of the
C+
60 9577 and 9632 Å DIBs. There are still some residuals, but
these are limited mostly to the deepest, (partially) saturated tel-
luric lines. This occurs particularly when these lines are slightly
Doppler shifted or broadened due to atmospheric pressure in a
way that is not fully predicted by the model. For a description
of such effects see Bertaux et al. (2014). Nevertheless, the DIBs
at 9577 and 9632 Å, assigned to be due to C+
60 as mentioned in
the introduction, stand out clearly in the telluric corrected spec-
trum. The three weaker DIBs between 9350 and 9450 Å re-
ported by Walker et al. (2015), though not yet confirmed in-
dependently (Galazutdinov et al. 2017; Cordiner et al. 2017)
present significant challenges for detection due to the pres-
ence of strong, saturated telluric water lines in this spectral
range. 6.1. Earth transmission spectrum The
1d-extracted spectra are ingested into the ESO archive as so-called
“Phase 3” data products. p
p
g
For regions of stronger telluric lines, division by deep lines
induces overshoot and the exact shape of the telluric profiles and 3 http://ether.ipsl.jussieu.fr/tapas/
4 www.pole-ether.fr Article number, page 9 of 30page.30 A&A proofs: manuscript no. ediblesI_v2
6300
6295
6290
6285
6280
6275
6270
Wavelength (Å)
20
15
10
Flux (Arbit. Unit)
40
30
20
10
0
Flux (Arb. Unit)
9660
9640
9620
9600
9580
9560
Wavelength (Å)
C60
+candidate
C60
+candidate
6270 DIB
6283 DIB
Fig. 9. (Left) Example of telluric line correction in the weak line regime by means of the rope-length method applied to the spectrum of HD 170740. Telluric lines of O2 are corrected first, then H2O lines. (Right) Example of telluric line correction in the strong line regime by means of a two-step
method and a composite instrumental profile adjustment (see text). Residuals remain at the location of the deepest lines, especially when the model
does not predict their shape and exact Doppler shift very accurately. The positions of the 9577 and 9632 Å DIBs attributed to C+
60 are indicated. A&A proofs: manuscript no. ediblesI_v2 A&A proofs: manuscript no. ediblesI_v2 6300
6295
6290
6285
6280
6275
6270
Wavelength (Å)
20
15
10
Flux (Arbit. Unit)
6270 DIB
6283 DIB 40
30
20
10
0
Flux (Arb. Unit)
9660
9640
9620
9600
9580
9560
Wavelength (Å)
C60
+candidate
C60
+candidate Wavelength (Å) Fig. 9. (Left) Example of telluric line correction in the weak line regime by means of the rope-length method applied to the spectrum of HD 170740. Telluric lines of O2 are corrected first, then H2O lines. (Right) Example of telluric line correction in the strong line regime by means of a two-step
method and a composite instrumental profile adjustment (see text). Residuals remain at the location of the deepest lines, especially when the model
does not predict their shape and exact Doppler shift very accurately. The positions of the 9577 and 9632 Å DIBs attributed to C+
60 are indicated. 6.2. Stellar spectra Our longer-term objective is a quan-
titative analysis of the stellar spectra to determine their phys-
ical parameters (effective temperature, gravities, rotational ve-
locities, mass-loss rates etc), employing tools developed specif-
ically for the analysis of early-type spectra (e.g. Mokiem et al. 2005; Simón-Díaz et al. 2011). Ultimately this will help removal Article number, page 10 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey 5780
5785
5790
5795
Wavelength (Å)
0.70
0.75
0.80
0.85
0.90
0.95
1.00
Normalized flux
HD 149757
HD 184915
HD 144470
HD 145502
HD 147165
6612
6613
6614
6615
6616
Wavelength (Å)
0.75
0.80
0.85
0.90
0.95
1.00
Normalized flux
HD 149757
HD 184915
HD 144470
HD 145502
HD 147165
Fig. 10. An illustration of the quality of the spectra for the 5780 and 5797 Å (left) and 6614 Å (right) DIBs. For each target all observed spectra
were co-added in the heliocentric reference frame. The five targets shown have comparable E(B-V) values (Tables A.1 and A.2), and thus
have similar 5797 Å DIB strengths. The well established, strongly variable 5780/5797 ratio can be seen in the spectra, with the intensity of
5780 absorption inversely related to the molecular gas fraction, fH2 (Tables A.1 and A.2). The five spectra are shown superimposed on each
other at the top of the panels. Note that because of generally poor observing conditions there are numerous weak and narrow atmospheric water
features present (particularly noticeable around 5788–5792 Å) that could influence the 5797 Å profile. In the future these features will be removed
using the method described in Sect. 6. 6612
6613
6614
6615
6616
Wavelength (Å)
0.75
0.80
0.85
0.90
0.95
1.00
Normalized flux
HD 149757
HD 184915
HD 144470
HD 145502
HD 147165 5780
5785
5790
5795
Wavelength (Å)
0.70
0.75
0.80
0.85
0.90
0.95
1.00
Normalized flux
HD 149757
HD 184915
HD 144470
HD 145502
HD 147165 Normalized flux Fig. 10. An illustration of the quality of the spectra for the 5780 and 5797 Å (left) and 6614 Å (right) DIBs. For each target all observed spectra
were co-added in the heliocentric reference frame. The five targets shown have comparable E(B-V) values (Tables A.1 and A.2), and thus
have similar 5797 Å DIB strengths. 6.2. Stellar spectra The well established, strongly variable 5780/5797 ratio can be seen in the spectra, with the intensity of
5780 absorption inversely related to the molecular gas fraction, fH2 (Tables A.1 and A.2). The five spectra are shown superimposed on each
other at the top of the panels. Note that because of generally poor observing conditions there are numerous weak and narrow atmospheric water
features present (particularly noticeable around 5788–5792 Å) that could influence the 5797 Å profile. In the future these features will be removed
using the method described in Sect. 6. of stellar features from the EDIBLES spectra to aid analysis of
the interstellar features. ular fraction, whereas the sightline with the strongest 5780 Å
DIB has the highest atomic column density. However, while the
6614 Å DIB of the former is also the weakest, the latter does
not exhibit the strongest 6614 Å DIB, thus indicating that addi-
tional parameters must play a role in determining the DIB carrier
column density. 7. Quality assessment: the interstellar spectrum In this Section we highlight the interstellar lines and bands ob-
served for a few selected lines-of-sight. 7.1. EDIBLES High-resolution (R = 220,000) spectra of the 6614 Å band
have been recorded using the Coude Échelle Spectrograph
(CES) fed by the fibre link with the Cassegrain focus of the
3.6 m telescope at La Silla Observatory (Galazutdinov et al. 2002). A signal-to-noise of ∼600–1000 was achieved. Com-
parison of the EDIBLES and CES data is shown in Fig. 11
for HD 184915, HD 144470 and HD 145502 and the main fea-
tures are in good agreement. Of the three principal absorp-
tion components, the shortest wavelength feature (compo-
nent 1) is relatively strong for HD 184915 in both studies,
whereas components 1 and 3 have comparable intensities for
the lines-of-sight towards HD 144470 and HD 145502. No sig-
nificant additional structure is evident in the higher resolu-
tion CES spectra. As noted above, the full spectrum of HD 170740 is shown as an
example in Figs. 8 and B.1. For initial guidance in identifying the
DIBs in this line-of-sight the average ISM DIB spectrum (Jen-
niskens & Désert 1994), scaled to E(B −V)= 0.5 mag, is shown
in the latter figure. This reference spectrum includes broad DIBs
not included in e.g. Hobbs et al. (2009) but which appear to be
present in the observed spectrum. In Fig. 10 we compare the three strong DIBs at 5780, 5797 Å
(Heger 1922; Merrill & Wilson 1938) and 6614 Å for single-
cloud lines-of-sight towards five EDIBLES targets, HD 149757,
HD 184915, HD 144470, HD 145502, and HD 147165. The line
of sight reddening, E(B −V), for these sightlines differs by less
than 0.17 mag (c.f. Table A.1). The spectra are averages of indi-
vidual exposures co-added in the heliocentric rest frame and sub-
sequently continuum normalised (but not otherwise scaled other
than to offset them in the figure). Article number, page 11 of 30page.30 7.3. EDIBLES versus AAT As expected, for sightlines with such small variations in red-
dening, the 5797 Å DIB profiles have similar central depths (in-
sensitive to fH2; Cami et al. 1997). The large variations in the
strength of the 5780 Å DIB are thought to be related to variations
in the local interstellar conditions. The sightline with the weak-
est 5780 Å DIB is more neutral as indicated by the large molec- To assess the quality of our spectra with respect to previous stud-
ies, we compared our EDIBLES spectrum of HD147888 (ob-
tained in a single exposure on 2016-08-08) with a spectrum ob-
tained using the Anglo-Australian Telescope (AAT) in June 2004
by Cordiner et al. (2013). The AAT spectra were obtained using
the UCLES instrument at a resolving power of 58 000 and have Article number, page 11 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 A&A proofs: manuscript no. ediblesI_v2
3354
3356
3358
Wavelength ( ˚A)
0.90
0.95
1.00
1.05
1.10
Normalised Intensity
EDIBLES
UVES POP
Telluric
3579
3580
3581
Wavelength ( ˚A)
0.85
0.90
0.95
1.00
1.05
1.10
1.15
Normalised Intensity
Fig. 13. Comparison of EDIBLES (black) and
UVES POP (red) spectra of HD 169454 for in-
terstellar lines of NH(λλ3353.92, 3358.05 Å)
(top) and CN(1-0) (λλ3579.45, 3579.96, and
3580.9 Å; Meyer et al. 1989) (bottom); the tel-
luric spectrum is shown in orange. HD 169454
is a blue supergiant (B1 Ia) – the broad stellar
line in the spectrum is He I λ3355 Å. 3301
3302
3303
3304
Wavelength ( ˚A)
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Normalised Intensity
5860
5880
5900
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Intensity
5480
5500
5520
5540
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised Intensity
EDIBLES
UVES POP
Telluric
Average ISM
6360 6370 6380 6390 6400
Wavelength ( ˚A)
0.9
1.0
1.1
1.2
Normalised Intensity
Fig. 14. Comparison of EDIBLES (black) and UVES POP (red) spectra of HD 148937
(O6 f?p) for the interstellar Na lines (UV, top left; D, top right) and DIBs at λλ5480–
5545 Å (lower left) and λλ6360–6379 Å (lower right). The telluric spectrum is shown in
orange, and the average ISM DIB spectrum in green. The apparent feature at 6384 Å in the
UVES POP data (bottom right panel) is related to the order merging, but the significant
change in the He I 5876 line (top right panel) appears astrophysical in nature. 7.3. EDIBLES versus AAT Comparison of EDIBLES (black) and
UVES POP (red) spectra of HD 169454 for in-
terstellar lines of NH(λλ3353.92, 3358.05 Å)
(top) and CN(1-0) (λλ3579.45, 3579.96, and
3580.9 Å; Meyer et al. 1989) (bottom); the tel-
luric spectrum is shown in orange. HD 169454
is a blue supergiant (B1 Ia) – the broad stellar
line in the spectrum is He I λ3355 Å. Fig. 13. Comparison of EDIBLES (black) and
UVES POP (red) spectra of HD 169454 for in- Fig. 13. Comparison of EDIBLES (black) and
UVES POP (red) spectra of HD 169454 for in-
terstellar lines of NH(λλ3353.92, 3358.05 Å)
(top) and CN(1-0) (λλ3579.45, 3579.96, and
3580.9 Å; Meyer et al. 1989) (bottom); the tel-
luric spectrum is shown in orange. HD 169454
is a blue supergiant (B1 Ia) – the broad stellar
line in the spectrum is He I λ3355 Å. (top) a d CN(
0) (λλ35 9. 5, 35 9.96, a d
3580.9 Å; Meyer et al. 1989) (bottom); the tel-
luric spectrum is shown in orange. HD 169454
is a blue supergiant (B1 Ia) – the broad stellar
line in the spectrum is He I λ3355 Å. 6611
6612
6613
6614
6615
6616
Wavelength ( ˚A)
0.95
1.00
1.05
1.10
Normalised Intensity
HD184915
HD144470
HD145502
EDIBLES
CES
1
2
3
Fig. 11. Comparison
of
the
6614
Å
DIB
for
HD 184915,
HD 144470, and HD 145502 obtained with EDIBLES (black solid
line; this work, R
∼
110 000) and the CES (blue solid line;
Galazutdinov et al. 2002; R ∼220 000). The sub-structure compo-
nents 1, 2, and 3 are labelled in the bottom trace (see text). S/N ∼900. Cordiner et al. (2013) reduced their data using a cus-
tom procedure, taking special care to optimally correct for CCD
non-linearity, scattered light subtraction and flat fielding, as well
as wavelength calibration. A comparison between the EDIBLES
and AAT spectra is shown in Fig 12 for the 6614 Å DIB which
6612
6614
6616
6618
Heliocentric Wavelength (˚A)
0.95
1.00
1.05
1.10
Normalised Intensity
EDIBLES
AAT
ADP
HD147888
Fig. 12. Comparison of the 6614 Å DIB for HD 147888 (ρ Oph D) ob-
tained with EDIBLES (this work, R ∼110 000) and the AAT (Cordiner
et al. 2013, R ∼58 000). The top spectrum labeled “ADP” is the spec-
trum obtained with the standard ESO archive pipeline processing (i.e. using the default number of 5 flat-field frames). 7.3. EDIBLES versus AAT &
p oo s:
a usc pt o. ed b es _v
3301
3302
3303
3304
Wavelength ( ˚A)
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Normalised Intensity
5860
5880
5900
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Intensity
5480
5500
5520
5540
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised Intensity
EDIBLES
UVES POP
Telluric
Average ISM
6360 6370 6380 6390 6400
Wavelength ( ˚A)
0.9
1.0
1.1
1.2
Normalised Intensity
Fig. 14. Comparison of EDIBLES (black) and UVES POP (red) spectra of HD 148937
(O6 f?p) for the interstellar Na lines (UV, top left; D, top right) and DIBs at λλ5480–
5545 Å (lower left) and λλ6360–6379 Å (lower right). The telluric spectrum is shown in
orange, and the average ISM DIB spectrum in green. The apparent feature at 6384 Å in the
UVES POP data (bottom right panel) is related to the order merging, but the significant
change in the He I 5876 line (top right panel) appears astrophysical in nature. 3301
3302
3303
3304
Wavelength ( ˚A)
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Normalised Intensity 5860
5880
5900
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Intensity 3354
3356
3358
Wavelength ( ˚A)
0.90
0.95
1.00
1.05
1.10
Normalised Intensity
EDIBLES
UVES POP
Telluric 0
Normalised Intensity Normalised Intensity Wavelength ( ˚A) 5480
5500
5520
5540
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised Intensity
EDIBLES
UVES POP
Telluric
Average ISM 3579
3580
3581
Wavelength ( ˚A)
0.85
0.90
0.95
1.00
1.05
1.10
1.15
Normalised Intensity 6360 6370 6380 6390 6400
Wavelength ( ˚A)
0.9
1.0
1.1
1.2
Normalised Intensity Normalised Intensity Wavelength ( ˚A) Wavelength ( ˚A) Wavelength ( ˚A) Wavelength ( ˚A) Fig. 14. Comparison of EDIBLES (black) and UVES POP (red) spectra of HD 148937
(O6 f?p) for the interstellar Na lines (UV, top left; D, top right) and DIBs at λλ5480–
5545 Å (lower left) and λλ6360–6379 Å (lower right). The telluric spectrum is shown in
orange, and the average ISM DIB spectrum in green. The apparent feature at 6384 Å in the
UVES POP data (bottom right panel) is related to the order merging, but the significant
change in the He I 5876 line (top right panel) appears astrophysical in nature. Fig. 13. 7.3. EDIBLES versus AAT The red dotted line
represents the telluric absorption spectrum, shifted to match the helio-
centric rest frame of each target; highlighting the presence of a small
telluric absorption feature at 6614/6614.5 Å. makes for a good general test case due to the presence of narrow
and broad features within its profile. Apart from differences in
the wavelengths of the telluric features and a slight difference in 6612
6614
6616
6618
Heliocentric Wavelength (˚A)
0.95
1.00
1.05
1.10
Normalised Intensity
EDIBLES
AAT
ADP
HD147888
Fig. 12. Comparison of the 6614 Å DIB for HD 147888 (ρ Oph D) ob-
tained with EDIBLES (this work, R ∼110 000) and the AAT (Cordiner
et al. 2013, R ∼58 000). The top spectrum labeled “ADP” is the spec-
trum obtained with the standard ESO archive pipeline processing (i.e. using the default number of 5 flat-field frames). The red dotted line
represents the telluric absorption spectrum, shifted to match the helio-
centric rest frame of each target; highlighting the presence of a small
telluric absorption feature at 6614/6614.5 Å. 6611
6612
6613
6614
6615
6616
Wavelength ( ˚A)
0.95
1.00
1.05
1.10
Normalised Intensity
HD184915
HD144470
HD145502
EDIBLES
CES
1
2
3 6612
6614
6616
6618
Heliocentric Wavelength (˚A)
0.95
1.00
1.05
1.10
Normalised Intensity
EDIBLES
AAT
ADP
HD147888 Fig. 12. Comparison of the 6614 Å DIB for HD 147888 (ρ Oph D) ob-
tained with EDIBLES (this work, R ∼110 000) and the AAT (Cordiner
et al. 2013, R ∼58 000). The top spectrum labeled “ADP” is the spec-
trum obtained with the standard ESO archive pipeline processing (i.e. using the default number of 5 flat-field frames). The red dotted line
represents the telluric absorption spectrum, shifted to match the helio-
centric rest frame of each target; highlighting the presence of a small
telluric absorption feature at 6614/6614.5 Å. Fig. 11. Comparison
of
the
6614
Å
DIB
for
HD 184915,
HD 144470, and HD 145502 obtained with EDIBLES (black solid
line; this work, R
∼
110 000) and the CES (blue solid line;
Galazutdinov et al. 2002; R ∼220 000). The sub-structure compo-
nents 1, 2, and 3 are labelled in the bottom trace (see text). S/N ∼900. Cordiner et al. (2013) reduced their data using a cus-
tom procedure, taking special care to optimally correct for CCD
non-linearity, scattered light subtraction and flat fielding, as well
as wavelength calibration. Article number, page 12 of 30page.30 References Abt, H. A. & Morgan, W. W. 1969, AJ, 74, 813
Bagnulo, S., Jehin, E., Ledoux, C., et al. 2003, The Messenger, 114, 10
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B
l
S J hi
E L d
C
l 2003 T Abt, H. A. & Morgan, W. W. 1969, AJ, 74, 813
Bagnulo, S., Jehin, E., Ledoux, C., et al. 2003, The Messenger, 114, 10
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4013 The two right-hand panels of Fig. 14 reveal apparent varia-
tions between the EDIBLES data compared to the UVES POP
spectrum. The strong absorption in the POP data at ∼6384 Å co-
incides with the order ends/overlap, and appears to be an artefact
of the order merging. In contrast, the significant change in the
He I 5876 absorption seems robust. In the context of HD 148937
being a peculiar magnetic star, this variation is quite remarkable. This aspect will be discussed elsewhere. Ballester, P., Modigliani, A., Boitquin, O., et al. 2000, The Messenger, 101, 31 Baron, D., Poznanski, D., Watson, D., et al. 2015, MNRAS, 451, Berné, O., Montillaud, J., & Joblin, C. 2015, A&A, 577, A133 Berné, O., Mulas, G., & Joblin, C. 2013, A&A, 550, L4 Berné, O. & Tielens, A. G. G. M. 2012, Proceedings of the National Academy
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Bréchignac, P. & Pino, T. 7.4. EDIBLES versus UVES POP To further illustrate the quality of the data we compared spectra
of two targets, HD 148937 (Fig. 13) and HD 169454 (Fig. 14),
which were both observed as part of the EDIBLES survey
and the UVES Paranal Observatory Project (POP; Bagnulo
et al. 2003). The UVES POP programme gathered a library
of high-resolution, high S/N spectra of (field) stars across the
Hertzsprung-Russell diagram. The spectra in the figures have
been scaled in intensity to facilitate comparison, but not con-
tinuum normalised. Acknowledgements. This work is based on observations collected at the Euro-
pean Organisation for Astronomical Research in the Southern Hemisphere un-
der ESO programmes 194.C-0833 and 266.D-5655. The EDIBLES project was
initiated at the IAU Symposium 297 "The Diffuse Interstellar Bands" (Cami &
Cox 2014); the support of the International Astronomical Union for this meeting
is gratefully acknowledged. NLJC thanks the Paranal Observatory staff and the
ESO User Support Department for their assistance with successfully executing
the observations. The authors thank both the Royal Astronomical Society and the
Lorentz Center for hosting workshops, and Jacek Krełowski for making avail-
able the CES (ESO) data. We thank the referee for constructive comments
that helped improve the paper. The research leading to these results has re-
ceived funding from the European Research Council under the European Union’s
Seventh Framework Programme (FP/2007-2013) ERC-2013-SyG, Grant Agree-
ment n. 610256 NANOCOSMOS. FS acknowledges the support of NASA
through the APRA SMD Program. AMI acknowledges support from Agence Na-
tionale de la Recherche through the STILISM project (ANR-12-BS05-0016-02)
and from the Spanish PNAYA through project AYA2015-68217-P. Fig. 13 shows the NH and OH+ lines in the sightline towards
HD 169454. The top panels in Fig. 14 compare the sodium dou-
blets at ∼3303 Å (see also Hunter et al. 2006) and ∼5895 Å for
the sightline towards HD 148937. The bottom panel compares
several weak and strong DIBs in the HD 148937 line-of-sight. In each panel the EDIBLES and UVES POP spectra are shown
in black and red, respectively. The average ISM synthetic DIB
spectrum (adapted from Jenniskens & Désert 1994) is shown in
green and a generic Paranal model telluric spectrum5 is shown in
orange. The agreement between stellar and interstellar features
in the EDIBLES and UVES POP is excellent, where the EDI-
BLES data generally reach a higher S/N. 7.3. EDIBLES versus AAT A comparison between the EDIBLES
and AAT spectra is shown in Fig. 12 for the 6614 Å DIB, which makes for a good general test case due to the presence of narrow
and broad features within its profile. Apart from differences in
the wavelengths of the telluric features, and a slight difference in Article number, page 12 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey illustrate the quality and scope of the new spectra we have com-
pared (1) EDIBLES and AAT spectra of the 6613 Å DIB towards
HD 147888, (2) EDIBLES and CES spectra of the 6613 Å
DIB for the sightlines towards HD 184915, HD 144470 and
HD 145502, and (3) EDIBLES and UVES POP spectra of
HD 148937 and HD 169454. the overall spectral slope (presumably due to uncorrected differ-
ences between the UVES and UCLES blaze functions), the spec-
tra are almost identical within the noise. Slight differences in the
depths and widths of the substructure peaks are attributable to
the higher resolution of the UVES spectrum. The overall excel-
lent match demonstrates the quality of our EDIBLES reduction
procedure. For reference we show also the ADP spectrum, which
uses only 5 flat-field frames; the large increase in S/N when us-
ing our custom flat-field processing is apparent. Upcoming papers in this series will present in detail the
array of scientific results that are being explored with the
EDIBLES data set. Once the program is completed the advanced
data products (merged and normalised spectra) will be released
to the community through the ESO Science Archive and the
CDS/ViZieR service. References 1999, A&A, 343, L49 These comparisons with existing data for selected sightlines
show the excellent data quality of the spectra acquired within
EDIBLES, illustrating its potential for detailed studies of physi-
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McCall, B. J., Drosback, M. M., Thorburn, J. A., et al. 2010, ApJ, 708, 1628 Whitmore, J. B., Murphy, M. T., & Griest, K. 2010, ApJ, 723, 89 Merrill, P. W. & Wilson, O. C. 1938, ApJ, 87, 9 Whittet, D. C. B., Martin, P. G., Hough, J. H., et al. 1992, ApJ, 386, 562 p
Meyer, D. M., Roth, K. C., & Hawkins, I. 1989, ApJ, 343, L1 Zasowski, G., Ménard, B., Bizyaev, D., et al. 2015, ApJ, 798, 35 Mokiem, M. R., de Koter, A., Puls, J., et al. 2005, A&A, 441, 711 Zhen, J., Castellanos, P., Paardekooper, D. M., Linnartz, H., & Tielens,
A. G. G. M. 2014, ApJ, 797, L30 Monreal-Ibero, A., Weilbacher, P. M., Wendt, M., et al. 2015, A&A, 576, L3
Morgan W W Code A D & Whitford A E 1955 ApJS 2 41 Monreal-Ibero, A., Weilbacher, P. M., Wendt, M., et al. 2015, A&A, 576, L3 Morgan, W. W., Code, A. D., & Whitford, A. E. 1955, ApJS, 2, 41 Article number, page 14 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey 1 Université de Toulouse, UPS-OMP, IRAP, 31028, Toulouse, France 2 CNRS, IRAP, 9 Av. colonel Roche, BP 44346, F-31028 Toulouse,
France 3 Department of Physics and Astronomy, The University of Western
Ontario, London, ON N6A 3K7, Canada 4 SETI Institute, 189 Bernardo Avenue, Suite 100, Mountain View,
CA 94043, USA 5 School of Astronomy, Institute for Research in Fundamental Sci-
ences, 19395-5531 Tehran, Iran 6 European Southern Observatory, Alonso de Cordova 3107, Vitacura,
Santiago, Chile 7 GEPI, Observatoire de Paris, PSL Research University, CNRS, Uni-
versité Paris-Diderot, Sorbonne Paris Cité, Place Jules Janssen,
92195 Meudon, France 8 Instituto de Astrofísica de Canarias (IAC), E-38205 La Laguna,
Tenerife, Spain ,
p
9 Universidad de La Laguna, Dpto. Article number, page 15 of 30page.30 Article number, page 16 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Astrofísica, E-38206 La Laguna,
Tenerife, Spain p
10 School of Chemistry, The University of Nottingham, University
Park, Nottingham NG7 2RD, UK g
11 Royal Astronomical Society, Burlington House, Piccadilly, London
W1J 0BQ, UK Q
12 AAAS Science International, Clarendon House, Clarendon Road,
Cambridge CB2 8FH, UK g
13 UK Astronomy Technology Centre, Royal Observatory, Blackford
Hill, Edinburgh, EH9 3HJ, UK g
14 Instituut voor Sterrenkunde, KU Leuven, Celestijnenlaan 200D, bus
2401, Leuven, Belgium g
15 Sackler Laboratory for Astrophysics, Leiden Observatory, Leiden
University, PO Box 9513, NL2300 RA Leiden, The Netherlands g
15 Sackler Laboratory for Astrophysics, Leiden Observatory, Leiden niversity, PO Box 9513, NL2300 RA Leiden, The Netherla y
16 Astrochemistry Laboratory, NASA Goddard Space Flight Center
Code 691, 8800 Greenbelt Road, Greenbelt, MD 20771, USA 16 Astrochemistry Laboratory, NASA Goddard Space Flight Center, Code 691, 8800 Greenbelt Road, Greenbelt, MD 20771, USA 17 Department of Physics, The Catholic University of America, Wash-
ington, DC 20064, USA 18 Lennard-Jones Laboratories, Keele University, ST5 5BG, UK
19 19 ESTEC, ESA, Noordwijk, The Netherlands
20 20 ICCM, Madrid, Spain
21 21 Anton Pannekoek Institute for Astronomy, University of Amster-
dam, NL-1090 GE Amsterdam, The Netherlands 22 George Washington University, Washington DC, USA 23 LERMA, Observatoire de Paris, PSL Research University, CNRS,
Sorbonne Universités, UPMC Université Paris 06, 92190 Meudon,
France 23 LERMA, Observatoire de Paris, PSL Research Unive 24 INAF - Osservatorio Astronomico di Cagliari, Via della Scienza 5,
I-09047 Selargius, Italy g
y
25 NASA Ames Research Center, Space Science & Astrobiology Divi-
sion, Moffett Field, California, USA sion, Moffett Field, California, USA 26 Kapteyn Institute, University of Groningen, Groningen, The Nether-
lands Article number, page 15 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Appendix A: Survey sample Table A.1 lists spectral classifications and line-of-sight parameters for the observed EDIBLES targets to date; Table A.2 lists the
additional observations that will supplement the data discussed here in due course. The RV and AV extinction values are taken (in
order of preference) from Valencic et al. (2004), Fitzpatrick & Massa (2007), or Wegner (2003). H I and H2 column densities are
from Jenkins (2009). The interstellar reddening, E(B −V), was computed from (B −V)J colours (taking the average from Tycho-2
and Simbad B −V colours; where Tycho-2 colours were converted to Johnson colours; Mamajek et al. 2002) and the published
spectral classifications, adopting intrinsic colours from Fitzgerald (1970). References for the spectral classifications in col. 4 of both tables are: M50 (Morgan & Roman 1950); S52 (Sharpless 1952); M55
(Morgan et al. 1955); H56 (Hiltner 1956); S56 (Stebbins & Kron 1956, classification originally from W. W. Morgan); F57 (Feast
et al. 1957); O59 (Osawa 1959); G68 (Guetter 1968); L68 (Lesh 1968); A69 (Abt & Morgan 1969); C69 (Cowley et al. 1969); H69
(Hiltner et al. 1969); S71 (Schild & Chaffee 1971); W71 (Walborn 1971); H73 (Humphreys 1973); M73 (Morgan & Keenan 1973);
L75 (Levato 1975); L76 (Levato & Abt 1976); W76 (Walborn 1976); G77 (Garrison et al. 1977); H78 (Houk 1978); H82 (Hendry
1982); G94 (Garrison & Gray 1994); M95 (Massey et al. 1995); P98 (Parsons & Ake 1998); S99 (Steele et al. 1999); G01 (Gray
et al. 2001); E05 (Evans et al. 2005); L06 (Levenhagen & Leister 2006); R09 (Renson & Manfroid 2009); S11 (Sota et al. 2011);
H13 (Holberg et al. 2013); S14 (Sota et al. 2014). References for the spectral classifications in col. 4 of both tables are: M50 (Morgan & Roman 1950); S52 (Sharpless 1952); M55
(Morgan et al. 1955); H56 (Hiltner 1956); S56 (Stebbins & Kron 1956, classification originally from W. W. Morgan); F57 (Feast
et al. 1957); O59 (Osawa 1959); G68 (Guetter 1968); L68 (Lesh 1968); A69 (Abt & Morgan 1969); C69 (Cowley et al. 1969); H69
(Hiltner et al. 1969); S71 (Schild & Chaffee 1971); W71 (Walborn 1971); H73 (Humphreys 1973); M73 (Morgan & Keenan 1973);
L75 (Levato 1975); L76 (Levato & Abt 1976); W76 (Walborn 1976); G77 (Garrison et al. 1977); H78 (Houk 1978); H82 (Hendry
1982); G94 (Garrison & Gray 1994); M95 (Massey et al. 1995); P98 (Parsons & Ake 1998); S99 (Steele et al. 1999); G01 (Gray
et al. Appendix A: Survey sample 2001); E05 (Evans et al. 2005); L06 (Levenhagen & Leister 2006); R09 (Renson & Manfroid 2009); S11 (Sota et al. 2011);
H13 (Holberg et al. 2013); S14 (Sota et al. 2014). N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey Identifier
RA
Dec
Spectral type
Ref. E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 22951
03:42:22.7
+33:57:54.1
B0.5 V
M55
0.23
–
–
21.04
20.46
0.35
HD 23016
03:42:19.0
+19:42:00.9
B8 Ve
S99
0.05
2.30
0.12
W03
–
–
–
HD 23180
03:44:19.1
+32:17:17.7
B1 III
G77
0.28
3.11
0.93
V04
20.88
20.60
0.51
HD 24398
03:54:07.9
+31:53:01.1
B1 Ib
L68
0.29
2.63
0.89
W03
20.8
20.67
0.60
HD 27778
04:23:59.8
+24:18:03.6
B3 V
O59
0.36
2.59
0.91
V04
20.35
20.72
0.82
HD 34748
05:19:35.3
−01:24:42.8
B1.5 Vn
L68
0.13
2.61
0.29
W03
21.16
–
HD 36486
05:32:00.4
−00:17:56.7
O9.5 II Nwk
S11
0.16
–
–
20.19
14.68
0.00
HD 36695
05:33:31.5
−01:09:21.9
B1 V
L68
0.08
2.45
0.15
W03
20.7
–
–
HD 36822
05:34:49.2
+09:29:22.5
B0 III
L75
0.14
2.64
0.21
W03
20.81
19.32
0.06
HD 36861
05:35:08.3
+09:56:03.0
O8 III ((f))
S11
0.13
2.46
0.22
W03
20.78
19.12
0.04
HD 37022
05:35:16.5
−05:23:23.2
O7 Vp
S11
0.31
5.73
1.78
V04
21.54
17.55
0.00
HD 37041
05:35:22.9
−05:24:57.8
O9.5 IVp
S11
0.21
5.67
1.13
V04
–
–
–
HD 37061
05:35:31.4
−05:16:02.6
B0.5 V
S71
0.52
4.29
2.40
V04
–
–
–
HD 37367
05:39:18.3
+29:12:54.8
B2 IV-V
L68
0.37
3.55
1.49
V04
21.17
20.53
0.31
HD 37903
05:41:38.4
−02:15:32.5
B1.5 V
S52
0.34
3.74
1.31
V04
21.12
20.85
0.60
HD 38771
05:47:45.4
−09:40:10.6
B0.5 Ia
W71
0.07
–
–
20.52
15.68
0.00
HD 40111
05:57:59.7
+25:57:14.1
B1 Ib
L68
0.11
3.05
0.37
W03
20.9
19.73
0.12
HD 41117
06:03:55.2
+20:08:18.4
B2 Ia
W71
0.41
3.12
1.25
V04
20.15
20.69
0.87
HD 43384
06:16:58.7
+23:44:27.3
B3 Iab
L68
0.54
3.29
1.91
W03
21.27
–
–
HD 53975
07:06:36.0
−12:23:38.0
O7.5 Vz
S11
0.20
3.25
0.62
W03
21.13
19.18
0.02
HD 54439
07:08:23.2
−11:51:08.6
B2 IIIn
M55
0.27
2.73
0.79
V04
–
–
–
HD 54662
07:09:20.3
−10:20:47.8
O7 Vz var? Appendix A: Survey sample (SB2)
S14
0.32
3.12
0.81
W03
21.38
20.00
0.08
HD 55879
07:14:28.3
−10:18:58.5
O9.7 III
S11
0.13
2.47
0.15
W03
20.9
–
–
HD 57061
07:18:42.5
−24:57:15.8
O9 II (SB2+SBE)
S14
0.12
3.57
0.36
W03
20.7
15.48
0.00
HD 66194
07:58:50.6
−60:49:28.1
B2 Vne
A69
0.15
2.25
0.41
W03
–
–
–
HD 66811
08:03:35.1
−40:00:11.3
O4 I(n)fp
S14
0.08
5.01
0.25
W03
19.96
14.45
0.01
HD 73882
08:39:09.5
−40:25:09.3
O8.5 IV (SB1+SBE)
S14
0.68
3.56
2.46
V04
21.11
21.08
0.65
HD 75309
08:47:28.0
−46:27:04.0
B2 Ib/II
H78
0.16
3.53
1.02
V04
21.07
20.20
0.21
HD 75860
08:50:53.2
−43:45:05.4
BC2 Iab
W76
0.87
3.44
3.10
W03
–
–
–
HD 79186
09:11:04.4
−44:52:04.4
B5 Ia
H69
0.28
3.21
1.28
V04
21.18
20.72
0.41
HD 80558
09:18:42.4
−51:33:38.3
B6 Ia
H69
0.57
3.35
2.01
W03
–
–
–
HD 81188
09:22:06.8
−55:00:38.4
B2 IV
L75
0.10
–
–
19.95
17.70
0.01
HD 91824
10:34:46.6
−58:09:22.0
O7 V((f))z (SB1)
S14
0.25
3.35
0.80
V04
21.12
19.85
0.10
HD 93030
10:42:57.4
−64:23:40.0
B0.2 Vp
W76
0.08
2.43
0.10
W03
20.26
15.02
0.00
HD 93205
10:44:33.7
−59:44:15.5
O3.5 V((f)) + O8 V
S14
0.34
3.25
1.24
V04
21.38
19.78
0.05
HD 93222
10:44:36.2
−60:05:29.0
O7 V((f))z
S14
0.34
4.76
1.71
V04
21.4
19.78
0.05
HD 93843
10:48:37.8
−60:13:25.5
O5 III(fc) SB1? S14
0.27
3.89
0.66
V04
21.33
19.61
0.04
HD 94493
10:53:15.1
−60:48:53.2
B0.5 Ib:
H73
0.23
3.57
0.82
V04
21.08
20.15
0.19
HD 99953
11:29:15.1
−63:33:14.2
B2 Ia
M55
0.45
3.69
1.77
V04
–
–
–
HD 103779
11:56:58.0
−63:14:56.7
B0.5 Iab
G77
0.24
3.29
0.69
V04
21.16
19.82
0.08
HD 104705
12:03:23.9
−62:41:45.8
B0.5 III
M55
0.23
2.81
0.65
V04
21.1
20.08
0.16
HD 109399
12:35:16.5
−72:43:00.8
B0.5 Ib
H73
0.26
3.26
0.68
W03
21.11
20.04
0.15
d Identifier
RA
Dec
Spectral type
Ref. Appendix A: Survey sample E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 22951
03:42:22.7
+33:57:54.1
B0.5 V
M55
0.23
–
–
21.04
20.46
0.35
HD 23016
03:42:19.0
+19:42:00.9
B8 Ve
S99
0.05
2.30
0.12
W03
–
–
–
HD 23180
03:44:19.1
+32:17:17.7
B1 III
G77
0.28
3.11
0.93
V04
20.88
20.60
0.51
HD 24398
03:54:07.9
+31:53:01.1
B1 Ib
L68
0.29
2.63
0.89
W03
20.8
20.67
0.60
HD 27778
04:23:59.8
+24:18:03.6
B3 V
O59
0.36
2.59
0.91
V04
20.35
20.72
0.82
HD 34748
05:19:35.3
−01:24:42.8
B1.5 Vn
L68
0.13
2.61
0.29
W03
21.16
–
HD 36486
05:32:00.4
−00:17:56.7
O9.5 II Nwk
S11
0.16
–
–
20.19
14.68
0.00
HD 36695
05:33:31.5
−01:09:21.9
B1 V
L68
0.08
2.45
0.15
W03
20.7
–
–
HD 36822
05:34:49.2
+09:29:22.5
B0 III
L75
0.14
2.64
0.21
W03
20.81
19.32
0.06
HD 36861
05:35:08.3
+09:56:03.0
O8 III ((f))
S11
0.13
2.46
0.22
W03
20.78
19.12
0.04
HD 37022
05:35:16.5
−05:23:23.2
O7 Vp
S11
0.31
5.73
1.78
V04
21.54
17.55
0.00
HD 37041
05:35:22.9
−05:24:57.8
O9.5 IVp
S11
0.21
5.67
1.13
V04
–
–
–
HD 37061
05:35:31.4
−05:16:02.6
B0.5 V
S71
0.52
4.29
2.40
V04
–
–
–
HD 37367
05:39:18.3
+29:12:54.8
B2 IV-V
L68
0.37
3.55
1.49
V04
21.17
20.53
0.31
HD 37903
05:41:38.4
−02:15:32.5
B1.5 V
S52
0.34
3.74
1.31
V04
21.12
20.85
0.60
HD 38771
05:47:45.4
−09:40:10.6
B0.5 Ia
W71
0.07
–
–
20.52
15.68
0.00
HD 40111
05:57:59.7
+25:57:14.1
B1 Ib
L68
0.11
3.05
0.37
W03
20.9
19.73
0.12
HD 41117
06:03:55.2
+20:08:18.4
B2 Ia
W71
0.41
3.12
1.25
V04
20.15
20.69
0.87
HD 43384
06:16:58.7
+23:44:27.3
B3 Iab
L68
0.54
3.29
1.91
W03
21.27
–
–
HD 53975
07:06:36.0
−12:23:38.0
O7.5 Vz
S11
0.20
3.25
0.62
W03
21.13
19.18
0.02
HD 54439
07:08:23.2
−11:51:08.6
B2 IIIn
M55
0.27
2.73
0.79
V04
–
–
–
HD 54662
07:09:20.3
−10:20:47.8
O7 Vz var? N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey Appendix A: Survey sample (SB2)
S14
0.32
3.12
0.81
W03
21.38
20.00
0.08
HD 55879
07:14:28.3
−10:18:58.5
O9.7 III
S11
0.13
2.47
0.15
W03
20.9
–
–
HD 57061
07:18:42.5
−24:57:15.8
O9 II (SB2+SBE)
S14
0.12
3.57
0.36
W03
20.7
15.48
0.00
HD 66194
07:58:50.6
−60:49:28.1
B2 Vne
A69
0.15
2.25
0.41
W03
–
–
–
HD 66811
08:03:35.1
−40:00:11.3
O4 I(n)fp
S14
0.08
5.01
0.25
W03
19.96
14.45
0.01
HD 73882
08:39:09.5
−40:25:09.3
O8.5 IV (SB1+SBE)
S14
0.68
3.56
2.46
V04
21.11
21.08
0.65
HD 75309
08:47:28.0
−46:27:04.0
B2 Ib/II
H78
0.16
3.53
1.02
V04
21.07
20.20
0.21
HD 75860
08:50:53.2
−43:45:05.4
BC2 Iab
W76
0.87
3.44
3.10
W03
–
–
–
HD 79186
09:11:04.4
−44:52:04.4
B5 Ia
H69
0.28
3.21
1.28
V04
21.18
20.72
0.41
HD 80558
09:18:42.4
−51:33:38.3
B6 Ia
H69
0.57
3.35
2.01
W03
–
–
–
HD 81188
09:22:06.8
−55:00:38.4
B2 IV
L75
0.10
–
–
19.95
17.70
0.01
HD 91824
10:34:46.6
−58:09:22.0
O7 V((f))z (SB1)
S14
0.25
3.35
0.80
V04
21.12
19.85
0.10
HD 93030
10:42:57.4
−64:23:40.0
B0.2 Vp
W76
0.08
2.43
0.10
W03
20.26
15.02
0.00
HD 93205
10:44:33.7
−59:44:15.5
O3.5 V((f)) + O8 V
S14
0.34
3.25
1.24
V04
21.38
19.78
0.05
HD 93222
10:44:36.2
−60:05:29.0
O7 V((f))z
S14
0.34
4.76
1.71
V04
21.4
19.78
0.05
HD 93843
10:48:37.8
−60:13:25.5
O5 III(fc) SB1? S14
0.27
3.89
0.66
V04
21.33
19.61
0.04
HD 94493
10:53:15.1
−60:48:53.2
B0.5 Ib:
H73
0.23
3.57
0.82
V04
21.08
20.15
0.19
HD 99953
11:29:15.1
−63:33:14.2
B2 Ia
M55
0.45
3.69
1.77
V04
–
–
–
HD 103779
11:56:58.0
−63:14:56.7
B0.5 Iab
G77
0.24
3.29
0.69
V04
21.16
19.82
0.08
HD 104705
12:03:23.9
−62:41:45.8
B0.5 III
M55
0.23
2.81
0.65
V04
21.1
20.08
0.16
HD 109399
12:35:16.5
−72:43:00.8
B0.5 Ib
H73
0.26
3.26
0.68
W03
21.11
20.04
0.15
d Article number, page 17 of 30page.30 continued on next page Identifier
RA
Dec
Spectral type
Ref. continued on next page E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 111934
12:53:37.6
−60:21:25.4
B1.5 Ib
E05
0.34
2.45
1.25
V04
–
–
–
HD 112272
12:56:33.7
−64:21:39.2
B0.5 Ia
M55
0.85
3.09
3.09
V04
–
–
–
HD 113904
13:08:07.2
−65:18:21.7
O9 III
S14
0.20
3.63
0.76
W03
21.08
19.83
0.10
HD 116852
13:30:23.5
−78:51:20.5
O8.5 II-III((f))
S14
0.20
2.42
0.51
V04
20.96
19.79
0.12
HD 122879
14:06:25.2
−59:42:57.3
B0 Ia
W76
0.34
3.15
1.13
V04
21.26
20.24
0.16
HD 124314
14:15:01.6
−61:42:24.4
O6 IV(n)((f))
S14
0.49
2.97
1.49
W03
21.41
20.52
0.21
HD 133518
15:06:56.0
−52:01:47.2
B2 Vp
G77
0.13
1.79
0.27
W03
–
–
–
HD 135591
15:18:49.1
−60:18:51.5
O8 IV((f))
S14
0.20
3.57
0.79
W03
21.08
19.77
0.09
HD 143275
16:00:20.0
−22:37:18.1
B0.3 IV
M73
0.19
3.09
0.43
W03
21.15
19.41
0.04
HD 144470
16:06:48.4
−20:40:09.1
B1 V
W71
0.21
3.37
0.74
V04
21.17
20.05
0.13
HD 145502
16:11:59.7
−19:27:38.5
B2 V (SB2)
L75
0.27
–
–
21.07
19.89
0.12
HD 147084
16:20:38.2
−24:10:09.8
A5 II
G01
0.78
–
–
–
–
–
HD 147165
16:21:11.3
−25:35:34.1
B1 III
W71
0.37
3.86
1.47
V04
21.17
20.05
0.13
HD 147683
16 24 43.7
−34 53 37.5
B3: Vn (SB2)
G77
0.29
–
–
21.20
20.68
0.38
HD 147888
16:25:24.3
−23:27:36.8
B3 V:
F57
0.44
3.89
1.98
V04
21.69
20.48
0.11
HD 147889
16:25:24.3
−24:21:07.1
B2 V
H56
1.03
3.95
4.35
V04
–
–
–
HD 147933
16:25:35.1
−23:26:48.7
B2 IV
H69
0.43
5.74
2.58
V04
21.64
20.57
0.15
HD 147934
16:25:35.0
−23:26:46.0
B2 V
H69
0.43
–
–
–
–
–
HD 148605
16:30:12.5
−25:06:54.8
B2 V
M73
0.07
3.18
0.25
W03
20.68
18.74
0.02
HD 148937
16:33:52.4
−48:06:40.5
O6 f?p
S14
0.65
3.28
2.20
W03
21.60
20.71
0.21
HD 149038
16:34:05.0
−44:02:43.1
O9.7 Iab
S14
0.31
4.92
1.08
V04
21.00
20.45
0.36
HD 149404
16:36:22.6
−42:51:31.9
O8.5 Iab(f)p (SB2)
S14
0.67
3.53
2.19
V04
–
–
–
HD 149757
16:37:09.5
−10:34:01.5
O9.2 IVnn
S14
0.32
2.55
0.82
V04
20.72
20.65
0.63
HD 150136
16:41:20.4
−48:45:46.8
O3.5-4 III(f*) + O6 IV
S14
0.43
3.27
1.64
W03
–
–
–
HD 151804
16:51:33.7
−41:13:49.9
O8 Iaf (SB2)
S14
0.34
4.33
1.30
V04
21.08
20.26
0.23
HD 152248
16:54:10.1
−41:49:30.1
O7 Iabf + O7 Ib(f)
S14
0.47
3.68
1.51
V04
–
20.92
–
HD 152408
16:54:58.5
−41:09:03.1
O8: Iafpe
S14
0.44
4.17
1.75
V04
21.27
20.38
0.21
HD 152424
16:55:03.3
−42:05:27.0
OC9.2 Ia (SB1)
S14
0.66
3.23
2.07
W03
–
–
–
HD 153919
17:03:56.8
−37:50:38.9
O6 Iafcp (SB1E)
S14
0.59
3.58
1.97
V04
–
–
–
HD 154043
17:05:18.9
−47:04:08.5
B2 Iab
H78
0.80
3.30
2.57
W03
–
–
–
HD 155806
17:15:19.3
−33:32:54.3
O7.5 V((f))z(e)
S14
0.27
2.48
0.57
W03
21.08
19.92
0.12
HD 157246
17:25:23.7
−56:22:39.8
B1 Ib
H69
0.06
2.82
0.17
W03
20.68
19.23
0.07
HD 157978
17:26:19.0
+07:35:44.3
G0/K0 II: + A1
P98
0.64
3.72
2.42
W03
–
–
–
HD 158926
17:33:36.5
−37:06:13.8
B2 IV + DA.79
H13
0.10
–
–
19.23
12.70
0.00
HD 161056
17:43:47.0
−07:04:46.6
B1.5 V
L68
0.58
–
–
21.23
–
–
HD 164073
18:02:00.6
−48:48:37.7
B3III/IV
H78
0.22
2.96
0.62
V04
–
–
–
HD 164353
18:00:39.0
+02:55:53.6
B5 Ib
M50
0.10
–
–
21.00
20.26
0.27
HD 166937
18:13:45.8
−21:03:31.8
B8 Iab(e)
G94
0.22
–
–
–
–
–
HD 167264
18:15:12.9
−20:43:41.8
O9.7 Iab
S14
0.24
3.26
0.98
V04
21.15
20.28
0.21
HD 167838
18:17:37.7
−15:25:50.6
B5 Ia
M55
0.55
3.39
2.13
V04
–
–
–
HD 167971
18:18:05.9
−12:14:33.3
O8 Iaf(n) + O4/5
S11
1.01
3.44
3.54
V04
21.6
20.85
0.26
HD 169454
18:25:15.2
−13:58:42.3
B1 Ia
W76
1.03
3.37
3.64
V04
–
–
–
HD 170740
18:31:25.7
−10:47:45.0
B2 V
S56
0.45
3.01
1.51
V04
21.03
20.86
0.58
i
d Identifier Table A.1. continued on next page E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 111934
12:53:37.6
−60:21:25.4
B1.5 Ib
E05
0.34
2.45
1.25
V04
–
–
–
HD 112272
12:56:33.7
−64:21:39.2
B0.5 Ia
M55
0.85
3.09
3.09
V04
–
–
–
HD 113904
13:08:07.2
−65:18:21.7
O9 III
S14
0.20
3.63
0.76
W03
21.08
19.83
0.10
HD 116852
13:30:23.5
−78:51:20.5
O8.5 II-III((f))
S14
0.20
2.42
0.51
V04
20.96
19.79
0.12
HD 122879
14:06:25.2
−59:42:57.3
B0 Ia
W76
0.34
3.15
1.13
V04
21.26
20.24
0.16
HD 124314
14:15:01.6
−61:42:24.4
O6 IV(n)((f))
S14
0.49
2.97
1.49
W03
21.41
20.52
0.21
HD 133518
15:06:56.0
−52:01:47.2
B2 Vp
G77
0.13
1.79
0.27
W03
–
–
–
HD 135591
15:18:49.1
−60:18:51.5
O8 IV((f))
S14
0.20
3.57
0.79
W03
21.08
19.77
0.09
HD 143275
16:00:20.0
−22:37:18.1
B0.3 IV
M73
0.19
3.09
0.43
W03
21.15
19.41
0.04
HD 144470
16:06:48.4
−20:40:09.1
B1 V
W71
0.21
3.37
0.74
V04
21.17
20.05
0.13
HD 145502
16:11:59.7
−19:27:38.5
B2 V (SB2)
L75
0.27
–
–
21.07
19.89
0.12
HD 147084
16:20:38.2
−24:10:09.8
A5 II
G01
0.78
–
–
–
–
–
HD 147165
16:21:11.3
−25:35:34.1
B1 III
W71
0.37
3.86
1.47
V04
21.17
20.05
0.13
HD 147683
16 24 43.7
−34 53 37.5
B3: Vn (SB2)
G77
0.29
–
–
21.20
20.68
0.38
HD 147888
16:25:24.3
−23:27:36.8
B3 V:
F57
0.44
3.89
1.98
V04
21.69
20.48
0.11
HD 147889
16:25:24.3
−24:21:07.1
B2 V
H56
1.03
3.95
4.35
V04
–
–
–
HD 147933
16:25:35.1
−23:26:48.7
B2 IV
H69
0.43
5.74
2.58
V04
21.64
20.57
0.15
HD 147934
16:25:35.0
−23:26:46.0
B2 V
H69
0.43
–
–
–
–
–
HD 148605
16:30:12.5
−25:06:54.8
B2 V
M73
0.07
3.18
0.25
W03
20.68
18.74
0.02
HD 148937
16:33:52.4
−48:06:40.5
O6 f?p
S14
0.65
3.28
2.20
W03
21.60
20.71
0.21
HD 149038
16:34:05.0
−44:02:43.1
O9.7 Iab
S14
0.31
4.92
1.08
V04
21.00
20.45
0.36
HD 149404
16:36:22.6
−42:51:31.9
O8.5 Iab(f)p (SB2)
S14
0.67
3.53
2.19
V04
–
–
–
HD 149757
16:37:09.5
−10:34:01.5
O9.2 IVnn
S14
0.32
2.55
0.82
V04
20.72
20.65
0.63
HD 150136
16:41:20.4
−48:45:46.8
O3.5-4 III(f*) + O6 IV
S14
0.43
3.27
1.64
W03
–
–
–
HD 151804
16:51:33.7
−41:13:49.9
O8 Iaf (SB2)
S14
0.34
4.33
1.30
V04
21.08
20.26
0.23
HD 152248
16:54:10.1
−41:49:30.1
O7 Iabf + O7 Ib(f)
S14
0.47
3.68
1.51
V04
–
20.92
–
HD 152408
16:54:58.5
−41:09:03.1
O8: Iafpe
S14
0.44
4.17
1.75
V04
21.27
20.38
0.21
HD 152424
16:55:03.3
−42:05:27.0
OC9.2 Ia (SB1)
S14
0.66
3.23
2.07
W03
–
–
–
HD 153919
17:03:56.8
−37:50:38.9
O6 Iafcp (SB1E)
S14
0.59
3.58
1.97
V04
–
–
–
HD 154043
17:05:18.9
−47:04:08.5
B2 Iab
H78
0.80
3.30
2.57
W03
–
–
–
HD 155806
17:15:19.3
−33:32:54.3
O7.5 V((f))z(e)
S14
0.27
2.48
0.57
W03
21.08
19.92
0.12
HD 157246
17:25:23.7
−56:22:39.8
B1 Ib
H69
0.06
2.82
0.17
W03
20.68
19.23
0.07
HD 157978
17:26:19.0
+07:35:44.3
G0/K0 II: + A1
P98
0.64
3.72
2.42
W03
–
–
–
HD 158926
17:33:36.5
−37:06:13.8
B2 IV + DA.79
H13
0.10
–
–
19.23
12.70
0.00
HD 161056
17:43:47.0
−07:04:46.6
B1.5 V
L68
0.58
–
–
21.23
–
–
HD 164073
18:02:00.6
−48:48:37.7
B3III/IV
H78
0.22
2.96
0.62
V04
–
–
–
HD 164353
18:00:39.0
+02:55:53.6
B5 Ib
M50
0.10
–
–
21.00
20.26
0.27
HD 166937
18:13:45.8
−21:03:31.8
B8 Iab(e)
G94
0.22
–
–
–
–
–
HD 167264
18:15:12.9
−20:43:41.8
O9.7 Iab
S14
0.24
3.26
0.98
V04
21.15
20.28
0.21
HD 167838
18:17:37.7
−15:25:50.6
B5 Ia
M55
0.55
3.39
2.13
V04
–
–
–
HD 167971
18:18:05.9
−12:14:33.3
O8 Iaf(n) + O4/5
S11
1.01
3.44
3.54
V04
21.6
20.85
0.26
HD 169454
18:25:15.2
−13:58:42.3
B1 Ia
W76
1.03
3.37
3.64
V04
–
–
–
HD 170740
18:31:25.7
−10:47:45.0
B2 V
S56
0.45
3.01
1.51
V04
21.03
20.86
0.58
i
d Article number, page 18 of 30page.30 continued on next page Table A.1. Targets observed for EDIBLES. Article number, page 17 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Identifier
RA
Dec
Spectral type
Ref. A&A proofs: manuscript no. ediblesI_v2 Article number, page 19 of 30page.30 continued on next page E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 36982
05:35:09.8
−05:27:53.3
B1.5 Vp
S52
0.31
4.84
1.74
V04
21.57
–
–
HD 37020
05:35:15.8
−05:23:14.3
B0.5 V
L76
0.28
6.33
1.77
V04
21.48
–
–
HD 37021
05:35:16.1
−05:23:07.3
B0 V
G68
0.30
5.84
2.80
V04
21.68
–
–
HD 37023
05:35:17.2
−05:23:15.6
B1.5 Vp
L06
0.37
4.82
1.74
V04
–
–
–
HD 37128
05:36:12.8
−01:12:06.9
B0 Ia
M50
0.09
–
–
20.45
16.57
0.04
HD 38087
05:43:00.6
−02:18:45.4
B5 V
S71
0.28
4.93
1.48
V04
20.91
20.64
0.52
HD 39680
05:54:44.7
+13:51:16.9
O6 V:[n]pe var
S11
0.33
4.73
1.51
W03
21.3
19.53
0.03
HD 45314
06:27:15.8
+14:53:21.1
O9: npe
S11
0.13
4.42
1.90
V04
21.04
20.60
0.42
HD 49787
06:49:55.5
−05:30:47.5
B1 V:pe
M55
0.20
2.39
0.43
W03
–
–
–
HD 50820
06:54:42.0
−01:45:23.4
B3 IVe + K2 II
H82
0.72
4.39
3.38
W03
–
–
–
HD 61827
07:39:49.3
−32:34:42.2
B3 Iab
G77
0.78
2.98
2.68
W03
–
–
HD 114886
13:14:44.4
−63:34:51.8
O9 III + O9.5 III
S14
0.28
2.90
0.84
W03
21.34
20.23
0.13
HD 156201
17:17:45.5
−35:13:27.0
B0.5 Ia
G77
0.91
3.23
2.78
W03
–
–
–
HD 164740
18:03:40.0
−24:22:43.0
O7: V + sec. S14
0.90
5.03
4.48
V04
21.95
20.19
0.03
HD 164906
18:04:25.8
−24:23:08.3
B0 Ve
L06
0.44
4.57
2.06
V04
–
–
–
HD 165319
18:05:58.8
−14:11:53.0
O9.7 Ib
S11
0.78
3.25
2.70
W03
–
–
–
HD 168076
18:18:36.4
−13:48:02.0
O4 III(f)
S11
0.76
3.47
2.64
V04
21.65
20.68
0.18
HD 183143
19:27:26.6
+18:17:45.2
B7 Ia
M55
1.19
–
–
–
–
– Table A.2. Observations planned to complete the EDIBLES sample. Identifier
RA
Dec
Spectral type
Ref. continued on next page continued Article number, page 18 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey Identifier
RA
Dec
Spectral type
Ref. E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 170938
18:32:37.8
−15:42:05.9
B1 Ia
M55
0.97
3.34
3.47
W03
–
–
–
HD 171957
18:38:04.5
−14:00:16.9
B9 IV
C69
0.27
–
–
–
–
–
HD 172694
18:42:16.6
−16:25:41.0
B1: Vne
G68
0.34
5.04
1.86
W03
–
–
–
HD 180554
19:16:13.0
+21:23:25.4
B4 IV
L68
–
–
–
–
–
HD 184915
19:36:53.5
−07:01:38.9
B0.5 IIIn
L68
0.24
3.07
0.68
W03
20.85
20.31
0.37
HD 185418
19:38:27.5
+17:15:26.1
B0.5 V
M55
0.42
2.54
1.27
V04
21.19
20.71
0.40
HD 185859
19:40:28.3
+20:28:37.5
B0.5 Ia
L68
0.56
2.74
1.64
V04
21.23
–
–
HD 186745
19:45:24.3
+23:56:34.4
B8 Ia
M55
0.88
3.10
2.98
W03
–
–
–
HD 186841
19:45:54.0
+24:05:47.0
B0.5 I
M95
0.95
2.89
3.01
W03
–
–
–
HD 203532
21:33:54.6
−82:40:59.1
B3 IV
H69
0.30
3.37
0.94
V04
20.22
20.64
0.84
HD 303308
10:45:05.9
−59:40:06.4
O4.5 V((fc)) (SB1?)
S14
0.43
3.02
1.36
V04
21.41
20.34
0.15 Identifier
RA
Dec
Spectral type
Ref. E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 170938
18:32:37.8
−15:42:05.9
B1 Ia
M55
0.97
3.34
3.47
W03
–
–
–
HD 171957
18:38:04.5
−14:00:16.9
B9 IV
C69
0.27
–
–
–
–
–
HD 172694
18:42:16.6
−16:25:41.0
B1: Vne
G68
0.34
5.04
1.86
W03
–
–
–
HD 180554
19:16:13.0
+21:23:25.4
B4 IV
L68
–
–
–
–
–
HD 184915
19:36:53.5
−07:01:38.9
B0.5 IIIn
L68
0.24
3.07
0.68
W03
20.85
20.31
0.37
HD 185418
19:38:27.5
+17:15:26.1
B0.5 V
M55
0.42
2.54
1.27
V04
21.19
20.71
0.40
HD 185859
19:40:28.3
+20:28:37.5
B0.5 Ia
L68
0.56
2.74
1.64
V04
21.23
–
–
HD 186745
19:45:24.3
+23:56:34.4
B8 Ia
M55
0.88
3.10
2.98
W03
–
–
–
HD 186841
19:45:54.0
+24:05:47.0
B0.5 I
M95
0.95
2.89
3.01
W03
–
–
–
HD 203532
21:33:54.6
−82:40:59.1
B3 IV
H69
0.30
3.37
0.94
V04
20.22
20.64
0.84
HD 303308
10:45:05.9
−59:40:06.4
O4.5 V((fc)) (SB1?)
S14
0.43
3.02
1.36
V04
21.41
20.34
0.15 Table A.1. continued Article number, page 19 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 Observations planned to complete the EDIBLES sample. Identifier
RA
Dec
Spectral type
Ref. Article number, page 20 of 30page.30 Article number, page 21 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
Appendix B: Spectrum of HD170740 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey continued on next page E(B −V)
RV
AV
Ref
log N(H I)
log N(H2)
fH2
J2000
J2000
mag
mag
cm−2
cm−2
HD 36982
05:35:09.8
−05:27:53.3
B1.5 Vp
S52
0.31
4.84
1.74
V04
21.57
–
–
HD 37020
05:35:15.8
−05:23:14.3
B0.5 V
L76
0.28
6.33
1.77
V04
21.48
–
–
HD 37021
05:35:16.1
−05:23:07.3
B0 V
G68
0.30
5.84
2.80
V04
21.68
–
–
HD 37023
05:35:17.2
−05:23:15.6
B1.5 Vp
L06
0.37
4.82
1.74
V04
–
–
–
HD 37128
05:36:12.8
−01:12:06.9
B0 Ia
M50
0.09
–
–
20.45
16.57
0.04
HD 38087
05:43:00.6
−02:18:45.4
B5 V
S71
0.28
4.93
1.48
V04
20.91
20.64
0.52
HD 39680
05:54:44.7
+13:51:16.9
O6 V:[n]pe var
S11
0.33
4.73
1.51
W03
21.3
19.53
0.03
HD 45314
06:27:15.8
+14:53:21.1
O9: npe
S11
0.13
4.42
1.90
V04
21.04
20.60
0.42
HD 49787
06:49:55.5
−05:30:47.5
B1 V:pe
M55
0.20
2.39
0.43
W03
–
–
–
HD 50820
06:54:42.0
−01:45:23.4
B3 IVe + K2 II
H82
0.72
4.39
3.38
W03
–
–
–
HD 61827
07:39:49.3
−32:34:42.2
B3 Iab
G77
0.78
2.98
2.68
W03
–
–
HD 114886
13:14:44.4
−63:34:51.8
O9 III + O9.5 III
S14
0.28
2.90
0.84
W03
21.34
20.23
0.13
HD 156201
17:17:45.5
−35:13:27.0
B0.5 Ia
G77
0.91
3.23
2.78
W03
–
–
–
HD 164740
18:03:40.0
−24:22:43.0
O7: V + sec. S14
0.90
5.03
4.48
V04
21.95
20.19
0.03
HD 164906
18:04:25.8
−24:23:08.3
B0 Ve
L06
0.44
4.57
2.06
V04
–
–
–
HD 165319
18:05:58.8
−14:11:53.0
O9.7 Ib
S11
0.78
3.25
2.70
W03
–
–
–
HD 168076
18:18:36.4
−13:48:02.0
O4 III(f)
S11
0.76
3.47
2.64
V04
21.65
20.68
0.18
HD 183143
19:27:26.6
+18:17:45.2
B7 Ia
M55
1.19
–
–
–
–
– Appendix B: Spectrum of HD170740 Appendix B: Spectrum of HD170740 Article number, page 21 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 3190
3200
3210
3220
3230
3240
3250
3260
3270
3280
3290
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
TiII
TiII
3290
3300
3310
3320
3330
3340
3350
3360
3370
3380
3390
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
NaI
NaI
TiII
NH
3390
3400
3410
3420
3430
3440
3450
3460
3470
3480
3490
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
FeI
3490
3500
3510
3520
3530
3540
3550
3560
3570
3580
3590
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
CH+
OH+
12CN
12CN
3590
3600
3610
3620
3630
3640
3650
3660
3670
3680
3690
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
CH
3760
3770
3780
3790
3800
3810
3820
3830
3840
3850
Wavelength ( ˚A)
0.6
0.8
1.0
Normalised intensity
3860
3870
3880
3890
3900
3910
3920
3930
3940
3950
Wavelength ( ˚A)
0.8
1.0
1.2
Normalised intensity
CaII
FeI
CH
12CN
12CN
12CN
3960
3970
3980
3990
4000
4010
4020
4030
4040
4050
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
CH+
CH+
g. B.1. EDIBLES spectrum for HD 170740 (solid blue). The preliminary normalisation shown here is from spline fits to broad regions of ea
VES settings, and order-merging residuals can also be seen – future quantitative analysis will employ tailored, local rectifications of the da
ectronic transitions of interstellar atomic and di-atomic species are labeled. Above 4000 Å a generic model of the telluric transmission spectru
trieved from the ESO Sky Calculator) is shown in yellow/orange as well as the Jenniskens & Désert (1994) average ISM DIB spectrum (scal
ticle number, page 22 of 30page.30 3190
3200
3210
3220
3230
3240
3250
3260
3270
3280
3290
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
TiII
TiII TiII NaI
NaI NaI
NaI 3870
3880
3890
3900
3910
3920
3930
3940
3950
Wavelength ( ˚A)
CaII
CH
12CN
12CN
12CN Fig. B.1. EDIBLES spectrum for HD 170740 (solid blue). The preliminary normalisation shown here is from spline fits to broad regions of each
UVES settings, and order-merging residuals can also be seen – future quantitative analysis will employ tailored, local rectifications of the data. Electronic transitions of interstellar atomic and di-atomic species are labeled. Appendix B: Spectrum of HD170740 Above 4000 Å a generic model of the telluric transmission spectrum
(retrieved from the ESO Sky Calculator) is shown in yellow/orange as well as the Jenniskens & Désert (1994) average ISM DIB spectrum (scaled
to E(B −V) = 0.5 mag) in solid green. Article number, page 22 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
4060
4070
4080
4090
4100
4110
4120
4130
4140
4150
Wavelength ( ˚A)
0.6
0.8
1.0
Normalised intensity
4160
4170
4180
4190
4200
4210
4220
4230
4240
4250
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
CaI
CH+
4260
4270
4280
4290
4300
4310
4320
4330
4340
4350
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
CH
CH
CH
4360
4370
4380
4390
4400
4410
4420
4430
4440
4450
Wavelength ( ˚A)
0.6
0.8
1.0
Normalised intensity
4460
4470
4480
4490
4500
4510
4520
4530
4540
4550
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
4560
4570
4580
4590
4600
4610
4620
4630
4640
4650
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
4630
4640
4650
4660
4670
4680
4690
4700
4710
4720
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
4730
4740
4750
4760
4770
4780
4790
4800
4810
4820
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 23 of 30page Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 23 of 30page.30 A&A proofs: manuscript no. Appendix B: Spectrum of HD170740 ediblesI_v2 p
p
_
4830
4840
4850
4860
4870
4880
4890
4900
4910
4920
Wavelength ( ˚A)
0.90
0.95
1.00
Normalised intensity
4930
4940
4950
4960
4970
4980
4990
5000
5010
5020
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5030
5040
5050
5060
5070
5080
5090
5100
5110
5120
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5130
5140
5150
5160
5170
5180
5190
5200
5210
5220
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5230
5240
5250
5260
5270
5280
5290
5300
5310
5320
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5330
5340
5350
5360
5370
5380
5390
5400
5410
5420
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5430
5440
5450
5460
5470
5480
5490
5500
5510
5520
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5530
5540
5550
5560
5570
5580
5590
5600
5610
5620
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
ig. B.1. EDIBLES spectrum for HD 170740 (continued). Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 24 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
5670
5680
5690
5700
5710
5720
5730
5740
5750
5760
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
5770
5780
5790
5800
5810
5820
5830
5840
5850
5860
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
5870
5880
5890
5900
5910
5920
5930
5940
5950
5960
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
NaI
NaI
5970
5980
5990
6000
6010
6020
6030
6040
6050
6060
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
6070
6080
6090
6100
6110
6120
6130
6140
6150
6160
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
6170
6180
6190
6200
6210
6220
6230
6240
6250
6260
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
6270
6280
6290
6300
6310
6320
6330
6340
6350
6360
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
6370
6380
6390
6400
6410
6420
6430
6440
6450
6460
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 25 of 30pag Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 25 of 30page.30 Article number, page 25 of 30page.30 A&A proofs: manuscript no. Appendix B: Spectrum of HD170740 ediblesI_v2 p
p
6470
6480
6490
6500
6510
6520
6530
6540
6550
6560
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
6570
6580
6590
6600
6610
6620
6630
6640
6650
6660
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
6700
6710
6720
6730
6740
6750
6760
6770
6780
6790
Wavelength ( ˚A)
0.95
1.00
1.05
Normalised intensity
6800
6810
6820
6830
6840
6850
6860
6870
6880
6890
Wavelength ( ˚A)
0.95
1.00
Normalised intensity
6900
6910
6920
6930
6940
6950
6960
6970
6980
6990
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7000
7010
7020
7030
7040
7050
7060
7070
7080
7090
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
7100
7110
7120
7130
7140
7150
7160
7170
7180
7190
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7200
7210
7220
7230
7240
7250
7260
7270
7280
7290
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). 6900
6910
6920
6930
6940
6950
6960
6970
6980
6990
Wavelength ( ˚A)
7000
7010
7020
7030
7040
7050
7060
7070
7080
7090
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
7100
7110
7120
7130
7140
7150
7160
7170
7180
7190
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7200
7210
7220
7230
7240
7250
7260
7270
7280
7290
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 26 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey N.L.J. Appendix B: Spectrum of HD170740 Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
7300
7310
7320
7330
7340
7350
7360
7370
7380
7390
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7400
7410
7420
7430
7440
7450
7460
7470
7480
7490
Wavelength ( ˚A)
0.95
1.00
1.05
Normalised intensity
7500
7510
7520
7530
7540
7550
7560
7570
7580
7590
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7600
7610
7620
7630
7640
7650
7660
7670
7680
7690
Wavelength ( ˚A)
0.4
0.6
0.8
1.0
Normalised intensity
KI
7700
7710
7720
7730
7740
7750
7760
7770
7780
7790
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
KI
7800
7810
7820
7830
7840
7850
7860
7870
7880
7890
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
12CN
7900
7910
7920
7930
7940
7950
7960
7970
7980
7990
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
8000
8010
8020
8030
8040
8050
8060
8070
8080
8090
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 27 of 30pag 7300
7310
7320
7330
7340
7350
7360
7370
7380
7390
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7400
7410
7420
7430
7440
7450
7460
7470
7480
7490
Wavelength ( ˚A)
0.95
1.00
1.05
Normalised intensity
7500
7510
7520
7530
7540
7550
7560
7570
7580
7590
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
7600
7610
7620
7630
7640
7650
7660
7670
7680
7690
Wavelength ( ˚A)
0.4
0.6
0.8
1.0
Normalised intensity
KI
1 1 7300
7310
7320
7330
7340
7350
7360
7370
7380
7390
Wavelength ( ˚A)
0.9
1.0
Normalised intensity Wavelength ( ˚A)
7700
7710
7720
7730
7740
7750
7760
7770
7780
7790
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
KI
7800
7810
7820
7830
7840
7850
7860
7870
7880
7890
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
12CN
7900
7910
7920
7930
7940
7950
7960
7970
7980
7990
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
8000
8010
8020
8030
8040
8050
8060
8070
8080
8090
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 27 of 30pag Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 27 of 30page.30 Article number, page 27 of 30page.30 Article number, page 27 of 30page.30 A&A proofs: manuscript no. Appendix B: Spectrum of HD170740 ediblesI_v2 8100
8110
8120
8130
8140
8150
8160
8170
8180
8190
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
8200
8210
8220
8230
8240
8250
8260
8270
8280
8290
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
8300
8310
8320
8330
8340
8350
8360
8370
8380
8390
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
8400
8410
8420
8430
8440
8450
8460
8470
8480
8490
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
8500
8510
8520
8530
8540
8550
8560
8570
8580
8590
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
8700
8710
8720
8730
8740
8750
8760
8770
8780
8790
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
C2
C2
C2
C2
8800
8810
8820
8830
8840
8850
8860
8870
8880
8890
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
8890
8900
8910
8920
8930
8940
8950
8960
8970
8980
8990
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). 8100
8110
8120
8130
8140
8150
8160
8170
8180
8190
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 28 of 30page.30 N.L.J. Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey
8990
9000
9010
9020
9030
9040
9050
9060
9070
9080
9090
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
9100
9110
9120
9130
9140
9150
9160
9170
9180
9190
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
12CN
9200
9210
9220
9230
9240
9250
9260
9270
9280
9290
Wavelength ( ˚A)
0.7
0.8
0.9
1.0
Normalised intensity
9290
9300
9310
9320
9330
9340
9350
9360
9370
9380
9390
Wavelength ( ˚A)
0.6
0.8
1.0
Normalised intensity
9390
9400
9410
9420
9430
9440
9450
9460
9470
9480
9490
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
9500
9510
9520
9530
9540
9550
9560
9570
9580
9590
Wavelength ( ˚A)
0.7
0.8
0.9
1.0
Normalised intensity
9600
9610
9620
9630
9640
9650
9660
9670
9680
9690
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
9690
9700
9710
9720
9730
9740
9750
9760
9770
9780
9790
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 29 of 30page N.L.J. Article number, page 30 of 30page.30 Appendix B: Spectrum of HD170740 Cox et al.: The ESO Diffuse Interstellar Bands Large Exploration Survey 8990
9000
9010
9020
9030
9040
9050
9060
9070
9080
9090
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity 9600
9610
9620
9630
9640
9650
9660
9670
9680
9690
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Article number, page 29 of 30page.30 Article number, page 29 of 30page.30 A&A proofs: manuscript no. ediblesI_v2 9800
9810
9820
9830
9840
9850
9860
9870
9880
9890
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity
9890
9900
9910
9920
9930
9940
9950
9960
9970
9980
9990
Wavelength ( ˚A)
0.9
1.0
Normalised intensity
9990
10000
10010
10020
10030
10040
10050
10060
10070
10080
10090
Wavelength ( ˚A)
0.7
0.8
0.9
1.0
Normalised intensity
10100
10110
10120
10130
10140
10150
10160
10170
10180
10190
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
C2
10190
10200
10210
10220
10230
10240
10250
10260
10270
10280
10290
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
10290
10300
10310
10320
10330
10340
10350
10360
10370
10380
10390
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
g. B.1. EDIBLES spectrum for HD 170740 (continued). 9800
9810
9820
9830
9840
9850
9860
9870
9880
9890
Wavelength ( ˚A)
0.8
0.9
1.0
Normalised intensity 9990
10000
10010
10020
10030
10040
10050
10060
10070
10080
10090
Wavelength ( ˚A)
10100
10110
10120
10130
10140
10150
10160
10170
10180
10190
Wavelength ( ˚A)
0.9
1.0
1.1
Normalised intensity
C2
10190
10200
10210
10220
10230
10240
10250
10260
10270
10280
10290
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
10290
10300
10310
10320
10330
10340
10350
10360
10370
10380
10390
Wavelength ( ˚A)
0.8
0.9
1.0
1.1
Normalised intensity
Fig. B.1. EDIBLES spectrum for HD 170740 (continued). Fig. B.1. EDIBLES spectrum for HD 170740 (continued).
|
https://openalex.org/W3033320577
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https://europepmc.org/articles/pmc7321436?pdf=render
|
English
| null |
Experimental Investigation of Material Properties and Self-Healing Ability in a Blended Cement Mortar with Blast Furnace Slag
|
Materials
| 2,020
|
cc-by
| 12,686
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Received: 7 May 2020; Accepted: 3 June 2020; Published: 4 June 2020 Abstract: This paper presents the results of an experimental investigation on the material properties
and self-healing ability of a blended cement mortar incorporating blast furnace slag (BFS). The effect of
different types and Blaine fineness of BFS on the material properties and self-healing was investigated. Thirteen cement mixtures with BFS of different types and degrees of Blaine fineness are tested to
evaluate the mechanical properties, namely compressive strength, bending strength, freeze–thaw,
and accelerated carbonation. The pore structure is examined by means of mercury intrusion
porosimetry. Seven blended mortar mixtures incorporating BFS for cement are used to evaluate
the mechanical properties after applying freeze–thaw cycles until the relative dynamic modulus
of elasticity reached 60%. The experimental results reveal that incorporating BFS improves the
mechanical properties and self-healing ability. In the investigation of self-healing, smaller particle
and high replacement ratios of BFS contribute to increasing the relative dynamic modulus of elasticity
and decreasing the carbonation coefficient in the mortar after re-water curing. Moreover, BFS’s larger
particles and high replacement ratio are found to provide better self-healing ability. A regression
equation is created to predict the relative dynamic modulus of elasticity in mortar considering the
Blaine fineness, BFS replacement ratio, and curing conditions. Keywords: frost resistance; self-healing ability; blast furnace slag; freeze–thaw cycles; relative
dynamic modulus of elasticity; carbonation coefficient materials materials Experimental Investigation of Material Properties and
Self-Healing Ability in a Blended Cement Mortar
with Blast Furnace Slag Seunghyun Na 1, Wenyan Zhang 2, Madoka Taniguchi 3, Nguyen Xuan Quy 4,5 and
Y ki
H
4 * Seunghyun Na 1, Wenyan Zhang 2, Madoka Taniguchi 3, Nguyen Xuan Quy 4,5 and
Yukio Hama 4,* 1
Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan;
nash1122@naver.com 1
Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan;
nash1122@naver.com 2
School of Material Science and Engineering, Henan Polytechnic University, Jiaozuo 454-000, China;
zhangwy@hpu.edu.cn 2
School of Material Science and Engineering, Henan Polytechnic University, Jiaozuo 454-000, China;
zhangwy@hpu.edu.cn
3 3
Environmental Engineering Division, Northern Regional Building Research Institute, Hokkaido Resear
Organization, Asahikawa, Hokkaido 078-8801, Japan; madoka@hro.or.jp 4
College of Environmental Technology, Graduate School of Engineering, Muroran Institute of Technology,
Muroran 050-8585, Japan; quynx@hau.edu.vn 4
College of Environmental Technology, Graduate School of Engineering, Muroran Institute of Technolog
Muroran 050-8585, Japan; quynx@hau.edu.vn J p
q y
5
Department of Civil Engineering, Hanoi Architectural University, Hanoi 100000, Vietnam 5
Department of Civil Engineering, Hanoi Architectural University, Hanoi 100000, Vietnam
*
Correspondence: hama@mmm.muroran-it.ac.jp 5
Department of Civil Engineering, Hanoi Architectural University, Hanoi 100000, Vietnam 5
Department of Civil Engineering, Hanoi Architectural University, Hanoi 100000, Vietnam
*
Correspondence: hama@mmm.muroran-it.ac.jp p
g
g,
y,
,
*
Correspondence: hama@mmm.muroran-it.ac.jp *
Correspondence: hama@mmm.muroran-it.ac.jp
Received: 7 May 2020; Accepted: 3 June 2020; Published: 4 June 2020 1. Introduction It reported that the fly ash
has the long-term pozzolanic reaction and fills microcracks due to hydration products of fly ash [9]. Ahn et al. investigated the self-healing behavior of cement-based materials with mineral admixtures
and reported that a crack of 0.15 mm was self-healed after three days of re-curing [8]. It was found that
the phenomenon is due to the swelling effect, expansion effect, and re-crystallization of used materials. This study focused on the blocking water leakage. Sisomphon and Copuroglu reported that ettringite
formation generates in the cracks [18]. In addition, the researchers have studied the self-healing
ability of cement-based materials that are damaged by freeze–thaw. Studies have been performed to
overcome the abovementioned problems and to investigate self-healing of cracks in cement mixtures
incorporating fly ash [9–11]. It was shown to be possible to heal micro cracks in a cement mixture after
frost damage. Incorporating fly ash in the mortar was reported to lead to not only an increase in the
relative dynamic modulus of elasticity but also a decrease in the carbonation coefficient, and it was
reported that cracks occurring after frost damage are filled by hydration products of the fly ash [12]. Therefore, it was found that the addition of fly ash seemed to be beneficial for filling cracks. On the other hand, research on the effective use of BFS in cement-based materials have been
performed to reduce the cracks [15]. In the self-healing of the addition of BFS on self-healing behavior,
it can be concluded that self-healing after damage depends on the high percentage of cementitious
material and smaller micro cracks that can be filled by unhydrated cement and BFS [14]. The main
hydration products were C–S–H gels from the hydration of BFS, thus high recovery with respect
to compressive strength can be achieved [17]. The use of BFS as a self-healing material has many
advantages such as reducing carbon dioxide emissions and reducing manufacturing cost, compared to
an expansive agent. However, there is a lack of information about the self-healing ability of blended
cement mixtures for different types and Blaine fineness of BFS and cements by taking into consideration
freeze–thaw cracks. Therefore, the purpose of this study is to investigate the material properties and self-healing ability
of blended cement mortar incorporating BFS of different types and degrees of Blaine fineness. 1. Introduction Concrete has been widely used for a long time in the civil engineering and construction fields,
and it has many benefits, such as high durability, low cost, and high compressive strength. However,
although there are many advantages, the damage of a concrete structure is inevitable. In cold
environments, the deterioration of a concrete structure depends on not only the moisture content but
also the weather conditions, such as the freezing temperature and the number of freeze–thaw cycles in
the literature [1,2]. Kamada pointed out that the freeze–thaw resistance depends on pore structure in
the concrete, pore volume of 40–2000 nm in diameter [3]. The deterioration of concrete by frost damage www.mdpi.com/journal/materials Materials 2020, 13, 2564; doi:10.3390/ma13112564 www.mdpi.com/journal/materials Materials 2020, 13, 2564 2 of 17 may decrease the compressive strength, bending strength, and carbonation resistance. Moreover,
the decreasing performance due to micro cracks generated by freeze–thaw cycles reduces the lifetime of
the concrete structure. Therefore, it is necessary to develop a technique capable of healing such cracks. y
p
q
p
g
Up to now, many techniques have proposed to increase frost resistance in concrete. It is generally
known that mixture design optimization for the reduction of water content to form much denser
microstructure, increasing the air content, and decreasing the air void spacing factor are some of the
successful techniques [4]. According to recent studies, Fujii and Ayano investigated the effect of the
addition of blast furnace slag (BFS) on the frost resistance of concrete without air entrain agent and
reported that freeze–thaw resistance of concrete increases when BFS and fly ash were used with fine
aggregate [5]. However, the effect of the type of BFS on the freeze–thaw resistance of cement-based
materials was not examined. Consequently, there has been research into self-healing abilities as a promising solution to the
issue of damaged concrete. It has been reported that self-healing was classified into natural healing,
autonomic healing, activated healing according to the Japan Concrete Institute [6]. Natural healing is
re-hydration of residual unhydrated cement with low water to cement ratio or re-hydration cracks
found in hydraulic structures. Furthermore, various materials for autonomic healing have been used, such as expansive
agent [7,8], fly ash [9–11], bacteria [12,13], blast-furnace slag (BFS) [14–17], chemical admixtures [8],
and engineered cementitious composite with fly ash, described by [6]. 1. Introduction Tests were
carried out on the mortar samples to evaluate the mechanical properties, namely the compressive
strength, bending strength, freeze–thaw, and accelerated carbonation, and the pore structure was
investigated. Seven blended mortar mixtures incorporating BFS for cement were used to evaluate the
mechanical properties after applying freeze–thaw cycles until the relative dynamic modulus of elasticity
reached 60%. Then, a regression equation was created to predict the relative dynamic modulus of
elasticity in mortar considering the Blaine fineness, BFS replacement ratio, and curing conditions. 3 of 17 Materials 2020, 13, 2564 2.1. Materials BFS and cement were used as binder materials in this study. Table 1 shows the chemical composition
and physical properties of cement and BFS. The experiments used ordinary Portland cement (Nippon
steel cement corporation, Muroran, Japan) N; specific gravity: 3.16 g/cm3; Blaine fineness: 3250 cm2/g). Three types of BFS were used, with fineness values of 3180, 4030, and 7300 cm2/g and densities
of 2.91, 2.91, and 2.90 g/cm3, respectively. Natural land sand was used as the fine aggregate. The surface-dry density and the water absorption rates of the sand were 2.65 g/cm3 and 0.42%,
respectively. The fineness modulus, surface-dry density, and the water absorption of the sand were
2.74, 2.65 g/cm3, and 0.42%, respectively. Table 1. Chemical composition and physical properties of ordinary Portland cement and blast furnace
slag (BFS). Sample
Specific
Gravity (g/cm3)
Blaine
(cm2/g)
Chemical Composition (%)
SiO2
Al2O3
Fe2O3
CaO
MgO
K2O
Na2O
SO3
N
3.16
3250
21.5
5.4
2.9
64.3
1.9
0.4
0.2
1.8
BFS3000
2.91
3180
33.8
15.1
0.9
40.8
6.5
–
–
–
BFS4000
2.90
4030
34.1
16.0
0.4
42.1
6.2
–
–
–
BFS8000
2.90
7300
33.6
16.2
0.6
42.1
5.3
–
–
– 1. Chemical composition and physical properties of ordinary Portland cement and blast furnace
S) Table 1. Chemical composition and physical properties of ordinary Portland cement and blast furnace
slag (BFS). 2.2. Mixture Proportions and Specimen Preparation 2.2. Mixture Proportions and Specimen Preparation The mixture proportions of the mortars are shown in Table 2. The three types of BFS were added
to the mortar at replacement levels of 45% and 70% by weight of cement and 20% and 45% by weight
of sand. The water-to-cement ratio (W/C = water/cement) was 0.55 and the water-to-binder ratios
(W/B = water/(cement + BFS)) were 0.38, 0.45, and 0.55 for the three types, respectively. The flow value
and air content of all the investigated mortars with and without BFS were examined in accordance
with JIS R 5201 and JIS A 1171 [19,20]. Then, they were mixed and cast as 40 mm × 40 mm × 160 mm
prisms to form the mortar test specimens. After 1 day, the samples were de-molded and cured at 20 ◦C
in water for 28 days. 4 of 17 Materials 2020, 13, 2564 Table 2. Mixture proportions of all the investigated mortars. Number
Symbol
W/C
W/B
BFS Blaine
Fineness (cm2/g)
BFS Replacement
Unit Mass (kg/m3)
Flow Value
(mm)
Air Content
(%)
Portions
wt %
W
OPC
BFS
S
1
N
0.55
0.55
-
-
-
342
621
0
1242
258.5
1.6
2
3S45C
0.55
0.55
3000
Cement
45
342
342
280
1242
259.0
1.8
3
3S70C
0.55
0.55
3000
Cement
70
342
186
435
1242
275.0
2.1
4
4S45C
0.55
0.55
4000
Cement
45
342
342
280
1242
237.5
1.4
5
4S70C
0.55
0.55
4000
Cement
70
342
186
435
1242
259.5
1.2
6
8S45C
0.55
0.55
8000
Cement
45
342
342
280
1242
219.5
0.6
7
8S70C
0.55
0.55
8000
Cement
70
342
186
435
1242
224.5
0.3
8
3S20S
0.55
0.45
3000
Sand
20
342
621
124
1118
220.5
0.9
9
3S45S
0.55
0.38
3000
Sand
45
342
621
280
963
219.5
5.0
10
4S20S
0.55
0.45
4000
Sand
20
342
621
124
1118
187.5
0.9
11
4S45S
0.55
0.38
4000
Sand
45
342
621
280
963
187.5
1.1
12
8S20S
0.55
0.45
8000
Sand
20
342
621
124
1118
211.0
1.7
13
8S45S
0.55
0.38
8000
Sand
45
342
621
280
963
154.5
0.9 Table 2. Mixture proportions of all the investigated mortars. 5 of 17 Materials 2020, 13, 2564 2.3.1. Compressive Strength and Bending Strength Tests 2.3.1. Compressive Strength and Bending Strength Tests The compressive strength and bending strength of the mortar test specimens were tested in
accordance with JIS R 5201 [19]. The compressive strength and bending strength of the mortar test specimens were tested in
accordance with JIS R 5201 [19]. 2.3. Testing Methods Mixtures
Bending
Strength
Compressive
Strength
Accelerated
Carbonation
Freezing/Thawing
Mercury Intrusion
Porosimetry (MIP)
N
√
√
√
√
√
3S45C
√
√
√
√
√
3S70C
√
√
√
√
4S45C
√
√
√
√
√
4S70C
√
√
√
√
8S45C
√
√
√
√
8S70C
√
√
√
√
√
3S20S
√
√
√
√
3S45S
√
√
√
4S20S
√
√
√
√
4S45S
√
√
√
8S20S
√
√
√
√
8S45S
√
√
√
Table 4 Test items for measurement of self healing properties Table 3. Test items for measurement of materials properties. Table 3. Test items for measurement of materials properties. Table 4. Test items for measurement of self-healing properties. Table 4. Test items for measurement of self-healing properties. g p
p
Mixtures
Bending Strength
Compressive
Strength
Accelerated
Carbonation
Relative Dynamic
Modulus of Elasticity
N
√
√
√
√
3S45C
√
√
√
√
3S70C
√
√
√
√
4S45C
√
√
√
√
4S70C
√
√
√
√
8S45C
√
√
√
√
8S70C
√
√
√
√ 2.3. Testing Methods Tables 3 and 4 list the test items for all the samples used in this study. As shown in Table 3,
the BFS-blended mortar samples were examined for their material properties, namely the compressive
strength, bending strength, accelerated carbonation, and freeze–thaw. Two replicates of each of the
mixtures for tests. Then, as shown in Table 4, seven mixtures were tested to evaluate the self-healing
ability of the mortars incorporating BFS. Details of the test methods are presented below. Table 3. Test items for measurement of materials properties. Mixtures
Bending
Strength
Compressive
Strength
Accelerated
Carbonation
Freezing/Thawing
Mercury Intrusion
Porosimetry (MIP)
N
√
√
√
√
√
3S45C
√
√
√
√
√
3S70C
√
√
√
√
4S45C
√
√
√
√
√
4S70C
√
√
√
√
8S45C
√
√
√
√
8S70C
√
√
√
√
√
3S20S
√
√
√
√
3S45S
√
√
√
4S20S
√
√
√
√
4S45S
√
√
√
8S20S
√
√
√
√
8S45S
√
√
√
T bl 4 T
t it
f
t f
lf h
li
ti Table 3. Test items for measurement of materials properties. Mixtures
Bending
Strength
Compressive
Strength
Accelerated
Carbonation
Freezing/Thawing
Mercury Intrusion
Porosimetry (MIP)
N
√
√
√
√
√
3S45C
√
√
√
√
√
3S70C
√
√
√
√
4S45C
√
√
√
√
√
4S70C
√
√
√
√
8S45C
√
√
√
√
8S70C
√
√
√
√
√
3S20S
√
√
√
√
3S45S
√
√
√
4S20S
√
√
√
√
4S45S
√
√
√
8S20S
√
√
√
√
8S45S
√
√
√
Table 4. Test items for measurement of self-healing properties. Mixtures
Bending Strength
Compressive
Strength
Accelerated
Carbonation
Relative Dynamic
Modulus of Elasticity
N
√
√
√
√
3S45C
√
√
√
√
3S70C
√
√
√
√
4S45C
√
√
√
√
4S70C
√
√
√
√
8S45C
√
√
√
√
8S70C
√
√
√
√
2.3.1. Compressive Strength and Bending Strength Tests
The compressive strength and bending strength of the mortar test specimens were tested in
accordance with JIS R 5201 [19]. 2.3.2. Accelerated Carbonation Depth Table 3. Test items for measurement of materials properties. 2.3.2. Accelerated Carbonation Depth The accelerated carbonation test was conducted according to JIS A 1153. All of the mortar samples
were cured to water during 28 days in water before testing [21]. Then, the samples were dried in a control
chamber at 20 ◦C and 60% relative humidity for 28 days. Afterward, the samples were dried and the
tests conducted with 5% carbon dioxide gas concentration at 1, 4, and 13 weeks, when the carbonation
depth was measured using the phenolphthalein test. The carbonation coefficient was calculated from
the carbonation depths measured from the investigated samples using the following equation: x = k
√
t
(1) (1) where x is the depth of carbonation (mm), t is the duration of carbonation (weeks), and k is the
carbonation coefficient (mm·week−0.5). where x is the depth of carbonation (mm), t is the duration of carbonation (weeks), and k is the
carbonation coefficient (mm·week−0.5). Materials 2020, 13, 2564 6 of 17 2.3.4. Mercury Intrusion Porosimetry The pore structure was investigated via mercury intrusion porosimetry (MIP) by means of an
Autopore Master 33 porosimeter (Quantachrome instruments, Anton paar Quanta Tec. Ltd., Tokyo,
Japan) to evaluate the pore size distribution in the mortar samples. Pressures ranged 0–220 MPa. The surface tension and the density of mercury were 0.480 N/m and 13.546 g/mL, respectively. The contact angle of mercury was 140◦. The mortar prisms after 28 days in water at 20◦were cut into
5-mm-wide cube-shaped samples and then dried to stop the cement hydration reaction with acetone,
according to D-drying pretreatment [24,25]. 2.3.3. Freeze–Thaw Test Freeze–thaw testing was performed for 300 cycles in accordance with JIS A 1148 [22]. The relative
dynamic modulus of elasticity was determined by measuring the resonance frequency and was
calculated as the percentage fraction of the square transverse frequency after the freeze–thaw test
to the square transverse frequency before the freeze–thaw test, in accordance with JIS A 1127 [23]
and the durability factor was calculated from the relative dynamic modulus of elasticity, using
following equation. Pn =
fn2
f0
2
× 100%
(2) (2) where Pn is the relative dynamic modulus of elasticity (%), fn is the fundamental transverse frequency
at n ycle (Hz), and f 0 is the fundamental transverse frequency at 0 cycles (Hz). bility factor was calculated from the results of freeze/thaw test of all investigated samples,
ation (3): The durability factor was calculated from the results of freeze/thaw test of all investigated samples,
as shown Equation (3): DF = P × N
M
(3) (3) where DF is the durability factor of the test specimen; P is the relative dynamic modulus of elasticity at
cycles (%); N is the number of cycles at which P reaches the specified minimum value less than 60% or
the relative dynamic modulus of elasticity after 300 cycles, and M is the relative dynamic modulus of
elasticity after 300 cycles. 3.1.1. Bending Strength Figure 1a,b show the results of bending strength and compressive strength tests for BFS-blended
cement mortar samples with different Blaine fineness values. The BFS replacement ratios were set at
45 wt % and 70 wt % for cement, and 20 wt % and 45 wt % for sand in the mortar. Thirteen mixtures
were prepared with the proportions summarized in Table 2. The bending strength of mortar sample N
was approximately 7 MPa. For BFS as a replacement for cement, as shown in Figure 1a, it can be seen
that mortar samples 3C45 and 3C70 produced with a low fineness of 3000 cm2/g have a lower bending
strength than that of sample N, which is the mortar without BFS. However, the bending strengths
of mortar samples 4C70C and 8C70C are higher than those of the other mortar samples, implying
that high replacement ratios and BFS fineness values had a positive effect on the bending strength of
the mortar. For BFS as a replacement for sand, as shown in Figure 1b, the bending strengths of all the mortar
samples made with BFS were higher than that of mortar N. These BFS-blended mortars have bending
strengths over 8 MPa, even though the BFS fineness values and replacement ratios were different. This indicates that incorporating BFS for sand can contribute to the increase in the bending strength
in mortar. (a)
(b)
Figure 1. Bending strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. 6.9
8.0
8.3
8.5
8.5
8.5
8.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Bending strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡
Figure 1. Bending strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. (a) (b)
6.9
8.0
8.3
8.5
8.5
8.5
8.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Bending strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡ (a) (b) (a) Figure 1. Bending strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. Figure 1. 3.1.1. Bending Strength Bending strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. 3. Results and Discussion 3.1. Materials Properties 2.4. Investigation of Self-Healing Ability To generate micro cracks in the samples, the mortar samples after 28 days of water curing
underwent freeze–thaw testing until the relative dynamic modulus of elasticity was 60%. The relative
dynamic modulus of elasticity was determined by measuring the resonance frequency and calculated
from the result. After deterioration of the samples, different curing conditions were applied: 20 ◦C
for 1 week to reflect curing conditions over 1 year and 40 ◦C for 4 weeks in water to investigate the
self-healing ability, as reported in a previous study [9]. Then, tests to measure the physical properties,
namely the resonance frequency, bending strength, compressive strength, accelerated carbonation
depth, of the samples at different testing ages were performed. Moreover, accelerated carbonation
samples after re-curing was dried to in a control chamber at 20 ◦C and 60% relative humidity for
28 days. Then, carbonation depths were measured according to Section 2.3.2. Moreover, general
regression analysis was performed to predict the relative dynamic modulus of elasticity of the mortar
samples, considering Blaine fineness, replacement ratio, and water curing conditions, using Minitab
software (Version 17, Minitab Inc., Seoul, Korea). 7 of 17 Materials 2020, 13, 2564 3 1 3 Mercury Intrusion Porosimetry
3.1.3. Mercury Intrusion Porosimetry
3.1.3. Mercury Intrusion Porosimetry 3.1.3. Mercury Intrusion Porosimetry
The MIP results are presented in Figure 3. The total pore volumes of mortars N, 3C45, 4C45, and
8C45 were found to be 0.0576, 0.0594, 0.0567, and 0.0398 cc/g, respectively. It can be seen from the
figure that the pore volume of mortar 8C45 was smaller than that of the other mortar samples, which
is due to the densification of the smaller fineness and the hydration of BFS that can fill the pore
volume in the BFS-blended mortar sample. Therefore, as mentioned above for Figure 2a, the
increasing compressive strength of 8C45 in comparison to mortar N is due to the smaller pore volume
in the BFS-blended mortar sample, which is similar to the results of a previous study [27]. The MIP results are presented in Figure 3. The total pore volumes of mortars N, 3C45, 4C45,
and 8C45 were found to be 0.0576, 0.0594, 0.0567, and 0.0398 cc/g, respectively. It can be seen from
the figure that the pore volume of mortar 8C45 was smaller than that of the other mortar samples,
which is due to the densification of the smaller fineness and the hydration of BFS that can fill the pore
volume in the BFS-blended mortar sample. Therefore, as mentioned above for Figure 2a, the increasing
compressive strength of 8C45 in comparison to mortar N is due to the smaller pore volume in the
BFS-blended mortar sample, which is similar to the results of a previous study [27]. The MIP results are presented in Figure 3. The total pore volumes of mortars N, 3C45, 4C45, and
8C45 were found to be 0.0576, 0.0594, 0.0567, and 0.0398 cc/g, respectively. It can be seen from the
figure that the pore volume of mortar 8C45 was smaller than that of the other mortar samples, which
is due to the densification of the smaller fineness and the hydration of BFS that can fill the pore
volume in the BFS-blended mortar sample. Therefore, as mentioned above for Figure 2a, the
increasing compressive strength of 8C45 in comparison to mortar N is due to the smaller pore volume
in the BFS-blended mortar sample, which is similar to the results of a previous study [27]. Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for
BFS by means of mercury intrusion porosimetry. 3.1.2. Compressive Strength Figure 2a,b present the results of compressive strength tests for the mortar samples. As shown in
Figure 2a, the compressive strength of mortar samples 8C45 and 8C70 with high Blaine fineness values
of 8000 cm2/g was considerably higher than that of mortar N, implying that the addition of BFS as
a replacement for cement with high Blaine fineness is effective for the development of compressive
strength in BFS-blended mortar. As shown in Figure 2b, when using BFS as a replacement for sand,
all mortar samples with BFS were found to have higher compressive strength compared to mortar N,
which also agrees with the results of the bending strength test in Section 3.1.1. Moreover, in the case of
BFS as a replacement for sand, a low W/C may cause an increase of compressive strength, owing to the
decrease of the total pore volume with respect to the MIP results [26]. Materials 2020, 13, 2564
compressive stren
[26]
80.0
a) 8 of 17
s 72.8 8 of 17
s 72.8 (a)
(b)
Figure 2. Compressive strengths of mixtures made with different BFS replacement ratios: (a) BFS
as a replacement for cement and (b) BFS as a replacement for sand. 40.5 41.9
34.1
43.0
41.5
54.6
56.0
0.0
20.0
40.0
60.0
80.0
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Compressive strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡
40.5
52.5
64.4
59.1
69.2
61.4
72.8
0.0
20.0
40.0
60.0
80.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Compressive strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡
Figure 2. Compressive strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. (a)
(b)
Figure 2. Compressive strengths of mixtures made with different BFS replacement ratios: (a) BFS
as a replacement for cement and (b) BFS as a replacement for sand. 40.5 41.9
34.1
43.0
41.5
0.0
20.0
40.0
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Compressive strength
3000
4000
8000 cm2/g
BFS fineness➡
40.5
0.0
20.0
40.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Compressive strength
3000
4000
8000 cm2/
BFS fineness➡ (a)
40.5 41.9
34.1
43.0
41.5
54.6
56.0
0.0
20.0
40.0
60.0
80.0
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Compressive strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡
(a)
Figure 2. 3.1.2. Compressive Strength Compressive strengths of mixtures ma
l
t f
t
d (b) BFS
40.5 41.9
34.1
43.0
41.5
0.0
20.0
40.0
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Compressive strength
3000
4000
8000 cm2/g
BFS fineness➡ (b)
40.5
52.5
64.4
59.1
69.2
61.4
72.8
0.0
20.0
40.0
60.0
80.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Compressive strength (MPa)
3000
4000
8000 cm2/g
BFS fineness➡
(b)
e with different BFS replacement ratios: (a) BFS
t f
d
40.5
0.0
20.0
40.0
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S4
Compressive strength
3000
4000
8000 cm
BFS fineness➡ (a)
ve str (b)
repla Figure 2. Compressive strengths of mixtures made with different BFS replacement ratios: (a) BFS
as a replacement for cement and (b) BFS as a replacement for sand. Figure 2. Compressive strengths of mixtures made with different BFS replacement ratios: (a) BFS as a
replacement for cement and (b) BFS as a replacement for sand. as a replacement for cement and (b) BFS as a replacement for sand. 3 1 3 Mercury Intrusion Porosimetry
3.1.3. Mercury Intrusion Porosimetry
3.1.3. Mercury Intrusion Porosimetry 0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
1
10
100
1,000
10,000
100,000
Cumulative pore volume (cc/g)
Pore diameter (nm)
N
3C45C
4C45C
8C45C
Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for
BFS by means of mercury intrusion porosimetry. 0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
1
10
100
1,000
10,000
100,000
Cumulative pore volume (cc/g)
Pore diameter (nm)
N
3C45C
4C45C
8C45C
Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for BFS
by means of mercury intrusion porosimetry. Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for
BFS by means of mercury intrusion porosimetry. Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for
BFS by means of mercury intrusion porosimetry
Figure 3. Cumulative pore volume of BFS-blended mortar with different Blaine fineness values for BFS
by means of mercury intrusion porosimetry. 3.1.4. Freeze-Thaw Tes
y
3.1.4. Freeze-Thaw Test Materials 2020, 13, x FOR PEER REVIEW
9 of 16 Relative dynamic modulus of elasticity (%) 0
20
40
60
80
100
120
0
100
200
300
N
3S45C
3S70C
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
60% 0
20
40
60
80
100
120
0
100
200
300
N
4S45C
4S70C
Freeze / thaw cycle (cy.)
Relative dynamic modulus of elasticity (%)
60% 0
20
40
60
80
100
120
0
100
200
300
N
8S45C
8S70C
Freeze / thaw cycle (cy.)
Relative dynamic modulus of elasticity (%)
60% Relative dynamic modulus of elasticity (%) (a)
(b)
0
20
40
60
80
100
120
0
10
N
0
20
40
60
80
100
120
0
100
200
300
N
4S20S
4S45S
0
20
40
60
80
100
120
0
100
200
300
N
3S20S
3S45S
Relative dynamic modulus of elasticity (%)
Relative dynamic modulus of elasticity (%)
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
Freeze / thaw cycle (cy.)
Freez
60%
60% (a) (a)
(b)
0
20
40
60
80
100
120
0
100
200
300
N
4S20S
4S45S
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
60% 0
20
40
60
80
100
120
0
100
200
300
N
3S20S
3S45S
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy )
60% Relative dynamic modulus of elasticity (%) 0
20
40
60
80
100
120
0
100
200
300
N
8S20S
8S45S
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
60% (b) Figure 4. Relative dynamic modulus of elasticity for different mortar mixtures with or without BFS
during freeze–thaw cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. Figure 4. Relative dynamic modulus of elasticity for different mortar mixtures with or without BFS
during freeze–thaw cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. Materials 2020, 13, x FOR PEER REVIEW
9 of 16 (b)
3.9
60.7
90.3
34.7
97.5
17.4
98.9
0
20
40
60
80
100
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Durability factor
3000
4000
8000 cm2/g
BFS fineness➡
g (a)
3.9
3.4
2.4
7.7
4.5
11
11
0
20
40
60
80
100
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Durability factor
3000
4000
8000 cm2/g
BFS fineness➡ (a)
(b)
Figure 5. 3.1.4. Freeze-Thaw Tes
y
3.1.4. Freeze-Thaw Test Moreover, with increasing BFS replacement ratio, a high relative Materials 2020, 13, 2564
before 100 cycles
samples. 9 of 17
r dynamic modulus of elasticity of 90–100% at 300 cycles was observed for the mortar samples, especially
for 3S45S, 4S45S, and 8S45S. These observations indicate that an increase in BFS replacement for sand
yielded significantly better frost resistance performance in BFS-blended mortar. This result is similar to
the study by Ayano and Fugii [5]. samples with 20 or 45 wt % of BFS replacement for sand does not decrease as the number of freeze–
thaw cycles increases, unlike mortar N. Moreover, with increasing BFS replacement ratio, a high
relative dynamic modulus of elasticity of 90–100% at 300 cycles was observed for the mortar samples,
especially for 3S45S, 4S45S, and 8S45S. These observations indicate that an increase in BFS
replacement for sand yielded significantly better frost resistance performance in BFS-blended mortar. Moreover, in the case of 45% of BFS with sand, effect of Blaine fineness of BFS on frost resistance
was not significant, which implies that frost resistance in the BFS blended mortar depends on the ratios
of BFS replacement for sand. This result is similar to the study by Ayano and Fugii [5]. Moreover, in the case of 45% of BFS with sand, effect of Blaine fineness of BFS on frost resistance
was not significant, which implies that frost resistance in the BFS blended mortar depends on the
ti
f BFS
l
t f
d The durability factor was calculated from the results of the relative dynamic modulus of elasticity
of each mixture and the results are shown in Figure 5. The durability factor of the sample with BFS
as a replacement for sand is significantly higher than that of the other mortar samples. Furthermore,
in the case of a BFS replacement ratio of 45 wt %, the durability factor was 90–100, implying that BFS
replacement for sand may improve the frost resistance of the BFS-blended mortar samples, which is
related to the higher compressive strength before the freeze–thaw test. ratios of BFS replacement for sand. The durability factor was calculated from the results of the relative dynamic modulus of
elasticity of each mixture and the results are shown in Figure 5. The durability factor of the sample
with BFS as a replacement for sand is significantly higher than that of the other mortar samples. 3.1.4. Freeze-Thaw Tes
y
3.1.4. Freeze-Thaw Test In general, as the number of freeze–thaw cycles increases, the decrease of the relative dynamic
modulus may cause an increase in the number of cracks within the mortar sample because of internal
cracking in the cement. This causes not only the increased weight of the mortar sample but also the
increased length change compared to the initial state. Moreover, the relative dynamic modulus of
3.1.4. Freeze-Thaw Test
In general, as the number of freeze–thaw cycles increases, the decrease of the relative dynamic
modulus may cause an increase in the number of cracks within the mortar sample because of internal
cracking in the cement. This causes not only the increased weight of the mortar sample but also the
increased length change compared to the initial state. Moreover, the relative dynamic modulus of
In general, as the number of freeze–thaw cycles increases, the decrease of the relative dynamic
modulus may cause an increase in the number of cracks within the mortar sample because of internal
cracking in the cement. This causes not only the increased weight of the mortar sample but also the
increased length change compared to the initial state. Moreover, the relative dynamic modulus of
elasticity was approximately 60% in the mortar, which is considered to be poor performance for frost
resistance [28]. Figure 4a,b present the results of freeze–thaw tests of the mortar samples. As can be seen from
Figure 4a, the relative dynamic modulus of elasticity for all mortar samples decreased below 60% before
100 cycles. Therefore, an air-entraining agent is needed to prevent frost damage in the mortar samples. However, it is clear from Figure 4b that the relative dynamic modulus of elasticity of the mortar
samples with 20 or 45 wt % of BFS replacement for sand does not decrease as the number of freeze–thaw Figure 4a,b present the results of freeze–thaw tests of the mortar samples. As can be seen from
Figure 4a, the relative dynamic modulus of elasticity for all mortar samples decreased below 60% before
100 cycles. Therefore, an air-entraining agent is needed to prevent frost damage in the mortar samples. However, it is clear from Figure 4b that the relative dynamic modulus of elasticity of the mortar
samples with 20 or 45 wt % of BFS replacement for sand does not decrease as the number of freeze–thaw
cycles increases, unlike mortar N. 3.1.4. Freeze-Thaw Tes
y
3.1.4. Freeze-Thaw Test Furthermore, in the case of a BFS replacement ratio of 45 wt %, the durability factor was 90–100,
implying that BFS replacement for sand may improve the frost resistance of the BFS-blended mortar
samples, which is related to the higher compressive strength before the freeze–thaw test. (a)
(b)
Figure 4. Relative dynamic modulus of elasticity for different mortar mixtures with or without BFS
during freeze–thaw cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. 0
20
40
60
80
100
120
0
100
200
300
N
4S45C
4S70C
0
20
40
60
80
100
120
0
100
200
300
N
8S45C
8S70C
0
20
40
60
80
100
120
0
100
200
300
N
3S45C
3S70C
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
Freeze / thaw cycle (cy.)
Freeze / thaw cycle (cy.)
Relative dynamic modulus of elasticity (%)
Relative dynamic modulus of elasticity (%)
60%
60%
60%
0
20
40
60
80
100
120
0
100
200
300
N
8S20S
8S45S
0
20
40
60
80
100
120
0
100
200
300
N
4S20S
4S45S
0
20
40
60
80
100
120
0
100
200
300
N
3S20S
3S45S
Relative dynamic modulus of elasticity (%)
Relative dynamic modulus of elasticity (%)
Relative dynamic modulus of elasticity (%)
Freeze / thaw cycle (cy.)
Freeze / thaw cycle (cy.)
Freeze / thaw cycle (cy.)
60%
60%
60%
Figure 4. Relative dynamic modulus of elasticity for different mortar mixtures with or without BFS
during freeze–thaw cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. 3.1.5. Accelerated Carbonation Coefficient Figure 6 presents the results for the carbonation coefficients of the mortar samples with and
without BFS. The carbonation coefficient of mortar N was 1.17. The carbonation coefficient tends to
increase with the BFS replacement ratio over that of mortar N. This is due to the fact that Ca(OH)2 in
the mortar incorporating BFS with cement is more consumed, which causes the carbonation process to
be accelerated. However, in the case of BFS replacement for sand, the carbonation coefficient of the
mortars was significantly lower than that of mortar N, implying that replacement of the slag for sand
in the BFS mortar samples is advantageous for the improvement carbonation resistance. Yoshida and
Saito reported that the concrete sample with BFS decreased the air permeability, which is related to
the transition zone around the particle of BFS due to the hydration of BFS with increasing age [29]. Therefore, as shown in Figure 6b, it is considered that the carbonation coefficient is reduced because
the matrices of the mortar with mixed BFS were densified, thus, invasion of CO2 ions in inhibited by
the hydration reaction of slag. 3.1.5. Accelerated Carbonation Coefficient
Figure 6 presents the results for the carbonation coefficients of the mortar samples with and
without BFS. The carbonation coefficient of mortar N was 1.17. The carbonation coefficient tends to
increase with the BFS replacement ratio over that of mortar N. This is due to the fact that Ca(OH)2 in
the mortar incorporating BFS with cement is more consumed, which causes the carbonation process
to be accelerated. However, in the case of BFS replacement for sand, the carbonation coefficient of the
mortars was significantly lower than that of mortar N, implying that replacement of the slag for sand
in the BFS mortar samples is advantageous for the improvement carbonation resistance. Yoshida and
Saito reported that the concrete sample with BFS decreased the air permeability, which is related to
the transition zone around the particle of BFS due to the hydration of BFS with increasing age [29]. Therefore, as shown in Figure 6b, it is considered that the carbonation coefficient is reduced because
the matrices of the mortar with mixed BFS were densified, thus, invasion of CO2 ions in inhibited by
the hydration reaction of slag. (a)
(b)
Figure 6. 3.1.4. Freeze-Thaw Tes
y
3.1.4. Freeze-Thaw Test Durability factors for different mortar mixtures with or without BFS during freeze–thaw
cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. 3.9
3.4
2.4
7.7
4.5
11
11
0
20
40
60
80
100
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Durability factor
3000
4000
8000 cm2/g
BFS fineness➡
3.9
60.7
90.3
34.7
97.5
17.4
98.9
0
20
40
60
80
100
N
3S20S 3S45S 4S20S 4S45S 8S20S 8S45S
Durability factor
3000
4000
8000 cm2/g
BFS fineness➡
g
Figure 5. Durability factors for different mortar mixtures with or without BFS during freeze–thaw
cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. (b) (a) Figure 5. Durability factors for different mortar mixtures with or without BFS during freeze–thaw
cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. Figure 5. Durability factors for different mortar mixtures with or without BFS during freeze–thaw
cycles: (a) replacement by BFS for cement and (b) replacement by BFS for sand. Materials 2020, 13, 2564
Figure 5. D 10 of 17 10 of 17 3.1.5. Accelerated Carbonation Coefficient Carbonation coefficients for different mortar mixtures with and without BFS: (a)
replacement of BFS for cement and (b) replacement of BFS for sand. 1.17
2.10
3.22
2.00
2.59
1.76
3.04
0.00
1.00
2.00
3.00
4.00
5.00
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡
1.17
0.01
0.21
0.22
0.00
1.00
2.00
3.00
4.00
5.00
N
3S20S
4S20S
8S20S
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡
Figure 6. Carbonation coefficients for different mortar mixtures with and without BFS: (a) replacement
of BFS for cement and (b) replacement of BFS for sand. (b)
1.17
0.01
0.21
0.22
0.00
1.00
2.00
3.00
4.00
5.00
N
3S20S
4S20S
8S20S
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡ (a)
1.17
2.10
3.22
2.00
2.59
1.76
3.04
0.00
1.00
2.00
3.00
4.00
5.00
N
3S45C 3S70C 4S45C 4S70C 8S45C 8S70C
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡ (a) (b) Figure 6. Carbonation coefficients for different mortar mixtures with and without BFS: (a)
replacement of BFS for cement and (b) replacement of BFS for sand. Figure 6. Carbonation coefficients for different mortar mixtures with and without BFS: (a) replacement
of BFS for cement and (b) replacement of BFS for sand. 3 2 Self Healing Effec
3.2. Self-Healing Effect 3 2 Self-Healing Effe
3.2. Self-Healing Effect f
g
ff
3 2 1 I
e ti atio
of De
ee of Da
a e
3.2.1. Investigation of Degree of Damage 11 of 17 Materials 2020, 13, 2564
damage. 3.2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
after Damage
3.2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
after Damage 3.2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
after Damage
2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
fter Damage 3.2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
after Damage
3.2.2. Bending Strength, Compressive Strength, and Carbonation Coefficient of Mortar Samples
after Damage The results for the bending strength and compressive strength of the mortar samples after damage
caused by freeze–thaw testing are shown in Figures 8 and 9. Figure 8 shows that the bending strengths
of all the samples have decreased after the damage. Similar results are shown in Figure 9. These results
indicate that micro cracks were formed in all the mortar samples and increased with the number of
freeze–thaw cycles. Figure 10 shows the results of the accelerated carbonation test for the mortar
samples. The carbonation coefficients of all the mortar samples after damage are greater than before
the damage. The above results clarify that all the mortar samples exhibit decreased bending strength
and compressive strength and increased carbonation coefficient due to the micro cracks formed during
freeze–thaw cycling [30]. The results for the bending strength and compressive strength of the mortar samples after
damage caused by freeze–thaw testing are shown in Figures 8 and 9. Figure 8 shows that the bending
strengths of all the samples have decreased after the damage. Similar results are shown in Figure 9. These results indicate that micro cracks were formed in all the mortar samples and increased with
the number of freeze–thaw cycles. Figure 10 shows the results of the accelerated carbonation test for
the mortar samples. The carbonation coefficients of all the mortar samples after damage are greater
than before the damage. The above results clarify that all the mortar samples exhibit decreased
bending strength and compressive strength and increased carbonation coefficient due to the micro
cracks formed during freeze–thaw cycling [30]. aw cycling [30]. med during freeze–thaw cycling [30]. Figure 8. Changes of bending strength in different mortar samples. Figure 8. Changes of bending strength in different mortar samples. 20, 13, x FOR PEER REVIEW
. Figure 9. Changes of compressive strength in different mortar samples. Figure 9. f
g
ff
3 2 1 I
e ti atio
of De
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a e
3.2.1. Investigation of Degree of Damage 3.2.1. Investigation of Degree of Damage
The freeze–thaw test was performed until a relative dynamic modulus of elasticity of 60% was
reached, in accordance with JIS A 1127 [23], in order to generate cracks in the mortar sample. Figure
7 presents the results for the relative dynamic modulus of elasticity for BFS-blended cement mortar
The freeze–thaw test was performed until a relative dynamic modulus of elasticity of 60% was
reached, in accordance with JIS A 1127 [23], in order to generate cracks in the mortar sample. Figure 7
presents the results for the relative dynamic modulus of elasticity for BFS-blended cement mortar
samples. The results for the relative dynamic modulus of elasticity of all the mortar samples range
between 58.1% and 67.2%, and were approximately 60%, implying that these mortar samples satisfied
the target relative dynamic modulus of elasticity. Then, after being damaged, the mortar samples were
stored under two types of curing conditions, namely curing in water at 40 ◦C for 4 weeks and in water
at 20 ◦C for 1 week [9]. After that, the compressive strength, bending strength, accelerated carbonation
depth, and relative dynamic modulus of elasticity of these mortar samples were examined. Materials 2020, 13, x FOR PEER REVIEW
10 of 16
samples. The results for the relative dynamic modulus of elasticity of all the mortar samples range
between 58.1% and 67.2%, and were approximately 60%, implying that these mortar samples satisfied
the target relative dynamic modulus of elasticity. Then, after being damaged, the mortar samples
were stored under two types of curing conditions, namely curing in water at 40 °C for 4 weeks and
in water at 20 °C for 1 week [9]. After that, the compressive strength, bending strength, accelerated
carbonation depth, and relative dynamic modulus of elasticity of these mortar samples were
examined. Figure 7. Results for the relative dynamic modulus of elasticity of the different mixtures after
damage. 60%
67.2
66.3
65.7
62.8
58.1
60.1
65.2
0
10
20
30
40
50
60
70
80
90
100
N
3S45C
3S70C
4S45C
4S70C
8S45C
8S70C
Mixtures
Relative dynamic modulus of
elasticity (%)
Figure 7. Results for the relative dynamic modulus of elasticity of the different mixtures after damage. Figure 7. Results for the relative dynamic modulus of elasticity of the different mixtures after
damage
Figure 7. Results for the relative dynamic modulus of elasticity of the different mixtures after damage. f
g
ff
3 2 1 I
e ti atio
of De
ee of Da
a e
3.2.1. Investigation of Degree of Damage Changes of compressive strength in different mortar samples. 0, 13, x FOR PEER REVIEW
. Figure 9. Changes of compressive strength in different mortar samples. Figure 10. Carbonation coefficients of different mortar samples after damage. Figure 10. Carbonation coefficients of different mortar samples after damage. Figure 8. Changes of bending strength in different mortar samples. Figure 8. Changes of bending strength in different mortar samples. , 13, x FOR PEER REVIEW
13, x FOR PEER REVIEW Figure 8 Changes of bending strength in different mortar samples
Figure 8. Changes of bending strength in different mortar samples. Figure 8. Changes of bending strength in different mortar samples. . Figure 9. Changes of compressive strength in different mortar samples. . Figure 9. Changes of compressive strength in different mortar samples. Figure 8. Changes of bending strength in diff
Figure 9. Changes of compressive strength in di Figure 9. Changes of compressive strength in different mortar samples. Figure 9. Changes of compressive strength in different mortar samples. Figure 9. Changes of compressive strength in different mortar samples. Figure 10. Carbonation coefficients of different mortar samples after damage. Figure 10. Carbonation coefficients of different mortar samples after damage. Figure 10. Carbonation coefficients of different mortar samples after damage. Figure 10. Carbonation coefficients of different mortar samples after damage. Materials 2020, 13, 2564 12 of 17 3.2.3. Relative Dynamic Modulus of Elasticity, Bending Strength, Compressive Strength,
and Carbonation Coefficient of Mortar Samples after Water Curing 3.2.3. Relative Dynamic Modulus of Elasticity, Bending Strength, Compressive Strength,
and Carbonation Coefficient of Mortar Samples after Water Curing As mentioned above, all the mortar samples were immediately stored for 28 days and then
damaged to approximately 60% during freeze–thaw testing. Each mortar sample was tested for three
cases: after damage, after curing in water at 20 ◦C for 1 week, and after curing in water at 40 ◦C for
4 weeks. Figure 11 shows the results of measuring the compressive strength of the mortar samples
for different BFS replacement ratios for evaluating the self-healing ability. As shown in Figure 11a,
for the sample without BFS, compressive strength recovery was confirmed in the samples cured
in water at each temperature; in particular, the 3S45 mortar sample incorporating BFS with lower
fineness has a remarkably high strength recovery. This is because the rate of BFS hydration is slow
and thus additional reaction occurs after deterioration. f
g
ff
3 2 1 I
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3.2.1. Investigation of Degree of Damage Compressive strength results after damage and after water curing for different (a) BFS
l
t
ti
d (b) BFS Bl i
fi
l
33.0
28.2
30.5
42.7
42.9
41.1
43.8
52.5
45.0
0
20
40
60
80
N
3S45C
3S70C
Compressive strength (MPa)
After
damage
20℃ for
1 week
40℃ for
4 weeks
After
damage
20℃ for
1 week
40℃ for
4 weeks
28.2
38.6
44.2
42.9
46.8
41.9
52.5
35.7
32.5
0
20
40
60
80
3S45C
4S45C
8S45C
Compressive strength (MPa) (b)
After
damage
20℃ for
1 week
40℃ for
4 weeks
28.2
38.6
44.2
42.9
46.8
41.9
52.5
35.7
32.5
0
20
40
60
80
3S45C
4S45C
8S45C
Compressive strength (MPa)
(b)
After
damage
20℃ for
1 week
40℃ for
4 weeks
28.2
38.6
44.2
42.9
46.8
41.9
52.5
35.7
32.5
0
20
40
60
80
3S45C
4S45C
8S45C
Compressive strength (MPa) (a)
33.0
28.2
30.5
42.7
42.9
41.1
43.8
52.5
45.0
0
20
40
60
80
N
3S45C
3S70C
Compressive strength (MPa)
After
damage
20℃ for
1 week
40℃ for
4 weeks
( )
33.0
28.2
30.5
42.7
42.9
41.1
43.8
52.5
45.0
0
20
40
60
80
N
3S45C
3S70C
Compressive strength (MPa)
After
damage
20℃ for
1 week
40℃ for
4 weeks (b)
C (a)
3 Figure 11. Compressive strength results after damage and after water curing for different (a) BFS
replacement ratios and (b) BFS Blaine fineness values. Figure 11. Compressive strength results after damage and after water curing for different (a) BFS
replacement ratios and (b) BFS Blaine fineness values. (a)
(b)
Figure 11. Compressive strength results after damage and after water curing for different (a) BFS
l
t
ti
d (b) BFS Bl i
fi
l Figure 12 shows the results of the accelerated carbonation test for the three stages (after
deterioration and after two types of curing condition). After water curing at 20 °C and at 40 °C, the
carbonation coefficients of all the mortar samples were decreased after water curing, which was
caused by the additional hydration of cement and BFS. In particular, the carbonation coefficient of
the mortar sample with BFS was significantly reduced after curing compared to mortar sample N. Therefore, the self-healing effect of BFS, especially with a low fineness of approximately 3000 cm2/g,
significantly contributes to the decrease of the carbonation coefficient. f
g
ff
3 2 1 I
e ti atio
of De
ee of Da
a e
3.2.1. Investigation of Degree of Damage This may be because the micro
cracks become denser as a result of the potential hydration of BFS. Figure 12 shows the results of the accelerated carbonation test for the three stages (after deterioration
and after two types of curing condition). After water curing at 20 ◦C and at 40 ◦C, the carbonation
coefficients of all the mortar samples were decreased after water curing, which was caused by the
additional hydration of cement and BFS. In particular, the carbonation coefficient of the mortar
sample with BFS was significantly reduced after curing compared to mortar sample N. Therefore,
the self-healing effect of BFS, especially with a low fineness of approximately 3000 cm2/g, significantly
contributes to the decrease of the carbonation coefficient. This may be because the micro cracks become
denser as a result of the potential hydration of BFS. Figure 12 shows the results of the accelerated carbonation test for the three stages (after
deterioration and after two types of curing condition). After water curing at 20 °C and at 40 °C, the
carbonation coefficients of all the mortar samples were decreased after water curing, which was
caused by the additional hydration of cement and BFS. In particular, the carbonation coefficient of
the mortar sample with BFS was significantly reduced after curing compared to mortar sample N. Therefore, the self-healing effect of BFS, especially with a low fineness of approximately 3000 cm2/g,
significantly contributes to the decrease of the carbonation coefficient. This may be because the micro
cracks become denser as a result of the potential hydration of BFS. Figure 12. Carbonation coefficients of the mortar samples after damage and after water curing. After
damage
20℃ for
1 week
40℃ for
4 weeks
1.94
2.95
3.64
2.41
3.33
2.55
3.17
2.05
2.30
2.94
1.92
2.94
2.28
2.94
1.61
1.39
2.11
1.76
2.53
2.14
2.37
0
1
2
3
4
N
3S45C
3S70C
4S45C
4S70C
8S45C
8S70C
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡
Figure 12. Carbonation coefficients of the mortar samples after damage and after water curing. After
damage
20℃ for
1 week
40℃ for
4 weeks
1.94
2.95
3.64
2.41
3.33
2.55
3.17
2.05
2.30
2.94
1.92
2.94
2.28
2.94
1.61
1.39
2.11
1.76
2.53
2.14
2.37
0
1
2
3
4
5
N
3S45C
3S70C
4S45C
4S70C
8S45C
8S70C
Carbonation coefficient
3000
4000
8000 cm2/g
BFS fineness➡
Figure 12. f
g
ff
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3.2.1. Investigation of Degree of Damage Figure 11b presents the effect of increasing
BFS replacement ratio on self-healing ability. It can be seen that for the samples 4S45C and 8S45C,
the carbonation coefficient decreases with curing in water at 40 ◦C, but there is no clear explanation for
this behavior. The higher Blaine fineness may cause the high temperature-expansion coefficient owing
to the hydration of BFS [31], which is needed to investigate the relationship between the self-healing
effect of BFS with higher Blaine fineness and the temperature-expansion coefficient. Materials 2020, 13, x FOR PEER REVIEW
12 of 16 (a)
(b)
Figure 11. Compressive strength results after damage and after water curing for different (a) BFS
replacement ratios and (b) BFS Blaine fineness values. 33.0
28.2
30.5
42.7
42.9
41.1
43.8
52.5
45.0
0
20
40
60
80
N
3S45C
3S70C
Compressive strength (MPa)
After
damage
20℃ for
1 week
40℃ for
4 weeks
After
damage
20℃ for
1 week
40℃ for
4 weeks
28.2
38.6
44.2
42.9
46.8
41.9
52.5
35.7
32.5
0
20
40
60
80
3S45C
4S45C
8S45C
Compressive strength (MPa)
Figure 11. Compressive strength results after damage and after water curing for different (a) BFS
replacement ratios and (b) BFS Blaine fineness values. Materials 2020, 13, x FOR PEER REVIEW
12 of 16
(a)
(b)
Figure 11. Materials 2020, 13, 2564 Prediction of Self-Healing Performance and Optimum BFS Fineness and Replacement Ratio 3.2.4. Prediction of Self-Healing Performance and Optimum BFS Fineness and Replacement Ratio
In this study, general regression analysis was used to predict the relative dynamic modulus of
In this study, general regression analysis was used to predict the relative dynamic modulus
of elasticity of the BFS-blended mortar samples by taking into consideration the Blaine fineness,
replacement ratio, and water curing conditions. The regression equation is blended mortar samples by taking into consideration the Blaine fineness,
water curing conditions. The regression equation is
Y = 53.7834 −0.003 × x1 + 0.573 × x2 + 0.8387 × x3
(4) ness,
(4) Y = 53.7834 − 0.003 × x1 + 0.573 × x2 + 0.8387 × x3
(4)
where Y is the calculated relative dynamic modulus of elasticity; x1 is the fineness of BFS, and can be
3000, 4000, or 8000 cm2/g; x2 is the BFS replacement ratio; and x3 is the curing temperature in water
(20 and 40 °C). Twenty-four mortar samples were used. Table 4 and Figure 14 show the results of
general regression analysis. Generally, P-value plays a key parameter to evaluate the suggested
model. It can be seen that the value of R2 was 0.806 and P-value was significant at the level of 5% (P
< 0.05) as shown in Table 5. These values implied that the predicted model can be suitably fitted and
can be estimated as 80.6% of variability in the relative dynamic modulus of elasticity between
measured and predicted value. The results of a comparison of the experimental results for the relative
dynamic modulus of elasticity with the predicted values are presented in Figure 14. The results imply
good agreement between the experimental values and the predicted values of the relative dynamic
where Y is the calculated relative dynamic modulus of elasticity; x1 is the fineness of BFS, and can be
3000, 4000, or 8000 cm2/g; x2 is the BFS replacement ratio; and x3 is the curing temperature in water
(20 and 40 ◦C). Twenty-four mortar samples were used. Table 4 and Figure 14 show the results of
general regression analysis. Generally, p-value plays a key parameter to evaluate the suggested model. It can be seen that the value of R2 was 0.806 and p-value was significant at the level of 5% (p < 0.05)
as shown in Table 5. f
g
ff
3 2 1 I
e ti atio
of De
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a e
3.2.1. Investigation of Degree of Damage Carbonation coefficients of the mortar samples after damage and after water curing. Figure 12. Carbonation coefficients of the mortar samples after damage and after water curing. Fi
C
b
ff
f h
l
f
d
d f
Figure 12. Carbonation coefficients of the mortar samples after damage and after water curing. Materials 2020, 13, 2564 Materials 2020, 13, 2564 13 of 17 Figure 13 shows the changes of the relative dynamic modulus of elasticity of the mortar samples. It can be seen from the lower fineness and higher replacement ratio of BFS, especially for 3S45C and
3S70C, that the relative dynamic modulus of elasticity increases in the mortar samples after water
curing owing to the remaining products of BFS, implying that incorporating BFS with larger particles
in the mixture contributes to the self-healing effect. Matsushita et al. investigated the self-healing
properties of concrete using a BFS replacement ratio of 50% and measured the ultrasonic pulse velocity
before and after damage [32]. It is reported that the ultrasonic pulse velocity of concrete with BFS after
re-curing increased compared to normal concrete. According to the hydration analysis on the self-healing properties of BFS, Darquennes et al. have
studied the early-age self-healing of cementitious materials incorporating BFS under water curing. X-ray tomography test and scanning electronic microscopy were performed. It is reported that volume
of healing products of BFS is doubled compared to cement and in new hydration products is mainly
C-S-H gel (low CaO/SiO2 ratio). Therefore, from Figure 13, increasing the relative dynamic modulus of
elasticity due to the presence of new type of C-S-H gel, which generates from the BFS and Ca(OH)2 in
the BFS mortar sample [33]. Materials 2020, 13, x FOR PEER REVIEW
13 of 16
modulus of elasticity due to the presence of new type of C-S-H gel, which generates from the BFS
and Ca(OH)2 in the BFS mortar sample [33]. Figure 13 Changes in relative dynamic modulus of elasticity after damage and after water curing
67.2
66.3
65.7
62.8
58.1
60.1
65.2
75.9
86.0
104.1
84.3
91.8
70.9
95.5
87.3
110.8
127.1
99.1
106.0
88.6
101.6
0
20
40
60
80
100
120
140
160
N
3S45C
3S70C
4S45C
4S70C
8S45C
8S70C
3000
4000
8000 cm2/g
BFS fineness➡
After
damage
20℃ for
1 week
40℃ for
4 weeks
Relative dynamic modulus of
elasticity (%)
Figure 13. Changes in relative dynamic modulus of elasticity after damage and after water curing. Fi
13 Ch
i
l
i
d
i
d l
f l
i i
f
d
d f
i
Figure 13. Changes in relative dynamic modulus of elasticity after damage and after water curing. Figure 13. Changes in relative dynamic modulus of elasticity after damage and after water curing. 3.2.4. Materials 2020, 13, 2564 These values implied that the predicted model can be suitably fitted and can
be estimated as 80.6% of variability in the relative dynamic modulus of elasticity between measured
and predicted value. The results of a comparison of the experimental results for the relative dynamic
modulus of elasticity with the predicted values are presented in Figure 14. The results imply good
agreement between the experimental values and the predicted values of the relative dynamic modulus
of elasticity. 14 of 17
14 of 16 Materials 2020, 13, 2564
Materials 2020, 13, x FOR Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
of elasticity
y = 0.994x
R² = 0.806
40
60
80
100
120
140
160
40
60
80
100
120
140
160
Predicted value(%)
Measured value (%)
Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
of elasticity. terials 2020, 13, x FOR PEER REVIEW
14 of
y = 0.994x
R² = 0.806
60
80
100
120
140
160
Predicted value(%) Predicted value(%)
EW Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
f l
ti it
Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
of elasticity. R 0.806
60
Pre Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
f l
i i
Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
of elasticity. R 0.806
60
Pre of elasticity. Table 5. Estimated regression coefficients and corresponding t and p-value from the experimental data. 40 of elasticity. Figure 15 shows the optimum Blaine fineness values and replacement ratios for BFS. A contour
aph is presented based on the results of the relative dynamic modulus of elasticity. The figure
ows that a lower Blaine fineness and a higher replacement ratio for BFS are desirable to achieve
lf-healing concrete. However, further investigation by taking into consideration the effect of W/B
d the presence of CaSO4 on the self-healing ability is needed to improve the self-healing mixture
oportion to meet design codes for BFS-blended cement. Table 5. Estimated regression coefficients and corresponding t and p-value from the experimental data. Materials 2020, 13, 2564 Parameter Estimate
Coefficient
Standard Error
t-Value
p-Value
Constant
53.7834
7.64141
7.03841
0.000
Blain fineness
−0.003
0.00059
−5.10849
0.000
Replacement ratio (%)
0.573
0.10116
5.69353
0.000
Curing conditions
0.8387
0.12645
6.63228
0.000
Figure 14. Comparison of the experimental and predicted values of the relative dynamic modulus
of elasticity. 40
40
60
80
100
120
140
160
Measured value (%) Figure 15 shows the optimum Blaine fineness values and replacement ratios for BFS. A contour
graph is presented based on the results of the relative dynamic modulus of elasticity. The figure
shows that a lower Blaine fineness and a higher replacement ratio for BFS are desirable to achieve
self-healing concrete. However, further investigation by taking into consideration the effect of W/B
and the presence of CaSO4 on the self-healing ability is needed to improve the self-healing mixture
proportion to meet design codes for BFS-blended cement. Figure 15 shows the optimum Blaine fineness values and replacement ratios for BFS. A contour
graph is presented based on the results of the relative dynamic modulus of elasticity. The figure
shows that a lower Blaine fineness and a higher replacement ratio for BFS are desirable to achieve
self-healing concrete. However, further investigation by taking into consideration the effect of W/B
and the presence of CaSO4 on the self-healing ability is needed to improve the self-healing mixture
proportion to meet design codes for BFS-blended cement. 15. Contour plot of the optimum fineness and replacement ratio for BFS. ns
rpose of this study was to investigate the material properties and self-heali
rporating blast furnace slag (BFS), while considering the influence of the
aine fineness of the BFS and the curing conditions. The performance of the
15. Contour plot of the optimum fineness and replacement ratio for BFS. Figure 15. Contour plot of the optimum fineness and replacement ratio for BFS. orating blast furnace slag (BFS), while considering the influence of the
ne fineness of the BFS and the curing conditions. The performance of the
5. Contour plot of the optimum fineness and replacement ratio for BFS. Figure 15. Contour plot of the optimum fineness and replacement ratio for BFS. ability of mortar
ti
Bl i
fi
4. Conclusions ratios, Blaine fineness values, and curing conditions. On the basis of the experimental results, which
cover the materials properties and self-healing behavior, the main findings of this study can be
summarized as follows:
(1) It was found that replacing cement or sand with BFS in the mortar would increase the bending
strength, compressive strength, carbonation coefficient, and durability factor, indicating that the
4. Conclusions
The purpose of this study was to investigate the material properties and self-healing ability of
mortar incorporating blast furnace slag (BFS), while considering the influence of the replacement
ratio and Blaine fineness of the BFS and the curing conditions. The performance of the self-healing
The purpose of this study was to investigate the material properties and self-healing ability of
mortar incorporating blast furnace slag (BFS), while considering the influence of the replacement ratio
and Blaine fineness of the BFS and the curing conditions. The performance of the self-healing ability of
mortar samples incorporating BFS was evaluated using mixtures with different replacement ratios, 15 of 17 Materials 2020, 13, 2564 Blaine fineness values, and curing conditions. On the basis of the experimental results, which cover
the materials properties and self-healing behavior, the main findings of this study can be summarized
as follows: Blaine fineness values, and curing conditions. On the basis of the experimental results, which cover
the materials properties and self-healing behavior, the main findings of this study can be summarized
as follows: (1)
It was found that replacing cement or sand with BFS in the mortar would increase the bending
strength, compressive strength, carbonation coefficient, and durability factor, indicating that the
materials properties are dependent on the BFS. The reason for this is the low water-to-binder
ratio (W/B) of the BFS-blended cement mixture. (2)
The mortar samples were damaged through freeze–thaw testing until a relative dynamic modulus
of elasticity of 60% was achieved. The BFS-blended cement samples may exhibit decreased
bending strength, compressive strength, and carbonation coefficient after frost damage. This is
because of the micro cracks formed in the mortar during freeze–thaw cycling. (3)
In the investigation of self-healing, it was confirmed that incorporating BFS with a low Blaine
fineness and moderate or high replacement ratios (45 or 70 wt %) can improve the compressive
strength and relative dynamic modulus of elasticity of the BFS mortar samples. ability of mortar
ti
Bl i
fi
4. Conclusions Moreover,
these mortar samples displayed higher resistance to carbonation, which was due to the hydration
products of BFS. (4)
The relative dynamic modulus of elasticity of the BFS mortar samples after water curing was
predicted by a general regression equation (R2 = 0.806). Further experimental investigation
is needed to improve this equation for a BFS-blended cement mortar mixture. An optimum
replacement ratio and Blaine fineness for the BFS was suggested by a contour graph. However,
this requires additional investigation regarding the W/B. Author Contributions: Investigation: S.N.; writing–original draft: S.N.; data Curation: S.N., N.X.Q., and W.Z.;
review and editing: M.T. and Y.H.; project Administration: Y.H. All authors have read and agreed to the published
version of the manuscript. Author Contributions: Investigation: S.N.; writing–original draft: S.N.; data Curation: S.N., N.X.Q., and W.Z.;
review and editing: M.T. and Y.H.; project Administration: Y.H. All authors have read and agreed to the published
version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Powers, T.C. Working hypothesis for further studies of frost resistance of concrete. Am. Concr. Inst. J. 1945,
16, 245–271. 1. Powers, T.C. Working hypothesis for further studies of frost resistance of concrete. Am. Concr. Inst. J. 1945,
16, 245–271. 2. Powers, T.C.; Helmuth, R.A. Theory of volume changes in hardened Portland-cement pasted during f
Highw. Res. Board 1953, 32, 285–297. 2. Powers, T.C.; Helmuth, R.A. Theory of volume changes in hardened Portland-cement pasted during freezing. Highw. Res. Board 1953, 32, 285–297. g
. Kamada, E. Frost damage and pore structure of concrete. Proc. Jpn. Concr. Inst. 1988, 10, 51–60. g
3. Kamada, E. Frost damage and pore structure of concrete. Proc. Jpn. Concr. Inst. 1988, 10, 51–60. 4. Hama, Y.; Hirano, A.; Tabata, M.; Atarashi, D. Affecting factors on air void system and frost resistance of
concrete. J. JSCE 2008, 73, 2061–2067. [CrossRef] 4. Hama, Y.; Hirano, A.; Tabata, M.; Atarashi, D. Affecting factors on air void system and frost resistance of
concrete. J. JSCE 2008, 73, 2061–2067. [CrossRef] 5. Ayano, T.; Fujii, T. Resisance to freezing and thawing attack of concrete with blast furnace slag fine aggregate. J. JSCE 2014, 70, 417–427. 5. Ayano, T.; Fujii, T. Resisance to freezing and thawing attack of concrete with blast furnace slag fine aggregate. J. JSCE 2014, 70, 417–427. 6. Japan Industrial Standards Committee. Technical Committee Reports on Autogeneous Healing in Cementitious
Materials; Japan Industrial Standards Committee: Tokyo, Japan, 2009. 6. Japan Industrial Standards Committee. Technical Committee Reports on Autogeneous Healing in Cementitious
Materials; Japan Industrial Standards Committee: Tokyo, Japan, 2009. 7. Kishi, T.; Ahn, T.H.; Hosoda, A.; Suzuki, S.; Takaoka, H. Self-healing behaviour by cementitious
recrystallization of cracked concrete incorporating expansive agent. In Proceedings of the 1st International
Conference on Self Healing Materials, Noordwijk aan Zee, The Netherlands, 18–20 April 2007. 7. Kishi, T.; Ahn, T.H.; Hosoda, A.; Suzuki, S.; Takaoka, H. Self-healing behaviour by cementitious
recrystallization of cracked concrete incorporating expansive agent. In Proceedings of the 1st International
Conference on Self Healing Materials, Noordwijk aan Zee, The Netherlands, 18–20 April 2007. 8. Ahn, T.H.; Kishi, T. Crack self-healing behaviour of cementitious composites incorporating various mineral
admixtures. J. Adv. Concr. Technol. 2010, 8, 171–186. [CrossRef] 8. Ahn, T.H.; Kishi, T. Crack self-healing behaviour of cementitious composites incorporating various mineral
admixtures. J. Adv. Concr. Technol. 2010, 8, 171–186. [CrossRef] 9. References Na, S.H.; Hama, Y.; Taniguchi, M.; Katsura, O.; Sagawa, T.; Zakaria, M. Experimental investigation on reaction
rate and self-healing ability in fly ash-blended cement mixtures. J. Adv. Concr. Technol. 2012, 10, 240–253. [CrossRef] Materials 2020, 13, 2564 16 of 17 16 of 17 10. Taniguchi, M.; Hama, Y.; Katsura, O.; Sagawa, T.; Na, S.H. Self healing of frost-damaged concrete incorporation
fly ash. In Proceedings of the 3rd International Conference on Self-Healing Materials, Bath, UK, 27–29 June
2011. 11. Termkhajornkit, P.; Nawa, T.; Yamashiro, Y.; Saito, T. Self-healing ability of fly ash–cement s
Cem. Concr. Comp. 2009, 31, 195–203. [CrossRef] 12. Pei, R.; Liu, J.; Wang, S.; Yang, M. Use of bacterial cell walls to improve the mechanical performance of
concrete. Cem. Concr. Comp. 2013, 39, 122–130. [CrossRef] 13. Wiktor, V.; Jonkers, H.M. Quantification of crack-healing in novel bacteria-based self-healing concrete. Cem. Concr. Comp. 2010, 33, 763–770. [CrossRef] 14. Huang, H.; Ye, G.; Damidot, D. Effect of blast furnace slag on self-healing of microcracks in cementitious
materials. Cem. Concr. Res. 2014, 60, 68–82. [CrossRef] 15. Hilloulin, B.; Hilloulin, D.; Grondin, F.; Loukili, A.; De Belie, N. Mechanical regains due to self-healing in
cementitious materials.: Experimental measurements and micro-mechanical model. Cem. Concr. Res. 2016,
80, 21–32. [CrossRef] 16. Qian, S.; Zhou, J.; de Rooij, M.R.; Schlangen, E.; Ye, G.; van Breugel, K. Self-healing behavior of strain
hardening cementitious composites incorporating local waste materials. Cem. Concr. Comp. 2009, 31, 613–621. [CrossRef] 17. Takagi, E.M.; Lima, M.G.; Helene, P. Self-healing of self-compacting concretes made with blast furnace slag
cements activated by crystalline admixture. Int. J. Mater. Prod. Technol. 2018, 56, 169–186. [CrossRef] 18. Sisomphon, K.; Copuroglu, O. Some characteristics of a self healing mortar incorporating calcium
sulfo-aluminate based agent. In Proceedings of the 2nd International Conference on Durability of Concrete
Structure, Sapporo, Japan, 24–26 November 2010; pp. 157–164. 19. Japan Industrial Standards Committee. Physical Testing Methods for Cement; Japan Industrial Standards
Committee: Tokyo, Japan, 2015. 20. Japan Industrial Standards Committee. Test Methods for Polymer-Modified Mortar; Japan Industrial Standards
Committee: Tokyo, Japan, 2010. 21. Japan Industrial Standards Committee. Method of Accelerated Carbonation Test for Concrete; Japan Industrial
Standards Committee: Tokyo, Japan, 2012. 22. Japan Industrial Standards Committee. Method of Test for Resistance of Concrete to Freezing and Thawing;
Japan Industrial Standards Committee: Tokyo, Japan, 2010. 23. Japan Industrial Standards Committee. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Methods of Test for Dynamic Modulus of Elasticity, Rigidity and Poisson’s
Ratio of Concrete by Resonance Vibration; Japan Industrial Standards Committee: Tokyo, Japan, 2010. 24. Zhang, W.; Zakaria, M.; Hama, Y. Influence of aggregate materials characteristics on the drying shrinkage
properties of mortar and concrete. Constr. Build. Mater. 2013, 49, 500–510. [CrossRef] 25. Zhang, W.; Hama, Y.; Na, S.H. Drying shrinkage and microstructure characteristics of mortar incorporating
ground granulated blast furnace slag and shrinkage reducing admixture. Constr. Build. Mater. 2015,
93, 267–277. [CrossRef] 26. Suyama, H.; Koyama, T.; Ito, K. Study on compressive strength of concrete mixed with various types of
powder at constant cement contents by taking account of pore structure. Concr. Res. Technol. 2013, 24, 89–100. [CrossRef] 27. Sakai, K.; Watanabe, H.; Shimobayashi, S. Low-heat high-strength on concrete with highly pul
blast-furnace slag. J. JSCE 1996, 544, 187–203. 28. Yoshiro, K.; Toshio, H. On the test method for resistance of concrete to freezing and thawing. Concr. J. 1981,
19, 16–22. 29. Yoshida, R.; Saito, K.; Yoshizawa, C. Verification of mechanism on improvement of drying shrinkage or air
permeability on concrete using blast furnace slag sand based on pore structure. Cem. Sci. Concr. Technol. 2018, 71, 587–594. [CrossRef] 30. Matsumura, T.; Katsura, O.; Yoshino, T. Properties of frost damaged concrete and the estimation of the degree
of frost damage. J. Struct. Constr. Eng. AIJ 2003, 563, 9–13. [CrossRef] 31. Mizobuchi, T.; Ishizeki, K.; Sagawa, T.; Kanda, T. Study on the influence of minor constituents in blast furnace
slag rich cement on the thermal and mechanical properties of concrete. J. Adv. Concr. Technol. 2019, 17, 46–61. [CrossRef] 17 of 17 17 of 17 Materials 2020, 13, 2564 32. Matsushita, H.; Chikada, T.; Maeda, Y.; Kawasaki, E. Study on blockade of micro crack in concrete including
fine powder of granulated blast-furnace slag. Cem. Sci. Concr. Technol. 1998, 52, 638–643. 33. Darquennes, A.; Olivier, K.; Benboudjema, F.; Gagné, R. Early-age self-healing of cementitious materials
containing ground granulated blast-furnace slag under water curing. J. Adv. Concr. Technol. 2016, 14, 717–727. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Rusycystyczne Studia Literaturoznawcze nr 31 (2021)
ISSN 2353-9674
https://doi.org/10.31261/RSL.2021.31.01 Rusycystyczne Studia Literaturoznawcze nr 31 (2021)
ISSN 2353-9674
https://doi.org/10.31261/RSL.2021.31.01 (Pro)testy Rosja to kraj, w którym skala protestów, manifestacji ulicznych
zdecydowanie zintensyfikowała się w XXI wieku. O protestujących
w Rosji jest głośno szczególnie w ostatniej dekadzie. Oczywiście
wciąż mówimy o protestach występujących lokalnie, punktowo. Najczęściej protestują mieszkańcy dużych miast, dla których al-
ternatywą wobec rządowego przekazu telewizyjnego i radiowego
pozostaje przede wszystkim przestrzeń internetu i mediów spo-
łecznościowych. Powody, którymi się oni kierują, najczęściej mają
związek z polityką, gospodarką (szczególnie na rosyjskim Dalekim
Wschodzie) i kwestiami społecznymi. Z jednej strony walczy się
w ten sposób o prawa człowieka, z drugiej o prawa mniejszości na-
rodowych i seksualnych. Tematyka i specyfika protestów są związa-
ne ze zmianami pokoleniowymi i nowymi kanałami komunikacji
i informacji. Potwierdzeniem może służyć zjawisko flash mobów,
które nadzwyczaj szybko rozprzestrzeniło się w Rosji czy na Bia-
łorusi. Dla błyskawicznie zebranych w jednym miejscu ludzi flash
moby stały się nie tylko przejawem rozrywki, happeningu, rodza-
jem eksperymentu społecznego i artystycznego, ale przede wszyst-
kim spontaniczną reakcją grupy ludzi, którzy w ten sposób chcą
zamanifestować sprzeciw, zwracając uwagę na palące problemy Beata Pawletko Słowo wstępne Beata Pawletko Słowo wstępne RSL.2021.31 s. 1 z 4 i potrzeby społeczno-polityczne. Stąd politmoby, socjomoby czy tę-
czowe flash moby będące, co ciekawe, bardziej bezpieczną alterna-
tywą dla dużych mityngów i manifestacji, które często są nielegalne
i grożą za nie poważne konsekwencje. Nawet krótkie akcje pozwa-
lają ludziom się zjednoczyć, są aktem solidarności i kreatywności,
gdy mowa o dyskredytacji władzy, przedstawieniu jej w krzywym
zwierciadle i, co za tym idzie, oswojeniu, przełamaniu strachu. O ile bowiem upadek Związku Radzieckiego przyniósł w głów-
nej mierze pokojowe ruchy rewolucyjne i akcje protestacyjne w by-
łych republikach radzieckich i krajach satelickich, o tyle w ostat-
nich latach to właśnie w Rosji niezadowolenie społeczeństwa coraz
częściej manifestuje się na ulicach. Tak było w latach 2011–2013,
okrzykniętych mianem błotnej rewolucji w związku z zarzutami
o sfałszowanie wyników wyborów do Dumy Państwowej w 2011
oraz prezydenckich w 2012 roku, wygranych przez Władimira Puti-
na w pierwszej turze. Symbolem tych niespotykanych dotąd na taką
skalę protestów była biała wstążka, a za ich twarze, jeśli chodzi o pi-
sarzy, należy z pewnością uznać Borisa Akunina i Ludmiłę Ulicką. ystyczne Studia Literaturoznawcze 2021 (31) Studia Literaturoznawcze 2021 (31) Rusycystyczne Studia Literaturoznawc y,
y
p
ą
ę
ą
Twórcy rosyjscy angażują się w protesty w różny sposób. Dawniej
orężem walki pozostawały głównie dzieła literackie, choć znaczące
miejsce zajmowała także publicystyka. (Pro)testy Lew Tołstoj w traktacie Nie
mogę milczeć zawarł swój sprzeciw wobec kary śmierci, choć słowa
te mogłyby stać się mottem również jego innych licznych protestów,
choćby przeciwko władzy, duchowieństwu, obowiązkowi przymu-
sowej służby wojskowej i działaniom wojennym. Utożsamić z nimi
można i współczesnych autorów, z tą różnicą, że spektrum aktywno-
ści protestacyjnych obejmuje dziś oprócz wciąż popularnych form
publicystycznych i literackich działania w internecie i w mediach
społecznościowych. I trzeba przyznać, że pisarze rosyjscy (wśród
nich prym wiodą Zachar Prilepin i Dmitrij Bykow) z powodzeniem
wykorzystują nowe kanały komunikacji z odbiorcą, które sprawdzi-
ły się w przypadku najbardziej znanego opozycjonisty rosyjskiego,
prawnika, demaskatora nadużyć władzy — Aleksieja Nawalnego,
dla którego podstawowymi formami komunikowania się ze świa-
tem oprócz bloga stały się dwa kanały na platformie YouTube. Inni
twórcy z kolei aktywnie uczestniczą w manifestacjach ulicznych, RSL.2021.31 s. 2 z 4 udzielając przy okazji wywiadów zachodnim mediom. Starają się
więc nagłośnić ważkie problemy przeszłości i teraźniejszości. Przy-
kładem pozostaje Ludmiła Ulicka, która prowadzi aktywną działal-
ność obywatelską, polegającą również na komentowaniu tego, co
dzieje się w Rosji, oraz dzieleniu się swoimi poglądami na łamach
zachodnich gazet i tygodników opinii. Wśród autorów, którzy two-
rzą dzieła zaangażowane, nie brakuje pisarzy (casus Zachara Prile-
pina czy Aleksandra Prochanowa), ale i twórców kultury jak reży-
ser Andriej Zwiagincew. W opozycyjne — artystyczne, literackie i paraliterackie — formy
sprzeciwu wpisują się inicjatywy odnoszące się do kultury wyso-
kiej, popkultury i folkloru. Przestrzenią chyba najbardziej kojarzo-
ną w ostatnich dekadach z opozycyjnością jest muzyka, do czego
szczególnie przyczynił się postpunkowy zespół Pussy Riot. Należy
jednak podkreślić, że nie tylko literatura, ale i sztuka rosyjska od
dawna w nadzwyczaj udany sposób utrwalają protesty społeczne,
polityczne i artystyczne, o czym przekonują zgromadzone w niniej-
szym numerze teksty. O ile jednak w wieku XIX czy jeszcze na prze-
łomie XIX i XX mieliśmy do czynienia z bardziej zawoalowanymi
z racji cenzury formami sprzeciwu, o tyle wiek XX i XXI przynoszą
w tym względzie prawdziwy przełom. Coraz śmielej i coraz bardziej
dosłownie twórcy protestują przeciwko ingerencji władzy, zarówno
w sferę prywatną, intymną jednostki, tj. jej wolności osobiste, jak
i prawa polityczne, ekonomiczne, socjalne i kulturalne. Z obszaru
klasycznych gatunków podlegających działaniu cenzury i autocen-
zury protest wkracza w świat kontrkultury, samizdatu, przynosząc
ze sobą teksty odważne, eksperymentatorskie, krytyczne, choć pi-
sane do szuflady. (Pro)testy Kolejnym zaś krokiem, gdy mowa o działaniach
kontestacyjnych, będzie zaistnienie dyskursu alternatywnego
w przestrzeni wirtualnej (w blogach, fotoblogach, vlogach) oraz
miejskiej (performanse, flash moby, graffiti). Beata Pawletko Słowo wstępne lfi
W kontekście tekstów zgromadzonych w 31. numerze „Rusycy-
stycznych Studiów Literaturoznawczych”, prezentujących zaledwie
wyimek z możliwych ujęć literatury i sztuki jako formy protestu,
nie można nie zadać pytania, czy artystyczne przekazy i akty sprze-
ciwu o często doraźnym, a jednocześnie uniwersalnym i profetycz- RSL.2021.31 s. 3 z 4 nym wydźwięku wystarczą do przebudzenia ludzi, do przełamania
ich inercji, do uzmysłowienia im, że po pięknych, choć trudnych
chwilach zrywu wszystko najczęściej wraca do punktu wyjścia,
tj. stanu pustki po wyzwolonej na chwilę energii. Z pewnością nie. Działania twórcze rzadko wywołują natychmiastową reakcję. Nie
sposób traktować ich jako panaceum na wszystkie bolączki. Ważne
jednak, że nie przemijają one zupełnie bez śladu, stając się utrwa-
lonym aktem oskarżenia wobec opresyjnych zachowań władzy
i systemu, a także prowokacją i inspiracją do kolejnych prób oporu,
buntu, katharsis. ystyczne Studia Literaturoznawcze 2021 (31) tudia Literaturoznawcze 2021 (31) Rusycystyczne Studia Literaturoznawcze 2021 (31) Nie ulega kwestii, że protesty, które w odniesieniu do Rosji i by-
łego ZSRR w niniejszym numerze „Rusycystycznych Studiów Lite-
raturoznawczych” próbujemy uchwycić w locie, to kwestia paląca
i aktualna. Żywimy przeświadczenie, że jej intensywność, podobnie
jak kreatywność protestujących będzie wzrastać, dając badaczom
nie tylko z dziedziny socjologii i politologii pole do działania. Dla
literaturoznawców i kulturoznawców ciekawe jest jak obecne pro-
testy zostaną utrwalone w literaturze i sztuce, po tym jak przetrawi
je już bieżąca publicystyka. Asumpt do konsekwentnego krocze-
nia drogą niezależności i wolności słowa, wierności wyznawanym
zasadom i ideałom, a także potwierdzenie, że świat interesuje się
aktami oporu w tej części globu, stanowi ogłoszona w 2021 roku
pokojowa Nagroda Nobla dla Dmitrija Muratowa, redaktora na-
czelnego „Nowoj Gaziety”, która od lat konsekwentnie piętnuje
wszelkie nadużycia i akty przemocy, dokumentując m.in. sytuację
w wojennej i powojennej Czeczenii. To, jak podkreślił Muratow,
nagroda dla dziennikarzy, ale i obrońców praw człowieka, którzy
za swoją nieprzejednaną postawę zapłacili najwyższą cenę — Anny
Politkowskiej, Natalii Estemirowej, Anastasii Baburowej, Igora Do-
mnikowa, Stanisława Markiełowa, Jurija Szczekoczichina. Rusycystyczne Studia Literaturoznawc Beata Pawletko RSL.2021.31 s. 4 z 4 RSL.2021.31 s. 4 z 4 RSL.2021.31 s. 4 z 4
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The Breadth, but Not the Magnitude, of Circulating Memory B Cell Responses to P. falciparum Increases with Age/Exposure in an Area of Low Transmission
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Abstract Competing Interests: The authors have declared that no competing interests exist Competing Interests: The authors have declared that no competing interests exist. * E-mail: Michael.Walther@nih.gov . These authors contributed equally to this work. ¤ Current address: National Institutes of Health, NIAID, LMIV, Rockville, Maryland, United States of America ¤ Current address: National Institutes of Health, NIAID, LMIV, Rockville, Maryland, United States of America Sarah I. Nogaro1,2, Julius C. Hafalla2, Brigitte Walther1, Edmond J. Remarque3, Kevin K. A. Tetteh4,
David J. Conway1,4, Eleanor M. Riley2., Michael Walther1*.¤ Sarah I. Nogaro1,2, Julius C. Hafalla2, Brigitte Walther1, Edmond J. Remarque3, Kevin K. A. Tetteh4,
David J. Conway1,4, Eleanor M. Riley2., Michael Walther1*.¤ 1 Medical Research Council Laboratories, Fajara, Banjul, The Gambia, 2 Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London
School of Hygiene and Tropical Medicine, London, United Kingdom, 3 Department of Parasitology, Biomedical Primate Research Centre, GJ Rijswijk, The Netherlands,
4 Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom The Breadth, but Not the Magnitude, of Circulating
Memory B Cell Responses to P. falciparum Increases with
Age/Exposure in an Area of Low Transmission PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 Abstract Background: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity
against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that
the development of effective immune responses to malaria may be impaired. Methods: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a
Gambian village where P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells
(MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot. Findings and Conclusions: The proportion of individuals with malaria-specific MBC and antibodies, and the average
number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these
two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence
of malaria exposure (defined as a positive response to at least one malarial antigen either by ELISA or ELISPOT), the median
number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating
memory cell pool for malaria antigens is of similar size to that for other antigens. Citation: Nogaro SI, Hafalla JC, Walther B, Remarque EJ, Tetteh KKA, et al. (2011) The Breadth, but Not the Magnitude, of Circulating M
P. falciparum Increases with Age/Exposure in an Area of Low Transmission. PLoS ONE 6(10): e25582. doi:10.1371/journal.pone.00255 Editor: Lisa Ng Fong Poh, Agency for Science, Technology and Research - Singapore Immunology Network, Singapore Received June 7, 2011; Accepted September 6, 2011; Published October 4, 2011 Received June 7, 2011; Accepted September 6, 2011; Published October 4, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was funded by the Medical Research Council (UK), The Gambia. J.C.H. was a recipient of a Wellcome Trust Visiting Fellowship (grant reference
number 079920) and is currently supported by a University Research Fellowship from The Royal Society (UK). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. Study procedures and baseline characteristics of the
study cohort 118 healthy volunteers from Brefet, Foni District, The Gambia,
were selected using age-stratified randomisation into six different
age categories (Table S1). Venous blood samples were collected as
part of a cross-sectional survey carried out at the end of the dry
season (May–June 2009) over a six-week period. At the time of
sample collections, study participants were healthy and afebrile. Using qualitative P.falciparum PCR, one individual in each of the
1–4, 15–24 and 25–39 year age groups and three individuals in the
10–14 year age group carried parasites, none of which were
detected by slide microscopy. Malaria-Specific MBC in Low Transmission Areas Malaria-Specific MBC in Low Transmission Areas long-lived in adults [14,15,16,17]. Technological advances -
particularly the development of the B cell ELISpot assay to
quantify antibody secreting cells (ASC) as a surrogate of circulating
memory B cells (MBC) [1,18,19] – are now allowing the cellular
basis of humoral immune memory in malaria to be investigated. In
this assay, peripheral blood mononuclear cells (PBMC) are
stimulated with a cocktail of B cell mitogens in order to stimulate
the differentiation of MBC into Ab producing plasma cells (PC) [1]
and the secreted Ig is detected by enzyme immunoassay. Using an
early version of this assay, Dorfman et al. [20], attempted to
identify malaria-specific MBC among PBMC sampled from
malaria-exposed Kenyan children but only very few cells were
detected [20]. Recent refinements of the technique [21], have
enhanced its sensitivity allowing detection of malaria-specific MBC
in children from a high transmission area in Mali [22]. Findings
from that study demonstrated that accumulation of MBC was both
gradual and occurred in a stepwise fashion over many years of
repeated exposure [22]. Using a similar method, malaria-specific
MBC were also detected in adults from a low endemicity setting in
Thailand [15] and these MBC were found to persist - in the
absence of re-exposure - for more than 7 years [15]. Despite the
enhanced sensitivity of the current ELISpot assay, in all of these
studies antigen-specific MBCs could not be detected in a
significant proportion of seropositive individuals. Whether this
reflects a real absence of MBC or insufficient sensitivity of the
assay is currently not clear. At the end of the transmission season, 8 individuals had shown
evidence of exposure based on a$1.5 fold increase to at least one
of the malaria specific Ab tested for (see methods section); one of
those individuals presented with clinical symptoms compatible
with malaria and tested positive by RDT. The proportion of individuals with malaria specific Ab
increases with age g
Samples collected in May 2009 from Brefet were assayed for Ab
specific to MSP-119, MSP-2, MSP-3, AMA-1 and diphtheria
toxoid (DT), and the percentages of individuals having Ab to the
different Ag were plotted for each age group (Figure 1). The
geometric mean anti-diphtheria IgG concentration of the study
population was 26.9 IU/ml (CI 95%:22.2 to 32.6) with all study
participants having .1.4 IU/ml. Since antibody titres .0.1 IU/
ml are regarded by the test manufacturer as a sign of ‘‘good
immunity’’, all participants were classified as responders with
regard to anti-diphtheria Ab. For all four malarial Ags the
proportion of individuals having malaria-specific Ab increased
significantly with age (Chi square test for trend for all Ags:
p,0.0001). AMA-1 was the only Ag for which by the age of $40
years, all individuals had Abs, confirming that AMA-1 is amongst
the most immunogenic of the malarial Ags [16,26]. In comparison,
for MSP-119, considered to be less immunogenic [16,27,28], the
increase with age was less pronounced with Ab prevalence
reaching 60% amongst adults aged $40 years. In this study, in an area in The Gambia where malaria
transmission has declined substantially over the last decade
[23,24], to unprecedentedly low prevalence [25], we find that
the magnitude of the malaria-specific memory B cell response in
children is very similar to that in older individuals and that, in all
age groups, malaria-specific MBC responses are of similar
magnitude to vaccine antigen-specific responses. However, the
prevalence of malaria-specific humoral responses and the breadth
of the response (as judged by the number of antigens recognised by
an individual) increases with age indicating that acquisition of
humoral
immunity
requires
repeated
exposure
to
malaria
infection. Inter-individual variation in the frequency of circulating
MBC Surprisingly little is known about how the total population of
circulating MBCs - as measured by their conversion into IgG ASC
in the B cell ELISpot assay - develops with increasing age and,
thus, cumulative Ag experience. We therefore plotted the number
of ASC per million cultured PBMC for each age group and
examined the data for any change in ASC numbers with
increasing age (Figure 2). Assuming that the number of ASC
detected after in vitro culture is a reliable reflection of the
precursor frequency of circulating MBCs, we find considerable
inter-individual variation in the frequency of MBC in all age
groups (Kruskal Wallis test: p = 0.0024). Although the 1–4 year
olds had significantly lower frequencies of MBC than the 25–39
year olds (p,0.05, Dunn’s post test), logistic regression across all
age groups failed to detect a significant increase with age
(p = 0.086); one reason for this may be the anomalous and
unexplained drop in total MBC numbers in the 15–24 year age
group. However, when data for children above 15 years of age
were pooled and the 10–14 year old age group was used as the
baseline group, the model suggests that the MBC frequency in 10–
14 year olds may be higher than in 1–4 year olds (p = 0.051) but
that there is no difference in MBC frequency between 10–14 year
olds and those aged 15 years or more (p = 0.802). PLoS ONE | www.plosone.org Introduction reducing clinical symptoms was highlighted several decades ago,
by passive immunoglobulin (Ig) transfer experiments [5,6]. A
number of studies have subsequently demonstrated an association
between protection from clinical symptoms of uncomplicated
disease and levels of anti-malarial Ab [7]. However, the frequency
of repeated malaria infections in some children in highly endemic
areas, together with anecdotal accounts of apparent loss of
immunity in the absence of continuing exposure and experimental
evidence of dysfunctional T cell responses, has raised legitimate
questions regarding the duration of immune memory to malaria
[3,8,9]. The immune system’s ability to mount an accelerated humoral
immune response upon repeated encounter of the same pathogen
allows for rapid reduction in disease severity or even complete
sterile immunity [1]. However, in the case of malaria, sterile
immunity that would prevent re-infection is rare; clinical immunity
is thought to be species and strain-specific and repeated infections
are required to develop immune responses specific for the
prevalent antigenic types in the area of residence [2]. The rapidity
with which effective immunity is acquired thus depends on the rate
of transmission [3]. Longitudinal studies showing that titres of many anti-malarial
Ab decline rapidly in children once parasitaemia is cleared after
acute infection [8,10,11,12,13] have contributed to the belief that
humoral memory to malaria may be defective. On the other hand,
there is a growing body of evidence to indicate that Ab responses
become increasingly stable with increasing age [12] and can be The immune control of malaria infection is multi-factorial, and
there is growing consensus that the synergistic action of antibodies
(Ab) and cell mediated effector mechanisms is required for both
anti-parasitic as well as clinical immunity [2,4]. The paramount
importance of Ab in clearing parasitized red blood cells and October 2011 | Volume 6 | Issue 10 | e25582 1 October 2011 | Volume 6 | Issue 10 | e25582 The proportion of individuals with malaria-specific MBC
increases with age Stool analysis to assess carriage of intestinal helminths and
pathogenic gut parasites was carried out on 46 samples, evenly
spread across the different age groups. Two (4.3%) had Giardia
lamblia cysts (individuals stemming from 0–4 and .39 years age
groups) and one (2.2%) had hookworm ova (13 years old). Previous studies that have looked at the frequency of malaria
Ag-specific MBC measured by ELISpot have reported their results
either as the number of malaria-specific spots per million PBMC
seeded into the well (MBC/PBMC) [20,22], or the proportion of
all IgG ASC (%MBC) that are malaria antigen-specific [15,29,30]. The considerable inter-individual variation we observed in overall
MBC frequencies and the possibility that the population of MBC
might increase during childhood, clearly indicates that the choice
of the denominator may affect the interpretation of the data. In Morbidity surveillance was performed during the transmission
season and study participants suspecting they had malaria, or
experiencing symptoms compatible with malaria were asked to
report to the village health worker who performed a rapid
diagnostic test (RDT). Out of eight participants presenting with
symptoms, only one had a positive RDT. PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 2 Malaria-Specific MBC in Low Transmission Areas Figure 1. The proportion of individuals with malaria-specific plasma antibodies increases with age. The percentage of individuals
having Ab levels above the cut-off for AMA-1, MSP-119, MSP-2, MSP-3 or DT are shown for each age group. Data are for samples collected in May–
June 2009, prior to the annual malaria transmission season. N is equal to the number of samples available per age group. P values indicate the result
for the Chi-squared test for trend. doi:10.1371/journal.pone.0025582.g001 Figure 1. The proportion of individuals with malaria-specific plasma antibodies increases with age. The percentage of individuals
having Ab levels above the cut-off for AMA-1, MSP-119, MSP-2, MSP-3 or DT are shown for each age group. Data are for samples collected in May–
June 2009, prior to the annual malaria transmission season. N is equal to the number of samples available per age group. P values indicate the result
for the Chi-squared test for trend. doi:10.1371/journal.pone.0025582.g001 level
of
agreement
with
regards
to
identifying
individual
responders, with kappa values ranging from 0.55 (MSP-2), 0.64
(MSP-119), 0.72 (DT), 0.78 (AMA-1), to 0.81 (MSP-3). this study, we therefore analysed the ELISpot data using both
methods of calculation. The proportion of individuals with malaria-specific MBC
increases with age Unfortunately, due to a temporary
shortage of IgG detection reagents, we were only able to
enumerate the total number of IgG producing cells (and therefore
calculate the % MBC) for 86 of the 118 participants. The breadth of the malaria-specific immune response
increases with age When the data were expressed as MBC/PBMC, the proportion
of individuals with malaria specific MBC above the cut-off
(hereafter referred to as responders) increased significantly with
age for all Ags (Figure 3A). Responder status was defined as
malaria Ag-specific MBC frequencies above the upper limit of the
99% CI of the median MBC/PBMC frequency for the negative
control Keyhole Limpet Hemocyanin (KLH), being 1.88 spots/
million PBMC. A similar trend was observed when data were
expressed as % MBC (Figure 3B; here responder status was
defined as values above the upper limit of the 99% CI of the
median % MBC determined for KLH, being 0.004%), but, apart
from
AMA-1,
the
trends
were
not
statistically
significant. Importantly, the two methods of analysis showed a substantial To assess how the breadth of the immune response to malaria
Ags develops with age, the average number of malaria Ags
recognised by each individual’s plasma or cells was stratified by
age. As shown in Figure 3C, the number of Ags against which
specific plasma IgG or MBC were detected increases significantly
with age (Chi-square test for trend p,0.0001 for ELISA and
MBC/PBMC; and p = 0.0083 for %MBC). Interestingly, from the
age of 10 years, the repertoire of Ags recognised by plasma IgG
was significantly broader than the repertoire recognized by MBC
(expressed as MBC/PBMC; p value for Chi square for each age
strata: ,0.012). When the same comparison was carried out
between ELISA data and ELISpot data expressed as %MBC, the
repertoire of Ags recognized by plasma IgG became significantly
broader from 25 years onwards (p value for Chi square for each
age strata: ,0.0015). Figure 2. Accumulation of MBC with age. The total number of
MBC/million PBMC (as measured by their conversion into IgG producing
cells after culture) were counted for each individual using the B cell
ELISpot technique, and are shown according to age group. The
horizontal lines show the median values for each age group. N equals
the number of tests that were performed per age group. doi:10.1371/journal.pone.0025582.g002 Similar frequencies of malaria-specific and diphtheria-
specific MBCs We were interested to explore i) whether the magnitude of the
malaria-specific humoral and B cell ELISpot responses increases
with age, and ii) whether the magnitude of malaria specific B cell
ELISpot responses differs from DT specific responses. This
requires defining each participant’s prior exposure to the antigens. Based on the 100% prevalence of IgG against diphtheria in the
study group, it can be assumed that every participant has
encountered diphtheria antigen in the past. Determining prior
exposure to malaria is less straightforward, especially in this
community where the very low levels of transmission over the last
10 years mean that the younger individuals may have had very few
(if any) previous malaria infections. Extensive polymorphism of
malarial blood stage antigens means that even among exposed
individuals, prior exposure to the precise antigenic variants used in
our assays cannot be assumed. Also, different individuals may
respond differently to a given antigen [14] and, as we have shown,
circulating IgG antibody may not be detectable despite the
presence of corresponding MBCs, and vice versa. Given the very
low levels of malaria transmission in Brefet (23) we believe it is
justifiable to consider individuals who are seronegative to all four
malaria antigens, and who lack detectable MBC to any of these
four antigens, to be malaria unexposed; conversely individuals who
have IgG or MBC to one or more malaria antigens were
considered to have been exposed. By this definition, 60–80% of
children and .90% of adults had evidence of prior malaria
exposure (Figure 5). Figure 6 shows the magnitude of malaria
specific IgG and MBC responses for all participants considered as
malaria exposed across age groups, and diphtheria responses for
all participants. Consistent with the high coverage of the childhood
immunisation programme in The Gambia [31], anti-diphtheria
plasma IgG concentrations declined during childhood from a peak
in children aged 1–4 years (Kruskal Wallis test, p = 0.004, with
significant p value for post test between 1–4 years and 10–14 years,
Figure 6A), but were relatively stable from age 10 years onwards. However, there was no significant difference in diphtheria-specific
MBC numbers between different age groups (Figure 6F and 6K). By
contrast,
and
as
expected
from
previous
studies,
the
concentration of all malaria-specific antibodies tested increased
with age (Figure 6B–E). F
ll h A
d A
ifi MBC f
i
(
d Figure 3. Lack of correlation between plasma IgG responses and
MBCs detectable by ELISpot y
IgG measured in plasma by ELISA is an indicator of the
presence of antibody-secreting effector cells (plasma cells) in
peripheral tissues or bone marrow whereas ASC detected by
cultured ELISpot assay indicate the presence of circulating MBC. To explore the relationship between these two very different
measures of the humoral immune response, IgG concentrations
were plotted against MBC numbers for all subjects for each Ag
(Figure 4A–E) and a correlation coefficient was calculated using
data points for which one or both variables were above the
respective cut-offs. No significant positive correlation was detected
between MBC and Ab for any of the Ags tested, regardless
whether ELISpot results were expressed as %MBC (Figure 4) or as
MBC/PBMC (data not shown). To explore this data further, we classified each individual’s
responses to all 4 malaria Ags into the following categories: MBC+
IgG+ (that is, a response in both the ELISpot and the ELISA was
seen), MBC+ IgG2 (ELISpot response but no Ab), MBC2 IgG+
(no ELISpot response but Ab) or MBC2 IgG2 (no response in
either of the tests). The frequency with which each category
occurred in each age group was then expressed as a proportion Figure 2. Accumulation of MBC with age. The total number of
MBC/million PBMC (as measured by their conversion into IgG producing
cells after culture) were counted for each individual using the B cell
ELISpot technique, and are shown according to age group. The
horizontal lines show the median values for each age group. N equals
the number of tests that were performed per age group. doi:10.1371/journal.pone.0025582.g002 PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 3 Malaria-Specific MBC in Low Transmission Areas Figure 3. Prevalence and antigenic breadth of the malaria-
specific MBC response increases with age. The percentage of
individuals in each age group with detectable MBC specific for the
different malaria Ags and for DT are shown as (A) number of MBC/
PBMC, and as (B) % of all MBC. P-values for a Chi-squared test for trend
are shown. N equals the number of samples tested per age group. The
average number of Ags for which individuals had specific Ab (open
bars) or MBC (hatched bars = MBC/PBMC; black bars = %ASC) are shown,
by age group, in (C). Lack of correlation between plasma IgG responses and
MBCs detectable by ELISpot For all three parameters, the Chi-squared test for
trends indicated a significant increase with age (p,0.0001 for Ab and
MBC/PBMC, and p = 0.0083 for %MBC). Positive responses to individual
antigens were defined as described in methods. doi:10.1371/journal.pone.0025582.g003 with increasing age (Chi squared test for trend: ,0.0001)
suggesting that antibody secreting plasma cells persist even though
the frequency of circulating MBC remains below the threshold of
detection. Similar analysis was performed for responses to DT (Figure 4G). Since all participants had anti-diphtheria Ab, only two categories
(MBC+ IgG+ and MBC2 IgG+) existed. With increasing age,
significantly more responses were classified as MBC+ IgG+ (Chi
squared test for trend: 0.009). Since diphtheria vaccination is only
given during childhood and natural exposure to diphtheria is now
extremely rare in The Gambia [31], we speculate that this change
reflects either age-dependent qualitative changes in the immune
response or recurrent bystander B cell activation (driving DT-
specific MBC into periodic clonal expansion) rather than repeated
exposure to diphtheria Ag. The magnitude of MBC responses expressed as % MBC were
only poorly correlated with numbers of MBC/PBMC (Table 1),
reflecting the considerable inter-individual variation in total MBC
numbers. Similar frequencies of malaria-specific and diphtheria-
specific MBCs Prevalence and antigenic breadth of the malaria-
specific MBC response increases with age. The percentage of
individuals in each age group with detectable MBC specific for the
different malaria Ags and for DT are shown as (A) number of MBC/
PBMC, and as (B) % of all MBC. P-values for a Chi-squared test for trend
are shown. N equals the number of samples tested per age group. The
average number of Ags for which individuals had specific Ab (open
bars) or MBC (hatched bars = MBC/PBMC; black bars = %ASC) are shown,
by age group, in (C). For all three parameters, the Chi-squared test for
trends indicated a significant increase with age (p,0.0001 for Ab and
MBC/PBMC, and p = 0.0083 for %MBC). Positive responses to individual
antigens were defined as described in methods. doi:10.1371/journal.pone.0025582.g003 and is plotted in Figure 4F for all the malaria Ags combined and
Figure 4G for DT, using MBC/PBMC for the ELISpot data. and is plotted in Figure 4F for all the malaria Ags combined and
Figure 4G for DT, using MBC/PBMC for the ELISpot data. The proportion of responses to malaria Ags categorized as
MBC+ IgG+ increases with age, while the proportion of responses
classified as MBC2 IgG2 decreases (Chi squared test for trend,
p,0.0001
for
both), which
presumably
reflects cumulative
exposure to malaria over time. Interestingly however, the
proportion of responses classified as MBC2 IgG+ also increased The proportion of responses to malaria Ags categorized as
MBC+ IgG+ increases with age, while the proportion of responses
classified as MBC2 IgG2 decreases (Chi squared test for trend,
p,0.0001
for
both), which
presumably
reflects cumulative
exposure to malaria over time. Interestingly however, the
proportion of responses classified as MBC2 IgG+ also increased For all the Ags tested, Ag-specific MBC frequencies (expressed
as %MBC) did not vary significantly across age groups (Figure 6F–
J). With the exception of MSP-119 (Figure 6M), for which a PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 4 Malaria-Specific MBC in Low Transmission Areas Malaria-Specific MBC in Low Tr Figure 4. Plasma Ab levels do not correlate with MBC numbers. A–E: correlation plots for Ab versus % MBC. The cut-off values
responders are shown by dotted lines parallel to each axis. The degree of correlation was assessed by calculating Spearman’s correlation coeffi
points where one or both values were above the cut-off. Similar frequencies of malaria-specific and diphtheria-
specific MBCs F–G: Each individual’s response to each Ag were classified as: MBC+ IgG+ (that is,
both the ELISpot and the ELISA was seen), MBC+ IgG2 (ELISpot response but no Ab), MBC2 IgG+ (no ELISpot response but Ab) or MBC
response in either of the tests). The frequency with which each category occurred in each age group was then expressed as a proportion and
F) malaria Ag and G) DT. Responder status for MBC was determined using MBC/PBMC. P test for trend values are indicated. doi:10.1371/journal.pone.0025582.g004
PL S ONE |
l
5
O t b
2011 | V l
6 | I Figure 4. Plasma Ab levels do not correlate with MBC numbers. A–E: correlation plots for Ab versus % MBC. The cut-off values for defining
responders are shown by dotted lines parallel to each axis. The degree of correlation was assessed by calculating Spearman’s correlation coefficient for data
points where one or both values were above the cut-off. F–G: Each individual’s response to each Ag were classified as: MBC+ IgG+ (that is, a response in
both the ELISpot and the ELISA was seen), MBC+ IgG2 (ELISpot response but no Ab), MBC2 IgG+ (no ELISpot response but Ab) or MBC2 IgG2 (no
response in either of the tests). The frequency with which each category occurred in each age group was then expressed as a proportion and is plotted for
F) malaria Ag and G) DT. Responder status for MBC was determined using MBC/PBMC. P test for trend values are indicated. doi:10.1371/journal.pone.0025582.g004 October 2011 | Volume 6 | Issue 10 | e25582 5 Malaria-Specific MBC in Low Transmission Areas Table 1. Correlation between the two methods of calculating
Ag-specific MBC. n
r2
p
AMA-1
33
0.297
0.090
MSP-119
30
0.237
0.207
MSP-2
29
0.462
0.012
MSP-3
22
0.135
0.550
DT
44
0.288
0.058
The magnitude of the MBC response, expressed either as MBC/PBMC or % MBC
was assessed for correlation. n: number of pairs tested for each Ag; r2:
Spearman correlation coefficient. doi:10.1371/journal.pone.0025582.t001 infections are assumed to have been asymptomatic. PCR carried
out at the end of the transmission period detected P. falciparum
DNA in only one of these 8 subjects. A ‘‘rising titre’’ of serum Ab is
widely used as a diagnostic tool and suggests that these 8
individuals were indeed infected with malaria. Effect of recent malaria exposure on malaria-specific
plasma IgG antibodies and MBCs A recent study reported an increased frequency of dysfunctional
and exhausted MBCs (designated atypical memory B cells, AMB)
in HIV-infected patients compared to healthy individuals [32]. B
cells with a similar surface phenotype (CD19+ CD272 CD212 and
CD102) have also been reported to be more prevalent in adults
from an area with high malaria transmission (Mali) than in
malaria-naı¨ve volunteers (from the USA), and their proportion was
higher among asymptomatic parasite carriers than among non-
parasitized individuals [21]. This prompted us to examine the
frequency of AMB with this phenotype in 16 children aged 1–15
years, who were followed up as part of an ongoing malaria case-
control study [33,34]. All children had a confirmed episode of
malaria 2 months previously, but were parasite free at the time of
blood collection, and live in an area of low malaria endemicity at
the Gambian coast [24,25]. The median proportion of B cells
expressing the AMB phenotype (CD19+ CD272 CD212 and
CD102; Figure 9A) was 4.71% (CI 95%: 2.48–6.41%) with no
significant difference between children below and above the age of
5 years (Figure 9B). This compares to AMB frequencies of 15.5%
in Malian adults, 9.8% in Malian children (aged 2–10 years) and
1.6% in U.S. adults [21], supporting the hypothesis that malaria
induces AMB in an exposure-dependent manner. Interestingly,
the proportion of classical MBCs (CD19+ CD27+ CD21+) amongst
all B cells was twice as high in children aged more than 5 years
than in children aged less than 5 years (Figure 9B), consistent with
the age-related increase in the repertoire of antigens recognised by
the MBC population. To capture evidence of malaria exposure during the transmis-
sion season we performed active morbidity surveillance of our
study population from August 2009 till December 2009. Only one
symptomatic case with a positive RDT was detected (in a 5 year
old child). In addition, we repeated the serological survey in
December 2009 and tested these samples together with those
collected in May 2009. Taking the study population as a whole we
observed small, but significant, decreases in the median concen-
trations of Abs to AMA-1, MSP-119, MSP-2 and MSP-3
(Figure 8A), confirming the lack of recent re-exposure to malaria
in the majority of subjects. Effect of recent malaria exposure on malaria-specific
plasma IgG antibodies and MBCs However, 8 (including the 1 RDT positive case) out of 99
participants that were tested showed a$1.5 fold increase in plasma
IgG levels to at least one malaria Ag suggesting that they may have
recently experienced a malaria infection; 7 out of 8 of these Figure 5. Evidence of malaria exposure in Brefet increases with
age. The percentage of individuals showing a response above the
respective cut off to at least one of the malarial antigens tested either in
the ELISA or the ELISpot assays is shown, stratified according to age. Chi-squared test for trends indicated a significant increase with age
(p = 0.0157). doi:10.1371/journal.pone.0025582.g005 Similar frequencies of malaria-specific and diphtheria-
specific MBCs However, despite
the fact that (apart from the one RDT positive case) none of them
presented with any signs of illness during the study period, we
cannot entirely rule out that the increased malaria Ab titres may
be the result of bystander activation of malaria-specific B cells by
another asymptomatic infection. Table 1. Correlation between the two methods of calculating
Ag-specific MBC. Nevertheless, when we compared the change in frequency (pre-
and post-malaria season) of malaria-specific MBCs among the 8
subjects who had evidence of a recent malaria infection (cases)
with the change in MBC frequency in 8 age-matched controls with
no evidence of recent infection, the post/pre-season ratio for
malaria-specific MBCs (calculated as a % of all MBC) tended to be
higher in cases than in controls (Figure 8B). While the low
numbers of cases precludes the drawing of any firm conclusions,
this analysis does suggest that MBC numbers can be boosted in the
same manner as serum IgG concentrations. Interestingly, the
post/pre-season ratio for diphtheria-specific MBCs was also
significantly higher in cases than in controls, suggesting that
recent infection may have resulted in polyclonal, antigen-
independent, ‘‘bystander’’ activation of diphtheria-specific MBCs. The magnitude of the MBC response, expressed either as MBC/PBMC or % MBC
was assessed for correlation. n: number of pairs tested for each Ag; r2:
Spearman correlation coefficient. doi:10.1371/journal.pone.0025582.t001 significant decrease in MBC numbers was observed between age
groups 10–14 and 15–24 years, the same result was obtained when
MBC were expressed as MBC/PBMC (Figure 6K, L, N and O). Of note, the magnitude of malarial Ag -specific MBC responses
amongst those with evidence of previous malaria exposure was not
different from that for diphtheria, irrespective of the chosen
denominator (Kruskal Wallis test p = 0.08 [MBC/PBMC], or
p = 0.178 [%MBC]; Figure 7A, B). significant decrease in MBC numbers was observed between age
groups 10–14 and 15–24 years, the same result was obtained when
MBC were expressed as MBC/PBMC (Figure 6K, L, N and O). Of note, the magnitude of malarial Ag -specific MBC responses
amongst those with evidence of previous malaria exposure was not
different from that for diphtheria, irrespective of the chosen
denominator (Kruskal Wallis test p = 0.08 [MBC/PBMC], or
p = 0.178 [%MBC]; Figure 7A, B). Discussion The advent of methods to enumerate circulating MBC [18] has
enabled researchers to begin to explore the induction and
maintenance of humoral immune responses to malaria in humans
[15,20,22]. While the B cell ELISpot may still benefit from further
improvements to its sensitivity, the assay is sensitive enough to
detect durable MBC responses to both P.falciparum and P.vivax in
infrequently exposed Thai adults [15] and, in a recent report from
a rural village in Mali where people are exposed to ,50 infective Figure 5. Evidence of malaria exposure in Brefet increases with
age. The percentage of individuals showing a response above the
respective cut off to at least one of the malarial antigens tested either in
the ELISA or the ELISpot assays is shown, stratified according to age. Chi-squared test for trends indicated a significant increase with age
(p = 0.0157). p
doi:10.1371/journal.pone.0025582.g005 PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 6 Malaria-Specific MBC in Low Transmission Areas Figure 6. The magnitude of the malaria antigen-specific MBC response does not increase with age. The magnitude of Ag-specific serum
IgG (A–E) and of Ag-specific MBC responses [expressed as %MBC (F–J) or expressed as MBC/PBMC (K–O)] are shown for individuals who showed
evidence of previous malaria exposure (as defined in Figure 5). Box plots indicate the 25th, 50st and 75th percentile, with whiskers representing the
Malaria-Specific MBC in Low Transmission Areas Figure 6. The magnitude of the malaria antigen-specific MBC response does not increase with age. The magnitude of Ag-specific serum
IgG (A–E) and of Ag-specific MBC responses [expressed as %MBC (F–J) or expressed as MBC/PBMC (K–O)] are shown for individuals who showed
evidence of previous malaria exposure (as defined in Figure 5). Box plots indicate the 25th, 50st and 75th percentile, with whiskers representing the
10th and 90th percentiles. Outliers are denoted by a spot. P values are given for Kruskal-Wallis tests. doi:10.1371/journal.pone.0025582.g006 Figure 6. The magnitude of the malaria antigen-specific MBC response does not increase with age. The magnitude of Ag-specific serum
IgG (A–E) and of Ag-specific MBC responses [expressed as %MBC (F–J) or expressed as MBC/PBMC (K–O)] are shown for individuals who showed
evidence of previous malaria exposure (as defined in Figure 5). Box plots indicate the 25th, 50st and 75th percentile, with whiskers representing the
10th and 90th percentiles. Outliers are denoted by a spot. P values are given for Kruskal-Wallis tests. Discussion Values below the antigen-specific cut-off (as
described in the methods) were given the cut-off value. Box plots
indicate the 25th, 50st and 75th percentile, with whiskers representing
the 10th and 90th percentiles. Outliers are indicated by spots. (B)
Comparisons of the ratio of post-season MBC to pre-season MBC for
cases (individuals having a.1.5 fold increased Ab concentration post
season for at least one of the malarial Ags) and controls. P values are
given for the Mann-Whitney test, adjusted for multiple comparisons
using the Bonferroni method. doi:10.1371/journal.pone.0025582.g008 Figure 7. Naturally acquired MBC responses to malaria
antigens are similar in magnitude to vaccine-induced MBC
responses to DT. Ag-specific ELISpot responses of individuals with
immunological evidence of prior malaria exposure are shown and
expressed as (A) MBC/PBMC and (B) % MBC, for each Ag. The median for
each group is indicated by the horizontal line. The p value (Kruskal
Wallis test) for (A) = 0.08, and (B) = 0.178. doi:10.1371/journal.pone.0025582.g007 Figure 8. Boosting of circulating MBC numbers by recent
malaria infection. (A) Pre- and post-malaria season plasma Ab
concentrations to the different malaria Ags are shown for all
participants for whom paired samples were available (n = 99), pooled
across all age groups. Values below the antigen-specific cut-off (as
described in the methods) were given the cut-off value. Box plots
indicate the 25th, 50st and 75th percentile, with whiskers representing
the 10th and 90th percentiles. Outliers are indicated by spots. (B)
Comparisons of the ratio of post-season MBC to pre-season MBC for
cases (individuals having a.1.5 fold increased Ab concentration post
season for at least one of the malarial Ags) and controls. P values are
given for the Mann-Whitney test, adjusted for multiple comparisons
using the Bonferroni method. doi:10.1371/journal.pone.0025582.g008 Figure 7. Naturally acquired MBC responses to malaria
antigens are similar in magnitude to vaccine-induced MBC
responses to DT. Ag-specific ELISpot responses of individuals with
immunological evidence of prior malaria exposure are shown and
expressed as (A) MBC/PBMC and (B) % MBC for each Ag The median fo Figure 7. Naturally acquired MBC responses to malaria
antigens are similar in magnitude to vaccine-induced MBC
responses to DT. Ag-specific ELISpot responses of individuals with
immunological evidence of prior malaria exposure are shown and
expressed as (A) MBC/PBMC and (B) % MBC, for each Ag. The median for
each group is indicated by the horizontal line. Discussion doi:10.1371/journal.pone.0025582.g006 October 2011 | Volume 6 | Issue 10 | e25582 PLoS ONE | www.plosone.org 7 7 Malaria-Specific MBC in Low Transmission Areas mosquito bites per month during the peak of the transmission
season, to demonstrate that the P. falciparum-specific MBC
compartment expands in a step-wise fashion with increasing age
and/or repeated malaria infections [22]. Using a similar assay, we
have now described the acquisition of humoral immunity to four
malaria blood stage antigens (selected on the basis of their
immunogenicity and potential functional relevance) in a rural
community in The Gambia where malaria transmission has
declined sharply in the last 15 years [23,35] and where only ,8%
of study subjects appear to have been exposed to malaria infection
in the 2009 rainy season. The absence of anti-MSP-119 specific
IgG in children ,10 years in December 2009 [23], the high
coverage with insecticide treated nets (ITNs), and the virtual
absence of clinical infections in the study cohort all support the
assumption that malaria transmission in this community has been
extremely low for several years. Rather surprisingly, given the huge differences in malaria
endemicity between the two sites, the age-specific prevalence of
both Ab and MBC in The Gambia was remarkably similar to the
prevalence reported in Mali [22]. Although there are some slight
differences in methodology between the studies (age grouping,
d fi i i
f
ff
l
f f
h
d
ll ) h
lation of humoral immunity, and the findings of Wipasa et al. [15]
of very long-lived B cell memory in areas of exceptionally low
transmission, it may be that relatively infrequent exposure to
malaria is as effective (or more effective) at inducing long-lived
Figure 7. Naturally acquired MBC responses to malaria
antigens are similar in magnitude to vaccine-induced MBC
responses to DT. Ag-specific ELISpot responses of individuals with
immunological evidence of prior malaria exposure are shown and
expressed as (A) MBC/PBMC and (B) % MBC, for each Ag. The median for
each group is indicated by the horizontal line. The p value (Kruskal
Wallis test) for (A) = 0.08, and (B) = 0.178. doi:10.1371/journal.pone.0025582.g007
Figure 8. Boosting of circulating MBC numbers by recent
malaria infection. (A) Pre- and post-malaria season plasma Ab
concentrations to the different malaria Ags are shown for all
participants for whom paired samples were available (n = 99), pooled
across all age groups. PLoS ONE | www.plosone.org Discussion The p value (Kruskal
Wallis test) for (A) = 0.08, and (B) = 0.178. doi:10.1371/journal.pone.0025582.g007 mosquito bites per month during the peak of the transmission
season, to demonstrate that the P. falciparum-specific MBC
compartment expands in a step-wise fashion with increasing age
and/or repeated malaria infections [22]. Using a similar assay, we
have now described the acquisition of humoral immunity to four
malaria blood stage antigens (selected on the basis of their
immunogenicity and potential functional relevance) in a rural
community in The Gambia where malaria transmission has
declined sharply in the last 15 years [23,35] and where only ,8%
of study subjects appear to have been exposed to malaria infection
in the 2009 rainy season. The absence of anti-MSP-119 specific
IgG in children ,10 years in December 2009 [23], the high
coverage with insecticide treated nets (ITNs), and the virtual
absence of clinical infections in the study cohort all support the
assumption that malaria transmission in this community has been
extremely low for several years. mosquito bites per month during the peak of the transmission
season, to demonstrate that the P. falciparum-specific MBC
compartment expands in a step-wise fashion with increasing age
and/or repeated malaria infections [22]. Using a similar assay, we
have now described the acquisition of humoral immunity to four
malaria blood stage antigens (selected on the basis of their
immunogenicity and potential functional relevance) in a rural
community in The Gambia where malaria transmission has
declined sharply in the last 15 years [23,35] and where only ,8%
of study subjects appear to have been exposed to malaria infection
in the 2009 rainy season. The absence of anti-MSP-119 specific
IgG in children ,10 years in December 2009 [23], the high
coverage with insecticide treated nets (ITNs), and the virtual
absence of clinical infections in the study cohort all support the
assumption that malaria transmission in this community has been
extremely low for several years. Figure 8. Boosting of circulating MBC numbers by recent
malaria infection. (A) Pre- and post-malaria season plasma Ab
concentrations to the different malaria Ags are shown for all
participants for whom paired samples were available (n = 99), pooled
across all age groups. Values below the antigen-specific cut-off (as
described in the methods) were given the cut-off value. Box plots
indicate the 25th, 50st and 75th percentile, with whiskers representing
the 10th and 90th percentiles. Outliers are indicated by spots. October 2011 | Volume 6 | Issue 10 | e25582 Discussion that we observed in the number of Ags recognised with increasing
age indicates that the antigenic breadth (and likely, therefore, the
functional relevance) of the B cell response requires at least some
degree of repeated exposure to malaria. The underlying biology of
these observations clearly deserves further investigation. A caveat to these interpretations, however, is that the similarity
of MBC responses in the different studies may reflect some
limitation of the assay system rather than a true biological
phenomenon. For example, we find for all Ags, including
diphtheria, that although the prevalence of responses varies, the
magnitude of the MBC responses (in those classified as exposed) is
similar in all age groups. One potential explanation that deserves
investigation is that, even in individuals with long-lived humoral
immune memory, the maximum precursor frequency of circulat-
ing MBCs for any individual Ag is in the order of 1 MBC per
million PBMCs and that the spot frequency of 5–10 spots per
million PBMC simply reflects the differentiation of these very few
MBCs during the 6 days of mitogenic stimulation into MBC. In
other words, it may be necessary to seed ELISpot cultures with
many more PBMCs in order to detect the true frequency and
prevalence of circulating Ag-specific MBCs. Nevertheless, the
similar magnitude of the MBC response to malaria Ags and to DT,
a protein of fairly similar length to the malaria Ags used, implies
that mounting a B cell response to malaria Ags is not necessarily
any more difficult than mounting a B cell response to other Ags. Although it is possible that numbers of DT-specific MBC may
have been much higher immediately after vaccination in infancy,
the lack of any detectable decline in DT-specific MBC frequency
with increasing age tends to argue against this possibility. As in most studies of human immune responses, access to the
tissues of real interest - in this case spleen and bone marrow - is
restricted and observations from studies of PBMC need to be
interpreted with caution. In our study, the presence of detectable
Abs and MBC did not correlate for any of the Ags tested, and a
significant proportion of individuals lacked detectable circulating
MBCs despite having significant titres of the corresponding plasma
Ab. Discussion To identify atypical MBCs, CD102 cells were subsequently selected from CD212CD272 cells, using a CD10 gate defined on the entire PBMC
population. (B) Percentages of CD19+ cells expressing the atypical and classical MBC phenotype are shown for children ,5 and .5 years of age. P-values are given for the Mann-Whitney test. doi:10.1371/journal.pone.0025582.g009 Figure 9. Atypical and classical memory B cells in children 2 months after a malaria episode. AMB were defined as being
CD19+CD212CD272CD102, classical MBC were defined as CD19+CD21+CD27+ [21]. The gating strategy is shown in (A). Viable PBMC were gated using
FSC/SSC (not shown) and then displayed according to CD19+ expression. The CD19+ population was displayed according to their expression of CD21
and CD27. To identify atypical MBCs, CD102 cells were subsequently selected from CD212CD272 cells, using a CD10 gate defined on the entire PBMC
population. (B) Percentages of CD19+ cells expressing the atypical and classical MBC phenotype are shown for children ,5 and .5 years of age. P-values are given for the Mann-Whitney test. doi:10.1371/journal.pone.0025582.g009 15–24 year age group. The reason for this drop is unclear but is
highly unlikely to be assay or operator dependent since a random
selection of participants across all age groups were tested on each
day of the study and the range of values on plates that included
low-responding 15–24 years olds did not differ systematically from
the ranges on other plates. Given the very low prevalence of HIV
infection in this community (,2%) [36] this drop is unlikely to be
related to undiagnosed HIV infection. Further studies in this age
group are required. In any event, increasing total MBC numbers
are likely to lead to an underestimation of Ag-specific responses
when expressed as %MBC. This may be of particular relevance in
diseases where the compartment of IgG-producing cells is
expanded by means of polyclonal stimulation as has been
suggested
for
malaria
[37,38]. For
instance,
in
the
high
transmission area in Mali, an extremely marked increase in total
MBC numbers with age was observed [22], and reporting results
from such a setting as % MBC would further distort the
relationship between age and malaria specific memory responses. The number of PBMC per ml of blood is more stable. For this
reason, we suspect that MBC/PBMC may better reflect the
precursor frequency of malaria specific MBC in the peripheral
blood, but further studies are required to be certain that this is the
case. Discussion Another caveat of the B cell ELISpot assay is that we do not
know whether the stimulation medium differentiates MBC into
ASC at a fixed ratio or whether this is a true reflection of what
happens in vivo upon encounter with an Ag. Moreover, for different
types of Ags the efficiency of the differentiation of MBC into ASC
may be different. A direct comparison of MBC frequencies derived
from the ELISpot assay with MBC precursor frequencies derived
from limiting dilution assays or from antigen-specific flow
cytometry (using chromophore-labled antigen) is urgently needed
to validate the accuracy and sensitivity of the B cell ELISpot assay. Related to this is the question of whether B cell ELISpot data
should be presented as the proportion of all MBCs that are specific
for the Ag of interest (% MBC) [15,29,30] or as the number of
MBCs among all the PBMCs seeded into the plate (MBC/PBMC)
[20,22]. Our data indicate that for a given number of PBMC,
there is considerable inter-individual variation in the total number
of MBC. Further, the total number of MBC may increase with age
during childhood, presumably as a result of exposure to an ever-
increasing number of Ags. Indeed, the total number of MBCs
detected in the ELISpot assay tended to be higher in slightly older
children than in very young children and, by flow cytometry, the
proportion of all B cells that are classical MBCs was also higher in
older children than in younger children. Although we could not
detect any further expansion of total MBC numbers from
adolescence into adulthood, suggesting that MBC numbers may
reach an equilibrium in late childhood, our power to detect such a
trend was limited by the anomalous drop in MBC numbers in the Related to this is the question of whether B cell ELISpot data
should be presented as the proportion of all MBCs that are specific
for the Ag of interest (% MBC) [15,29,30] or as the number of
MBCs among all the PBMCs seeded into the plate (MBC/PBMC)
[20,22]. Our data indicate that for a given number of PBMC,
there is considerable inter-individual variation in the total number
of MBC. Further, the total number of MBC may increase with age
during childhood, presumably as a result of exposure to an ever-
increasing number of Ags. Discussion (B)
Comparisons of the ratio of post-season MBC to pre-season MBC for
cases (individuals having a.1.5 fold increased Ab concentration post
season for at least one of the malarial Ags) and controls. P values are
given for the Mann-Whitney test, adjusted for multiple comparisons
using the Bonferroni method. d i 10 1371/j
l
0025582 008 Rather surprisingly, given the huge differences in malaria
endemicity between the two sites, the age-specific prevalence of
both Ab and MBC in The Gambia was remarkably similar to the
prevalence reported in Mali [22]. Although there are some slight
differences in methodology between the studies (age grouping,
definition of cut off values, use of fresh or cryopreserved cells) the
similarity in the results is striking. Given the findings of Weiss et al. [22] that acute malaria infections lead to only transient expansion
of the malaria-specific MBC pool and that the subsequent
contraction of the MBC pool results in very inefficient accumu- lation of humoral immunity, and the findings of Wipasa et al. [15]
of very long-lived B cell memory in areas of exceptionally low
transmission, it may be that relatively infrequent exposure to
malaria is as effective (or more effective) at inducing long-lived
humoral immunity than is persistent re-infection. If so, we perhaps
need to consider the possibility that repeated exposure to antigen
gradually drives MBC to differentiate into tissue resident cells
(either ASCs or non-circulating memory cells) or – possibly –
causes clonal exhaustion. On the other hand, the clear increase October 2011 | Volume 6 | Issue 10 | e25582 October 2011 | Volume 6 | Issue 10 | e25582 8 Malaria-Specific MBC in Low Transmission Areas PLoS ONE | www.plosone.org
9
October 2011 | Volume 6 | October 2011 | Volume 6 | Issue 10 | e25582 October 2011 | Volume 6 | Issue 10 | e25582 PLoS ONE | www.plosone.org Malaria-Specific MBC in Low Transmission Areas Figure 9. Atypical and classical memory B cells in children 2 months after a malaria episode. AMB were defined as being
CD19+CD212CD272CD102, classical MBC were defined as CD19+CD21+CD27+ [21]. The gating strategy is shown in (A). Viable PBMC were gated using
FSC/SSC (not shown) and then displayed according to CD19+ expression. The CD19+ population was displayed according to their expression of CD21
and CD27. Discussion While this may reflect insufficient sensitivity of the assay, it is
also highly plausible that ASCs present in tissues (either as short-
lived plasma cells (SLPC) in spleen or as SLPC or long-lived
plasma cells (LLPC) in bone marrow) continue to secrete Abs even
though the frequency of circulating MBCs has fallen below the
lower limit of detection of the ELISpot assay. A similar lack of
correlation between MBC and serum Abs was observed in the
Thai study [15], where Ag exposure is infrequent, but not in Mali
[22], where re-exposure to Ag is very common. These observa-
tions, together with the evidence from our comparison of malaria
cases and controls in The Gambia, suggest that both Abs and
circulating MBC numbers are boosted by re-infection but that, in
the absence of boosting, circulating MBC numbers decline to
undetectable levels even though tissue resident LLPC continue to
secrete Ab. Our observation that the proportion of individuals
with Abs but no detectable circulating MBC increases with age - at
least for the blood stage malaria Ags tested here - suggests that
older people are less likely to have been recently infected, perhaps
indicating
a
degree
of pre-erythrocytic
immunity
in these
individuals. However, age-dependent differences in immune
responses may also contribute to this effect. Furthermore, due to
the marked reduction in malaria endemicity in The Gambia in the
last decade, the malaria exposure of children in this community is
not only of shorter duration than in adults but is also qualitatively
very different from that experienced in childhood by those who are
now adults. Another caveat of the B cell ELISpot assay is that we do not
know whether the stimulation medium differentiates MBC into
ASC at a fixed ratio or whether this is a true reflection of what
happens in vivo upon encounter with an Ag. Moreover, for different
types of Ags the efficiency of the differentiation of MBC into ASC
may be different. A direct comparison of MBC frequencies derived
from the ELISpot assay with MBC precursor frequencies derived
from limiting dilution assays or from antigen-specific flow
cytometry (using chromophore-labled antigen) is urgently needed
to validate the accuracy and sensitivity of the B cell ELISpot assay. PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 Malaria-Specific MBC in Low Transmission Areas Adult HIV prevalence in rural areas of The Gambia is estimated
to be approx 2% in 2009 [36]; thus undiagnosed HIV infection is
unlikely to have markedly affected the immune responses reported
here. Stool samples from 38% of randomly selected study
participants were also collected to assess worm carriage (Table S1). In summary, although the prevalence of individuals with
malaria-specific MBCs increases with increasing age (and/or
malaria exposure) the magnitude of an individual’s malaria specific
MBC response is similar in children with minimal prior malaria
exposure to adults with a considerably higher cumulative malaria
exposure. However, the antigenic repertoire of the B cell response
is more limited in children than in adults and only increases with
increasing exposure. Since the magnitude of the malaria-specific B
cell response is similar to that induced by diphtheria vaccination,
we find no evidence that mounting a B cell response to malarial
antigens is more difficult than to other antigens. Nevertheless, the
possibility remains that the development and/or retention of
malaria specific memory B cells is more efficient in areas of low
malaria transmission, such as The Gambia or Thailand, than in
very highly endemic areas such as Mali. If so, this might be
explained either by competition for limited numbers of environ-
mental niches for MBC survival, or by preferential induction of
SLPC rather than LLPCs, in frequently infected individuals. A village health worker was present in Brefet throughout the
transmission season to diagnose malaria infections by use of RDT
(OptiMALH) for malaria. Study participants had been asked to
report to the village health centre if they suspected they had
malaria. Anti-malarial treatment was only given if the rapid
diagnostic test was positive. At the end of the transmission season (December 2009), a finger
prick sample from each study participant was obtained to assess i)
anti-malarial Ab levels, and ii) parasitaemia levels by PCR. Samples from 20 individuals were missing (18 had travelled away
from the village and 2 had withdrawn their consent). A week later,
a further venous sample for B cell ELISpot was requested from
those individuals who showed a$1.5 fold increase in Ab
concentration for at least 1 Ag in comparison to the value
recorded at the start of the malaria season in May and/or for
whom a clinical episode of malaria during the last transmission
season was confirmed by a positive RDT. Malaria-Specific MBC in Low Transmission Areas Malaria-Specific MBC in Low Transmission Areas B cell mitogenic activity has long been ascribed to P. falciparum
[37] and has been invoked as the underlying cause of bystander
activation of unrelated B cells [38] and of the hypergammaglob-
ulinaemia commonly described in highly malaria endemic areas
[39–40]. While both the Malian study by Weiss et al. [22] and the
present study confirm a bystander effect of acute malaria (boosting
tetanus and diphtheria responses respectively), it is far from clear
that this is due to the activity of a parasite-derived mitogen as
opposed to generalised cytokine-mediated B cell activation. November [23]. The entomological inoculation rate in the area of
Brefet was estimated as 3.24 infective mosquito bites per person
per year in 1991 [42], as 0.92 in 2001 [43], and as 0.62 in 2006
[35]. At the time of this survey (in 2009), 82% of beds in Brefet
were covered with an insecticide treated bed net. Villagers were grouped according to age into the following six
age categories (1–4, 5–9, 10–14, 15–24, 25–39 and .40 years,
respectively) and 20 individuals from each group were randomly
selected. To avoid household clustering, all volunteers in each
group received consecutive numbers, the total number of villagers
per group was noted and out of this figure a random number
generator drew 20 numbers to identify 20 individuals per age
group. For individuals absent on the day of sampling, a new
number was drawn. There was an equal distribution of males and
females in each group. The functional significance of the relatively high frequencies of
cells with the phenotype of so-called ‘‘atypical MBCs’’ in malaria-
exposed individuals is not known. One potential explanation is
that these cells - which have a substantially shorter life span than
classical MBC [32] – have been displaced from bone marrow
niches as a result of intermittent polyclonal bystander activation of
B cells (after a transient malaria infection, for example) and are
destined to die. The observation that such atypical cells are present
at much higher frequencies in the high transmission area in Mali
[22] than in our low transmission setting or in another area of low
transmission in Peru [41] further supports this notion. Malaria-Specific MBC in Low Transmission Areas The fact
that the age-related expansion of classical MBCs that we observed
is not accompanied by a similar expansion of atypical MBCs in a
low endemicity setting tends to support the hypothesis that the
frequency of atypical MBCs reflects cumulative malaria exposure
and is not just a function of increasing age but, currently, it is not
possible to rule out other chronic or recurrent infections (such as
helminths, for example), nutritional status or other environmental
exposures as contributory causes of this atypical B cell phenotype. females in each group. A venous blood sample of 5 to 20 ml was obtained from each
participant in accordance with the age-specific guidance provided
by the Gambian Government / MRC Joint Ethics Committee. A
thick blood film was prepared to test for presence of parasitaemia
by slide microscopy, 0.5 ml of blood was collected into EDTA
tubes for parasite detection by PCR and the remainder of the
sample was collected into heparinised tubes and used for B cell
ELISpot and Ab measurement by ELISA. HIV testing was not
done on any of the samples, and HIV status was not reported. Adult HIV prevalence in rural areas of The Gambia is estimated
to be approx 2% in 2009 [36]; thus undiagnosed HIV infection is
unlikely to have markedly affected the immune responses reported
here. Stool samples from 38% of randomly selected study
participants were also collected to assess worm carriage (Table S1). A village health worker was present in Brefet throughout the
transmission season to diagnose malaria infections by use of RDT
(OptiMALH) for malaria. Study participants had been asked to
report to the village health centre if they suspected they had
malaria. Anti-malarial treatment was only given if the rapid
diagnostic test was positive. A venous blood sample of 5 to 20 ml was obtained from each
participant in accordance with the age-specific guidance provided
by the Gambian Government / MRC Joint Ethics Committee. A
thick blood film was prepared to test for presence of parasitaemia
by slide microscopy, 0.5 ml of blood was collected into EDTA
tubes for parasite detection by PCR and the remainder of the
sample was collected into heparinised tubes and used for B cell
ELISpot and Ab measurement by ELISA. HIV testing was not
done on any of the samples, and HIV status was not reported. Ethical Approval The studies were approved by both the Gambia Government/
Medical Research Council (MRC) Joint Ethics Committee and the
Ethics Committee at the London School of Hygiene and Tropical
Medicine. Participants were enrolled after individual written
informed consent was obtained from the participant or their
parent/guardian. For the measurement of AMB, blood collected from children
enrolled in an ongoing malaria case-control study based in the
coastal area of The Gambia [33,34] was used. Malaria-Specific MBC in Low Transmission Areas Individuals with
increased Ab concentrations and/or a clinical episode of malaria
are collectively referred to as cases. Age-matched (plus/minus 1
year) controls (study participants without evidence of malaria
exposure during the transmission season) were selected from the
study cohort for comparison. Antigens The MSP-119 and MSP-2 Ags used were glutathione s-
transferase (GST) fusion proteins representing amino acids
1631–1726 of the Wellcome allele of MSP-119 [44] and amino
acids 22–247 of the Dd2 allele of MSP-2 [45], respectively. MSP-
119 is highly conserved among P. falciparum isolates with minor
sequence
variations
having
a
minimal
effect
on
antibody
recognition [28]. Parasites belonging to the MSP2-Dd2 family
are widely represented amongst parasite isolates in The Gambia PLoS ONE | www.plosone.org Discussion Indeed, the total number of MBCs
detected in the ELISpot assay tended to be higher in slightly older
children than in very young children and, by flow cytometry, the
proportion of all B cells that are classical MBCs was also higher in
older children than in younger children. Although we could not
detect any further expansion of total MBC numbers from
adolescence into adulthood, suggesting that MBC numbers may
reach an equilibrium in late childhood, our power to detect such a
trend was limited by the anomalous drop in MBC numbers in the PLoS ONE | www.plosone.org PLoS ONE | www. October 2011 | Volume 6 | Issue 10 | e25582 PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25582 Study site and sample collection Blood samples from 118 healthy individuals were collected in
May–June 2009 (prior to the transmission season) from Brefet,
Foni District, The Gambia. Brefet is a rural village situated 55 km
inland from the Atlantic coast and 1 km from the river Gambia. In
The Gambia, malaria transmission is seasonal, starting in July and
ending in December with the peak of transmission being in PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 11 Malaria-Specific MBC in Low Transmission Areas [46]. The MSP-3 Ag used is a maltose binding protein (MBP)
fusion protein representing amino acids 2–354 of the 3D7 allele of
MSP-3 [47]. The AMA-1 Ag is a 66Histidine tagged protein
representing amino acids 22–545 of the 3D7 allele sequence
[48,49]. Although there is almost an infinite array of AMA-1
alleles, differing slightly from each other in sequence and
antigenicity, polyclonal antibody responses of individuals living
in endemic areas appear to cross-react extensively with heterol-
ogous AMA-1 sequences [50]. All the Ags were further diluted in
16PBS at the appropriate working concentrations (0.002 mg/ml). Purified diphtheria toxoid (DT) (NIBSC, UK), derived by
formaldehyde deactivation of diphtheria toxin, a 535 amino acid
long polypeptide, was used at 0.002 mg/ml to establish diphtheria
vaccine induced responses. KLH (from Calbiochem) was used as a
control Ag at 0.002 mg/ml. [46]. The MSP-3 Ag used is a maltose binding protein (MBP)
fusion protein representing amino acids 2–354 of the 3D7 allele of
MSP-3 [47]. The AMA-1 Ag is a 66Histidine tagged protein
representing amino acids 22–545 of the 3D7 allele sequence
[48,49]. Although there is almost an infinite array of AMA-1
alleles, differing slightly from each other in sequence and
antigenicity, polyclonal antibody responses of individuals living
in endemic areas appear to cross-react extensively with heterol-
ogous AMA-1 sequences [50]. All the Ags were further diluted in
16PBS at the appropriate working concentrations (0.002 mg/ml). Purified diphtheria toxoid (DT) (NIBSC, UK), derived by
formaldehyde deactivation of diphtheria toxin, a 535 amino acid
long polypeptide, was used at 0.002 mg/ml to establish diphtheria
vaccine induced responses. KLH (from Calbiochem) was used as a
control Ag at 0.002 mg/ml. plasma samples obtained from 20 malaria naı¨ve tourists from
Europe. Only individuals which were above the cut-off value were
termed responders. LISA for serum antibodies to malaria antigens Concentrations of IgG binding to MSP-119, MSP-2, MSP-3 and
AMA-1 were measured by ELISA, using the method described
elsewhere [12,51]. Ab levels to the different malaria antigens tested
were compared to a pool of sera samples collected in Brefet in
2008, and expressed as arbitrary units. Results obtained with a 50
fold dilution of the pooled sample were defined as 20 arbitrary
units. ELISpot assay y
PBMC were isolated from the participant’s whole blood as
described elsewhere [33]. Following a protocol adapted from
Crotty et al. [1,19], 16106 PBMCs in RPMI containing 10%
fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin
and 2 mM L-glutamine (all Sigma) were added to each well of a
24-well culture plate, and supplemented with 50 mM b-Mercap-
toethanol, 0.5 mg/ml Phytolacca Americana pokeweed mitogen
(PWE, Sigma), Staphylococcus aureus Cowan (SAC, Sigma),
3 mg/ml CpG-2006 (Eurofins MWG-Operon- 59TCG TCG
TTT
TGT
CGT
TTT
GTC
GTT
39)
and
25 ng/ml
recombinant human IL-10 (R&D Systems) and placed in the
incubator (37uC, 5% CO2) for six days. On day 5, quadruplicate
wells of multiscreen-HA plates (Millipore, MAHAS4510) were
coated overnight with either unbiotinylated AffiniPure F(ab9)2
fragment donkey anti-human IgG (Jackson ImmunoResearch
Laboratories), or with one of the four malarial Ags, DT or KLH,
respectively. On day six, 400,000 cultured cells were added to
each antigen coated well (2000 cells for IgG coated wells), and
incubated for 6 hours (37uC, 5% CO2). Cells were then
incubated overnight (at 4uC) with biotin-SP-conjugated Affini-
Pure fragment donkey anti-human IgG (Jackson ImmunoRe-
search). The
next
day,
strepavidin-AKP
(BD
Biosciences)
followed by detection solution (AP conjugate substrate kit
(BioRad)) was added and plates were read using the AID
ELISpot reader. All samples in the IgG coated wells yielded more
than 1000 spots/million PBMC, a threshold previously defined
for a valid assay [22]. The median of the coefficients of variation
for the replicates from all subjects and all antigens was 8.03%. Malaria-specific ASC are presented either as the percentage of
the average number of spots counted in the IgG coated wells (%
MBC), or as spots per million PBMC (MBC/PBMC) seeded onto
the ELISpot plate after the 6 day culture. Flow cytometry analysis Surface staining of AMB was performed on freshly isolated
PBMC. The cells were stained with the following fluorochrome-
labelled mouse anti-human antibodies: ECD-anti-CD19 (Beckman
Coulter), and APC-anti-CD21, APC-Cy7-anti-CD27, Pe-Cy7-
anti-CD10 (all from eBioscience). AMB were identified as follows:
CD19+CD212CD272CD102. All samples were acquired on a 9-
colour CyAn ADP flow cytometer and were analysed by FlowJo
software (TreeStar Inc., Ashland, OR, USA). PCR DNA was extracted from 150 ml of EDTA blood using the X-
tractor GeneTM robot, according to the manufacturer’s instruc-
tions (Corbett Robotics) [35]. PCR amplification of Plasmodium
falciparum ribosomal DNA was performed as described previously
[52]. PCR products were resolved by 2% agarose gel electropho-
resis (30 mins, 100 v); gels were stained with ethidium bromide
and read by a trans-illuminator (BioRad). Stool analysis Stool samples were collected into fixative (10% formalin) and
processed using the ParasiTrap fecal diagnostic system (Biosepar)
and read by the routine microbiology laboratories at the MRC in
Fajara, The Gambia. Diphtheria toxoid IgG ELISA Ab levels to DT were quantified using a commercial kit (MP
Biomedicals Diphtheria Toxoid IgG antibody ELISA kit catalogue
number 071-524002) following the manufacturer’s instructions. OD values are converted to International Units (IU/ml) by
comparison with the standard curve. The manufacturer considers
antibody titres .0.1 IU/ml sufficient for protection. Statistical analysis Data were analysed using GraphPad Prism 5 and STATA 10.1. Chi-squared tests for trend were used to assess whether the
percentage
of
individuals
having
Ab
and
MBC
changed
significantly with age for each Ag as well as for measuring the
breadth of the immune response. Kruskal-Wallis tests were used to
assess whether Ab concentration or MBC numbers differed
significantly between age groups, and to test for differences in
MBC numbers for the different Ags. As appropriate, Wilcoxon
matched paired tests or Mann-Whitney tests were performed to
compare two groups. Where multiple tests were performed on the
same individuals for multiple responses to different malarial Ags, p
values were adjusted for multiple comparisons using the Bonferoni
correction. To assess the agreement between the two read outs
used for the ELISpot results (% MBC or MBC/PBMC) in
identifying ‘‘responders’’ Kappa statistics were calculated, and the
results graded using the classification by Landis & Koch [53]. Correlation
of
two
quantitative
variables
was
assessed
by
calculating the Spearman’s correlation coefficient. A logistic
regression model was used to analyse the changes in the frequency
of MBC with age. Only values above the upper limit of the 99% confidence
interval of the median obtained for the negative control KLH
(0.004% or more than 1.88 spots/million PBMC) were considered
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recognition of malaria antigens by human serum antibodies is not genetically
determined but demonstrates some features of clonal imprinting. International
Immunology 8: 905–915. 34. Acknowledgments We express our deepest appreciation for the village community of Brefet, in
The Gambia for their participation in our study. A special thank you goes
to our study nurse Kebba Jobe, our field worker Lamin Manneh and to
Ousman Sanyang our village coordinator for ensuring the smooth running
of the study. We are grateful to David Cavanagh and Jana McBride for
provision of the plasmid for MSP-2 protein expression. Finally, we would We express our deepest appreciation for the village community of Brefet, in
The Gambia for their participation in our study. A special thank you goes
to our study nurse Kebba Jobe, our field worker Lamin Manneh and to Supporting Information Table S1
Baseline characteristics of the study cohort. (DOC) Table S1
Baseline characteristics of the study coho A positive response was defined by a value above the upper limit
of the 99% confidence interval obtained for this Ag with a pool of October 2011 | Volume 6 | Issue 10 | e25582 PLoS ONE | www.plosone.org 12 Malaria-Specific MBC in Low Transmission Areas like to thank Simon Correa, Madi Njie and Idrissa Sambou for their
laboratory assistance. Author Contributions Conceived and designed the experiments: MW EMR. Performed the
experiments: SN. Analyzed the data: SN MW EMR BW. Contributed
reagents/materials/analysis tools: JCH EJR KT DJC. Wrote the paper:
SN MW EMR. y
J
Ousman Sanyang our village coordinator for ensuring the smooth running
of the study. We are grateful to David Cavanagh and Jana McBride for
provision of the plasmid for MSP-2 protein expression. Finally, we would References gy
20. Dorfman JR, Bejon P, Ndungu FM, Langhorne J, Kortok MM, et al. (2005) B
cell memory to 3 Plasmodium falciparum blood-stage antigens in a malaria-
endemic area. Journal of Infectious Diseases 191: 1623–1630. 21. Weiss GE, Crompton PD, Li S, Walsh LA, Moir S, et al. (2009) Atypical
memory B cells are greatly expanded in individuals living in a malaria-endemic
area. Journal of Immunology 183: 2176–2182. 42. Thomson MC, D’Alessandro U, Bennett S, Connor SJ, Langerock P, et al. (1994) Malaria prevalence is inversely related to vector density in The Gambia,
West Africa. Transactions of the Royal Society of Tropical Medicine and
Hygiene 88: 638–643. 22. Weiss G, Traore B, Kayentao K, Ongoiba A, Doumbo S, et al. (2010) The
Plasmodium falciparum-specific human memory B cell compartment expands
gradually with repeated malaria infections. PloS Pathogens 6: e1000912. 43. Clarke SE, Bogh C, Brown RC, Walraven GE, Thomas CJ, et al. (2002) Risk of
malaria attacks in Gambian children is greater away from malaria vector PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 13 Malaria-Specific MBC in Low Transmission Areas breeding sites. Transactions of the Royal Society of Tropical Medicine and
Hygiene 96: 499–506. antigen 1 induces broader allelic recognition and growth inhibition responses in
rabbits. Infection and Immunity 76: 2660–2670. antigen 1 induces broader allelic recognition and growth inhibition responses in
rabbits. Infection and Immunity 76: 2660–2670. yg
44. Burghaus PA, Holder AA (1994) Expression of the 19-kilodalton carboxy-
terminal fragment of the Plasmodium falciparum merozoite surface protein-1 in
Escherichia coli as a correctly folded protein. Molecular and Biochemical
Parasitology 67: 343. y
49. Osier FH, Weedall GD, Verra F, Murungi L, Tetteh KKA, et al. (2011) Allelic
diversity and naturally acquired allele-specific antibody responses to Plasmodium
falciparum apical membrane antigen 1 in Kenya. Infection and Immunity. (In
press). 45. Polley SD, Conway DJ, Cavanagh DR, McBride JA, Lowe BS, et al. (2006) High
levels of serum antibodies to merozoite surface protein 2 of Plasmodium
falciparum are associated with reduced risk of clinical malaria in coastal Kenya. Vaccine 24: 4233–4246. 50. Hodder AN, Crewther PE, Anders RF (2001) Specificity of the protective
antibody response to apical membrane antigen 1. Infection and Immunity 69:
3286–3294. 51. Okech BA, Corran P, Todd J, Joynson-Hicks A, Uthaipibull C, et al. References (2004) Fine
specificity of serum antibodies to Plasmodium falciparum merozoite surface
protein, PfMSP-119, predicts protection from malaria infection and high-density
parasitemia. Infection and Immunity 72: 1557–1567. 46. Conway DJ (1997) Natural selection on polymorphic malaria antigens and the
search for a vaccine. Parasitology today 13: 26–29. 47. Polley SD, Tetteh KKA, Lloyd JM, Akpogheneta OJ, Greenwood BM, et al. (2007) Plasmodium falciparum merozoite surface protein 3 is a target of allele-
specific immunity and alleles are maintained by natural selection. The Journal of
Infectious Diseases 195: 279–287. parasitemia. Infection and Immunity 72: 1557–1567. 52. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, et al. (1993) High
sensitivity of detection of human malaria parasites by the use of nested
polymerase chain reaction Molecular Biochemical. Parasitology 61: 315–320. lymerase chain reaction Molecular Biochemical. Parasitology 61: 315 48. Remarque EJ, Faber BW, Kochen CH, Thomas AW (2008) A diversity-covering
approach to immunization with Plasmodium falciparum apical membrane 53. Landis JR, Koch GG (1977) The measurement of observer agreement for
categorical data. Biometrics 33: 159. PLoS ONE | www.plosone.org October 2011 | Volume 6 | Issue 10 | e25582 14
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https://openalex.org/W3199310460
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https://www.researchsquare.com/article/rs-885747/latest.pdf
|
English
| null |
Is it necessary to monitor the serum luteinizing hormone(LH) concentration on the human chorionic gonadotropin (HCG) day among young women during the follicular-phase long protocol? ：A retrospective cohort study
|
Research Square (Research Square)
| 2,021
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cc-by
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Results Divided the LHHCG into five groups: Group A (LH ≤ 0.5), Group B (0.5 IU/L < LH ≤ 1.2 IU/L), Group C (1.2
IU/L < LH ≤ 2.0 IU/L), Group D (2.0 IU/L < LH ≤ 5.0 IU/L), Group E (LH > 5 IU/L). In terms of the numbers of
retrieved eggs, embryos, high-quality embryos and diploid fertilized oocytes, an increase of the LHHCG
level showed a trend of a gradual decrease. However, there was no significant difference in clinical
outcomes among the groups.By adjusting for confounding factors, with an increase in LHHCG, the number
of retrieved eggs decreased. Conclusion In the follicular-phase long protocol among young women, monitoring of LHHCG are recommended in the
clinical guidelines. What’s more, those who undergo Preimplantation Genetic Testing(PGT) may benefit
more when the LH level is controlled within a certain range. Methods A retrospective cohort study included 4503 cycles of in vitro fertilization (IVF)/intracytoplasmic sperm
injection (ICSI) units from January 1, 2016, to June 30, 2019, in a single department. The main outcome
measures included retrieved eggs, available embryos, live birth rate. Background The normal physiological function of LH requires a certain concentration range, but because of pituitary
desensitization, even on the HCG day, endogenous levels of LH are low in the follicular-phase long
protocol. So our study aimed to determine whether it is necessary to monitor serum LH concentrations
and to determine whether there is an optimal LH range to achieve the desired clinical outcome. Research Article Keywords: luteinizing hormone, pituitary desensitization, retrieved eggs, live birth
Posted Date: September 16th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-885747/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Reproductive Biology and Endocrinology on
February 2nd, 2022. See the published version at https://doi.org/10.1186/s12958-022-00888-4. Page 1/14 Page 1/14 Introduction Appropriate protocols of controlled ovarian stimulation (COS) are critical for assisted reproductive
technology (ART) outcomes. The discovery of gonadotropin-releasing hormone (GnRH) analogs has
offered multiple options in assisted reproduction and improved in vitro fertilization (IVF) success rates
(1). Effective control of the premature luteinizing hormone (LH) peak, reduction of the cycle cancellation
rate and more mature oocytes make the GnRH agonist protocol popular in many reproductive centers. In
China, the follicular-phase long protocol has gradually become mainstream because of its simple and
convenient single administration, good follicular homogeneity, and excellent fresh cycle outcomes (2). In Page 2/14 Page 2/14 the GnRH agonist protocol, because of pituitary desensitization, endogenous levels of LH are very low
during the late stimulation phase (3). Moreover, approximately 50% of patients undergoing
IVF/intracytoplasmic sperm injection (ICSI) using a GnRH agonist are LH deficient (3). Thus, it would
seem logical that LH supplementation would be beneficial. According to the “two-cell, two-gonadotrophin theory”, both follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) are important for follicle development in humans. This model explains our
understanding of folliculogenesis (4). LH stimulates theca cells, promoting androgen production, and
FSH regulates the proliferation of granulosa cells (GCs) and promotes E2 synthesis. Whereas FSH is the
main regulator of antral follicular growth, LH plays key roles in promoting steroidogenesis and in the
development of the leading follicle. Moreover, LH exerts different functions during the different stages of
both natural and stimulated cycles (5). During the menstrual cycle, LH not only promotes the growth of
larger follicles but also increases granulosa cell FSH activity by increasing androgen synthesis, and then
LH promotes the recruitment of follicles. The normal physiological function of LH requires a certain concentration range, namely, the “LH window”
(6). When below the LH threshold, the eggs cannot fully mature, and there is not enough androgen and
estrogen synthesis. Moreover, there is a lack of paracrine signals between granulosa cells and membrane
cells. In contrast, when beyond the upper limit, granulosa cell proliferation is inhibited, which can lead to a
series of problems in egg development. Therefore, we speculate that it is necessary to monitor the level of
LH during the early follicular phase long protocol. Introduction An increasing amount of evidence has demonstrated that downregulation with a GnRH agonist in some
normogonadotrophic women may result in profound suppression of LH, which in turn impairs adequate
estradiol synthesis during FSH stimulation for IVF/ICSI (4–7), and reduces the fertilization rate (8), the
number of clinical pregnancies (8), and the pregnancy outcomes (9). However, there is some controversy
about this conclusion. Consequently, this study aimed to determine whether it is necessary to monitor the serum LH
concentration on the HCG day(LHHCG), to identify whether the LH level on the human chorionic
gonadotropin (HCG) day (LHHCG), have an impact on the clinical outcome and to determine whether there
is an optimal LH range to achieve the expected clinical outcome. Stimulation protocols Follicular-phase single-dose GnRH agonist protocol: A single dose of 3.75 mg long-acting GnRH agonist
triptorelin (Diphereline, IPSEN, Paris, France) was administered on day 2 or 3 of the menstrual cycle. Twenty-eight days later, serum FSH, LH, E2, and P were examined. When FSH and LH were both < 5 IU/L,
P < 1 µg/L and E2 < 50 pg/ml, gonadotropin (Gn) was initiated daily until ovulation induction. HMG
(Livzon, China, Zhuhai) or recombinant LH (rLH Luveris Merck Serono S. A, Beijing) was added according
to follicular development. When three follicles reached a mean diameter of 17 mm or two follicles
reached a mean diameter of 18 mm, 0.25 mg of recombinant LH (Ovidrel, Merck Serono S.A., Beijing) was
administered intramuscularly. Oocyte retrieval was performed 36 h after hCG injection by transvaginal
ultrasound-guided single-lumen/double-lumen needle aspiration. Intracytoplasmic sperm injection (ICSI)
was performed only for severe male factor infertility or previous fertilization failure. Luteal phase support
was initiated on day1 after oocyte retrieval. The cleavage stage embryo transfer time was 3 days after
egg retrieval. The blastocyst transfer time was 5 days after egg retrieval. Morphologic criteria were used
for embryo scoring (10). S
i
i
l
l
i Study design and participants The subjects of this retrospective study underwent 4503 cycles of IVF or ICSI units from January 1, 2016
to June 30, 2019 in our department. The following inclusion criteria were applied: 1) age ≤ 40 years, 2)
follicular-phase single-dose GnRH agonist protocol, 3) fresh cycle transplants, HCG endometrial thinning,
recurrent miscarriage, and endometriosis. Page 3/14 Page 3/14 The study was approved by the ethics committee of the Third Affiliated Hospital of Zhengzhou
University(2021 − 105). Data on patient age and infertility treatment-related characteristics were collected from the files. Basic
sex hormone levels (follicle stimulating hormone [FSH], luteinizing hormone [LH], estradiol [E2],
progesterone [P]), Gn dose, duration of Gn stimulation, number of oocytes obtained, number of oocytes
retrieved, the two pronuclear zygote (2PN) embryo rate, the high-quality embryo rate, the number of
embryos transferred, and the LH level on day1 of ovarian stimulation/the HCG day were recorded. Data on patient age and infertility treatment-related characteristics were collected from the files. Basic
sex hormone levels (follicle stimulating hormone [FSH], luteinizing hormone [LH], estradiol [E2],
progesterone [P]), Gn dose, duration of Gn stimulation, number of oocytes obtained, number of oocytes
retrieved, the two pronuclear zygote (2PN) embryo rate, the high-quality embryo rate, the number of
embryos transferred, and the LH level on day1 of ovarian stimulation/the HCG day were recorded. E2/P/on the day of hCG administration was recorded. Clinical pregnancy was defined as visualization of
both a gestational sac and fetal cardiac activity on transvaginal ultrasound. Early spontaneous abortion
refers to pregnancy failure that occurred before 12 weeks. E2/P/on the day of hCG administration was recorded. Clinical pregnancy was defined as visualization of
both a gestational sac and fetal cardiac activity on transvaginal ultrasound. Early spontaneous abortion
refers to pregnancy failure that occurred before 12 weeks. Statistical analysis Differences in variables between the groups were statistically analyzed with Student’s t-test and chi-
squared tests when appropriate. A bilateral p value < 0.05 was considered to be significant. The results
are presented as the mean ± standard deviation. If the variances were not uniform, we used the rank sum
test for comparison. Linear regression analysis was performed to assess the association between the LH level on the HCG day
and the number of eggs retrieved, whereas logistic regression analysis was used to evaluate the
correlation with the rate of conception. The same set of potential confounders was introduced into the
regression models for adjustment by the enter method, regardless of whether significant differences
between groups were observed. Statistical analysis was performed with the Statistical Package for the
Social Sciences (version 24.0; SPSS Inc, USA). All P < 0.05 on one-sided tests was considered to be
statistically significant. Page 4/14 Page 4/14 The influence of LHHCG level on the outcome of IVF/ICSI-ET When the serum LH level is lower than 0.5 IU/L, both the fertilization rate and the number of embryos
decreases significantly. Some clinicians believe that the threshold of LH can be set to 1.2 IU/L; if the LH is
lower than this level, follicular development and endometrial growth will be severely insufficient (11). When the serum LH level is greater than 5.0 IU/L on the day of hCG in the follicular-phase long protocol,
the pregnancy rate significantly decreased (12). Thus, we divided the LHHCG into five groups: Group A (LH
≤ 0.5), Group B (0.5 IU/L < LH ≤ 1.2 IU/L), Group C (1.2 IU/L < LH ≤ 2.0 IU/L), Group D (2.0 IU/L < LH ≤ 5.0
IU/L), Group E (LH > 5 IU/L). There were 9 cycles with LHHCG ≥ 10 IU/L in the follicular-phase long protocol, of which only 1 cycle was
greater than 20 IU/L. There was no obvious follicle luteinization in these cycles. According to the basic information and clinical characteristics of the patients, the age of group D was
slightly lower than that of group C. The BMI of groups D and E was lower. The basal FSH and LH in
groups C, D and E were higher than those in groups A and B (Table 1). The influence of LHHCG level on the outcome of IVF/ICSI-ET Page 5/14 Table 1
Patient characteristics of clinical
Group
A(LH ≤
0.5)
Group B
(0.5༜LH ≤
1.2)
Group C
(1.2༜LH ≤
2)
Group D
(2༜LH ≤ 5)
Group E
(LH༞5)
P
No.of cycles
225
2143
1495
580
60
Age of
woman(years)
29.80 ±
3.93
29.69 ±
3.89
29.92 ±
3.82
29.30 ±
3.80c
29.65 ±
3.92
0.025
Body mass
index(kg/m2)
23.91 ±
3.50
23.80 ±
3.22
23.56 ±
3.17
22.49 ±
3.18abc
22.52 ±
3.37ab
0.000
Basic hormone
concentrations
FSH(IU/L)
6.38 ±
1.86
6.64 ± 1.90
7.06 ±
2.12ab
7.35 ±
2.13ab
7.73 ±
2.33ab
0.000
LH(IU/L)
4.89 ±
3.60
5.38 ± 3.77
5.99 ±
3.90ab
6.80 ±
5.67abc
6.71 ±
4.81ab
0.000
P(nmol/L)
145.60 ±
64.69
152.96 ±
109.13
155.76 ±
146.55
159.43 ±
169.57
164.23 ±
120.90
0.615
E2(pmol/L)
1.52 ±
2.17
1.42 ± 1.79
1.45 ±
3.03ab
1.57 ± 3.65
1.90 ± 2.63
0.000
AMH(pmol/L)
37.00 ±
23.12
33.16 ±
22.03
31.90 ±
22.52a
32.82 ±
23.50
32.98 ±
23.32
0.029
Start-up Gn
does(IU)
163.90 ±
52.36
167.20 ±
56.47
169.17 ±
60.02
157.46 ±
58.74bc
157.71 ±
62.91
0.000
Total Gn dose (IU)
2532.36
± 993.04
2479.22 ±
1012.23
2529.74 ±
1080.28
2351.01 ±
1083.11abc
2308.48 ±
1117.57
0.000
LHday 1(IU/L)
0.59 ±
0.29
0.72 ±
0.38a
0.97 ±
1.01ab
1.06 ±
0.73abc
1.05 ±
0.69ab
0.000
Hormone
t
ti Table 1 Table 1
Patient characteristics of clinical Patient characteristics of clinical Page 6/14
aCompared with group A, P༜0.05
bCompared with group B, P༜0.05
cCompared with group C, P༜0.05 Group
A(LH ≤
0.5)
Group B
(0.5༜LH ≤
1.2)
Group C
(1.2༜LH ≤
2)
Group D
(2༜LH ≤ 5)
Group E
(LH༞5)
P
E2(pmol/L)
1367.07
± 6341.47
12884.09 ±
5950.26ad
13131.38 ±
5834.56ad
14517.02 ±
6740.33bc
13151.15
± 6015.93
0.000
P(nmol/L)
3.89 ±
1.42bcd
3.56 ±
1.44cd
3.35 ± 1.49
3.21 ± 1.42
3.30 ± 1.34
0.000
Endometrial
thickness on HCG
11.35 ±
6.85
11.29 ±
2.26
11.41 ±
2.29ae
11.40 ± 2.09
10.56 ±
2.01
0.002
aCompared with group A, P༜0.05
bCompared with group B, P༜0.05
cCompared with group C, P༜0.05
The level of P in groups A and B was higher than that in the other groups. The AMH value of group C was
significantly lower than that of group A. The start-up Gn dose in group D was significantly lower than that
in groups B and C. The influence of LHHCG level on the outcome of IVF/ICSI-ET The total Gn dose in group D was significantly lower than that in the other groups. For
the hormone concentrations of HCG, the E2 level of group B and group C was lower, and the P level in
group A was the highest. On HCG day, the endometrium in group C was thicker than that in groups A and
E (Table 1). In terms of the numbers of retrieved eggs, embryos, high-quality embryos and diploid fertilized oocytes,
an increase of the LH
level showed a trend of a gradual decrease Group E had the highest rate of Group
A(LH ≤
0.5)
Group B
(0.5༜LH ≤
1.2)
Group C
(1.2༜LH ≤
2)
Group D
(2༜LH ≤ 5)
Group E
(LH༞5)
P
E2(pmol/L)
1367.07
± 6341.47
12884.09 ±
5950.26ad
13131.38 ±
5834.56ad
14517.02 ±
6740.33bc
13151.15
± 6015.93
0.000
P(nmol/L)
3.89 ±
1.42bcd
3.56 ±
1.44cd
3.35 ± 1.49
3.21 ± 1.42
3.30 ± 1.34
0.000
Endometrial
thickness on HCG
11.35 ±
6.85
11.29 ±
2.26
11.41 ±
2.29ae
11.40 ± 2.09
10.56 ±
2.01
0.002
aCompared with group A, P༜0.05
bCompared with group B, P༜0.05
cCompared with group C, P༜0.05 The level of P in groups A and B was higher than that in the other groups. The AMH value of group C was
significantly lower than that of group A. The start-up Gn dose in group D was significantly lower than that
in groups B and C. The total Gn dose in group D was significantly lower than that in the other groups. For
the hormone concentrations of HCG, the E2 level of group B and group C was lower, and the P level in
group A was the highest. On HCG day, the endometrium in group C was thicker than that in groups A and
E (Table 1). In terms of the numbers of retrieved eggs, embryos, high-quality embryos and diploid fertilized oocytes,
an increase of the LHHCG level showed a trend of a gradual decrease. Group E had the highest rate of
embryo transfer at the cleavage stage. However, there was no significant difference in clinical outcomes
among the groups (Table 2). The influence of LHHCG level on the outcome of IVF/ICSI-ET Page 7/14 Page 7/14 Page 7/14 Table 2
Embryological and clinical outcomes Table 2
Embryological and clinical outcomes
Group A(LH
≤ 0.5)
Group B
(0.5༜LH
≤ 1.2)
Group C
(1.2༜LH
≤ 2)
Group D
(2༜LH ≤
5)
Group E
(LH༞5)
P
No.of oocytes
15.22 ± 5.66
13.54 ±
5.23a
12.90 ±
5.05ab
12.30 ±
4.88ab
9.6 ±
4.09abcd
0.000
No.of diploid
fertilized oocytes
9.85 ± 4.70
8.69 ±
4.41a
8.39 ±
4.33a
7.78 ±
3.96ab
5.92 ±
2.78abcd
0.000
No.of embryos
7.90 ± 4.48
6.83 ±
4.03a
6.44 ±
3.88ab
6.22 ±
3.62ab
4.40 ±
2.55abcd
0.000
No.of high quality
embryos
4.32 ± 3.71
3.97 ±
3.42
3.76 ±
3.19
3.71 ±
3.04
2.52 ±
2.27abcd
0.007
No.of embryos
transferred
1.58 ± 0.49
1.63 ±
0.48
1.65 ±
0.48
1.66 ±
0.48
1.70 ± 0.46
0.203
Types of embryos
transferred
Cleavage stage
embryo rate
62.22%
68.60%
70.03%
70.34%
88.33%abcd
0.002
Blastocysts rate
37.78%
31.40%
29.97%
29.66%
11.67%
OHSS rate
3.56%
2.80%
3.95%
4.66%
6.67%
0.090
Biochemical
pregnancy rate
69.33%
67.48%
67.56%
68.62%
66.67%
0.964
Clinical pregnancy
rate
66.67%
64.35%
63.21%
64.48%
63.33%
0.867
Ectopic pregnancy
rate
2.67%
1.16%
1.59%
1.07%
0
0.484
Spontaneous
abortion rate
10.67%
9.64%
8.47%
9.09%
2.63%
0.509
Live birth rate
56.44%
55.58%
54.65%
56.03%
60%
0.898
Premature birth rate
21.26%
15.70%
14.44%
17.54%
22.22%
0.216
Logistic regression analysis was used to determine whether the LHHCG level was related to the number of
oocytes retrieved. The results showed that in the follicular-phase long protocol, with the increase in
LHHCG, the number of retrieved eggs decreased (Table 3 and Fig. 1). In the follicular-phase long protocol,
BMI
d AMH
t
l t d t th
b
f
t i
d Logistic regression analysis was used to determine whether the LHHCG level was related to the number of
oocytes retrieved. The results showed that in the follicular-phase long protocol, with the increase in
LHHCG, the number of retrieved eggs decreased (Table 3 and Fig. 1). In the follicular-phase long protocol,
BMI and AMH were not related to the number of eggs retrieved. The influence of LHHCG level on the outcome of IVF/ICSI-ET Page 8/14 Table 3
Linear regression analysis between serum LH concentration of HCG day and
eggs retrieved
Adjusted OR
95% CI
P
LHHCG
-0.264
0.939–1.065
0.000
Age of woman(years)
-0.041
0.954–1.049
0.006
BMI
-0.034
0.937–1.067
0.060
Basic LH (IU/L)
-0.019
0.871–1.148
0.192
AMH
0.001
0.778–1.285
0.732
Hormone concentrations on HCG
0.000
0.487–2.052
0.000
E2(pmol/L)
0.088
0.559–1.788
0.010
P(nmol/L)
0.797
0.749–1.335
0.000
P(nmol/L)
-0.034
0.937–1.067
0.060
No. of ≥ 14mm folliculars
-0.019
0.871–1.148
0.192 Table 3 Discussion The Asia-Pacific consensus on the application of LH in assisted reproductive
treatment published in 2011 concluded that the reasonable threshold window for LH ranges from 1.2–5
IU/L (16). However, some studies found that in the process of COH, when the LH level was lower than 1.2
IU/L on the HCG day, it did not affect the clinical outcome (17), whereas for the follicular-phase long
protocol, LH was very rarely elevated due to suppression. In this study, the LH concentration was greater
than 10 IU/L in only 10 cases, and there was only 1 case with LH > 20 IU/L, and the P concentrations on
the HCG day were all normal. Our study found that in the follicular-phase long protocol, with an LH ≤ 0.5 IU/L on the hCG day, patients
had a higher number of oocytes retrieved, a better embryo rate, and more embryos, but there was no
significant difference in the live birth rate compared with the other groups. So we speculate that when the
LH ≤ 0.5 IU/L, although there may be pituitary hypersuppression leading to inadequate LH secretion in
the late follicular phase, it does not affect the outcome. This is similar to the study of Brjercke et al. (18),
that is, a lower LH can satisfy the development of follicles. As long as less than 1% of LH receptors are
occupied, they can play a normal physiological role (18). However, when there are older women, a low
response, a slow response, etc. in the COH cycle, adding LH may be beneficial. With a normal ovarian
response, a low LH concentration does not affect the fertilization outcome (19). However, as LH increases, the numbers of oocytes retrieved, embryos, and high-quality embryos and the
number of 2PN decreased gradually, which may be because excessive LH levels may cause the ovaries to
secrete more androgens, resulting in follicular atresia and then fewer oocytes retrieved. However, the
pregnancy rate did not decrease, which may be because the increase in LH level only affected the number
of oocytes retrieved but did not affect the quality of the oocytes, the embryo development potential or
endometrial receptivity. For PGT patients, it may be more suitable to control the LH at a lower level on the
HCG day to retrieve more oocytes. The main limitations of this article are as follows: 1. Discussion The findings of our study suggest that the level of LHHCG can affect the IVF/ICSI outcome in the follicular-
phase long protocol in a young population. The ‘‘two cell–two gonadotropin’’ model has been the key to our understanding of folliculogenesis (4). According to this model, LH stimulates theca cells, thereby advancing androgen production, and FSH
governs the proliferation of granulosa cells (GCs) and promotes E2 synthesis. Both FSH and LH play an
important role in folliculogenesis. The details of the specific role of LH in folliculogenesis have not been
fully studied. The follicular-phase long protocol uses a single dose of 3.75 mg GnRH-a in the early follicular phase for
pituitary downregulation. Two weeks after a single injection of 3.75 mg GnRH-a, endogenous sex
hormones are almost completely inhibited. The FSH levels gradually recover after 3–4 weeks, and the E2
levels begin to rise after 7–8 weeks, whereas the inhibitory effect on LH lasts up to 8 weeks after
administration (13). After 30 days, as the FSH level gradually recovers, gonadotropin (Gn) is used to
stimulate the development of multiple follicles in the ovary. In the follicular-phase long protocol, due to
pituitary desensitization, endogenous LH levels are very low in the later stage of stimulation. In addition,
approximately 50% of patients undergoing IVF/ICSI using GnRH-A are LH deficient. Therefore, adding
recombinant LH (rLH) to the process of COH may be beneficial to the outcome (3). Therefore, when the
serum LH concentration after pituitary downregulation is between 0.5–2.5 U/L, we can obtain a normal
ovarian response (14). Too high or too low is not ideal. Page 9/14 LH, which is well known for its importance in the late follicular phase, supports theca cells in the ovary to
provide androgens and hormonal precursors for estradiol production. LH not only promotes the growth of
larger follicles but also increases FSH activity in granulosa cells by increasing androgen synthesis (15). During the development of a dominant follicle, its dependence on FSH gradually declines, whereas its
dependence on LH gradually increases. FSH’s partial role is replaced by LH in the late follicular phase. This facilitates the selection and maintenance of the dominant follicle. Physiological levels of LH are clearly important for follicle development. If the LH level is abnormal, it will
lead to abnormal follicular development. Therefore, it was speculated that there should be an LH
threshold window. The authors are grateful to physicians and coordinators who enrolled
patients and collected data all women who participated in this study. The authors are grateful to physicians and coordinators who enrolled
patients and collected data all women who participated in this study. Discussion The current measurement of LH levels may not
accurately reflect the true level of internal LH; 2. Our study mainly enrolled young people, which is a
specific group. This may have a certain influence on the outcome; 3. According to follicular development
in the later stage, HMG or rLH may be added, which may cause the conclusion to be inaccurate. The
cumulative pregnancy rate and cumulative live birth rate were not calculated. The data sample is still not
large enough, and additional prospective large-sample studies are needed to confirm this idea. Page 10/14 Page 10/14 In conclusion, in the follicular-phase long protocol, the LH level is maintained at a low level throughout
COH. From the data of our study, we should pay more attention to LHHCG. Elevated LH levels on the hCG
day may lead to a reduction in oocyte retrieval. However, even when LH ≥ 5 IU/L, approximately 10
oocytes are retrieved, which, for clinical purposes, is sufficient. However, those who undergo PGT may
benefit more when the LH level is controlled within a certain range. Therefore, in the follicular-phase long
protocol among young women, monitoring of LHHCG has clinical applications. In conclusion, in the follicular-phase long protocol, the LH level is maintained at a low level throughout
COH. From the data of our study, we should pay more attention to LHHCG. Elevated LH levels on the hCG
day may lead to a reduction in oocyte retrieval. However, even when LH ≥ 5 IU/L, approximately 10
oocytes are retrieved, which, for clinical purposes, is sufficient. However, those who undergo PGT may
benefit more when the LH level is controlled within a certain range. Therefore, in the follicular-phase long
protocol among young women, monitoring of LHHCG has clinical applications. Ethics approval and consent to participate The study was approved by the ethics committee of the Third Affiliated Hospital of Zhengzhou University
（2021-105） Consent for publication Not applicable. Author’ contributions WJZ, ZZL. contributed to the conception and design of the study. MML JHL were involved in acquisition
of date collection and analyzed data. YCG drafted the manuscript. All authors revised the article and gave
their final approval of the submitted version. Availability of data and materials The data sets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors have nothing to disclose Page 11/14 References 1. Shrestha D, La X, Feng HL. Comparison of different stimulation protocols used in in vitro fertilization:
a review. Ann Transl Med. 2015;3:137. 2. Liu S, Deng C. Is LH supplementation necessary in the follicular-phase long protocol? J Reprod Med. 2018;9:29. 2. Liu S, Deng C. Is LH supplementation necessary in the follicular-phase long protocol? J Reprod Med. 2018;9:29. 3. Westergaard LG, Laursen SB, Andersen CY. Increased risk of early pregnancy loss by profound
suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women
undergoing assisted reproduction. Hum Reprod. 2000;15:1003–8. 4. Falck B. Site of production of oestrogen in rat ovary as studied in micro-transplants. Acta Physiol
Scand Suppl. 1959;47:1–101. 4. Falck B. Site of production of oestrogen in rat ovary as studied in micro-transplants. Acta Physiol
Scand Suppl. 1959;47:1–101. 5. Fritz MA, Speroff L. Clinical gynecologic endocrinology and infertility. Philadelphia: Wolters Kluwer
Health/Lippincott Williams & Wilkins; 2011. 6. Balasch J, Fábregues F. Is luteinizing hormone needed for optimal ovulation induction? Curr Opin
Obstet Gynecol. 2002;14:265–74. 6. Balasch J, Fábregues F. Is luteinizing hormone needed for optimal ovulation induction? Curr Opin
Obstet Gynecol. 2002;14:265–74. 7. Westergaard LG, Erb K, Laursen S, Rasmussen PE, Rex S. The effect of human menopausal
gonadotrophin and highly purified, urine-derived follicle stimulating hormone on the outcome of in-
vitro fertilization in down-regulated normogonadotrophic women. Hum Reprod. 1996;11:1209–13. 7. Westergaard LG, Erb K, Laursen S, Rasmussen PE, Rex S. The effect of human menopausal
gonadotrophin and highly purified, urine-derived follicle stimulating hormone on the outcome of in-
vitro fertilization in down-regulated normogonadotrophic women. Hum Reprod. 1996;11:1209–13. 8. Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T, Murdoch A. Effects of profound suppression of
luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in
cycles stimulated with purified follicle stimulating hormone. Hum Reprod. 1998;13:1788–92. 8. Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T, Murdoch A. Effects of profound suppression of
luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in
cycles stimulated with purified follicle stimulating hormone. Hum Reprod. 1998;13:1788–92. 9. Fleming R, Rehka P, Deshpande N, Jamieson ME, Yates RW, Lyall H. Suppression of LH during
ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant
FSH. Hum Reprod. 2000;15:1440–5. 9. Fleming R, Rehka P, Deshpande N, Jamieson ME, Yates RW, Lyall H. References Suppression of LH during
ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant
FSH. Hum Reprod. 2000;15:1440–5. 10. Nasiri N, Eftekhari-Yazdi P. An overview of the available methods for morphological scoring of pre-
implantation embryos in in vitro fertilization. Cell J. 2015;16:392–405. 10. Nasiri N, Eftekhari-Yazdi P. An overview of the available methods for morphological scoring of pre-
implantation embryos in in vitro fertilization. Cell J. 2015;16:392–405. 11. Liang X. Clinical practice and improvement of assisted reproductive technology. China: People's
Health Publishing House; 2018. 11. Liang X. Clinical practice and improvement of assisted reproductive technology. China: People's
Health Publishing House; 2018. 12. Wu XX, Chen DL, Zheng YP. The effect of LH concentration on hCG day on the outcome of in vitro
fertilization in the follicular-phase long protocol. J Huazhong Univ Sci Technol (Health Sci). 2019;48:704–7. 12. Wu XX, Chen DL, Zheng YP. The effect of LH concentration on hCG day on the outcome of in vitro
fertilization in the follicular-phase long protocol. J Huazhong Univ Sci Technol (Health Sci). 2019;48:704–7. 13. Broekmans FJ, Bernardus RE, Berkhout G, Schoemaker J. Pituitary and ovarian suppression after
early follicular and mid-luteal administration of a LHRH agonist in a depot formulation: decapeptyl
CR. Gynecol Endocrinol. 1992;6:153–61. 14. Alviggi C, Clarizia R, Mollo A, Ranieri A, De Placido G. Outlook: who needs LH in ovarian stimulation? Reprod Biomed Online. 2006;12:599–607. 14. Alviggi C, Clarizia R, Mollo A, Ranieri A, De Placido G. Outlook: who needs LH in ovarian stimulation? Reprod Biomed Online. 2006;12:599–607. Page 12/14
15. Sungurtekin U, Jansen RP. Profound luteinizing hormone suppression after stopping the
gonadotropin-releasing hormone-agonist leuprolide acetate. Fertil Steril. 1995;63:663–5. Page 12/14
15. Sungurtekin U, Jansen RP. Profound luteinizing hormone suppression after stopping the
gonadotropin-releasing hormone-agonist leuprolide acetate. Fertil Steril. 1995;63:663–5. Page 12/14 Page 12/14 16. Wong PC, Qiao J, Ho C, Ramaraju GA, Wiweko B, Takehara Y, et al. Current opinion on use of
luteinizing hormone supplementation in assisted reproduction therapy: an Asian perspective. Reprod
Biomed Online. 2011;23:81–90. 17. Lossl K, Andersen AN, Loft A, Freiesleben NL, Bangsbøll S, Andersen CY. Androgen priming using
aromatase inhibitor and hCG during early-follicular-phase GnRH antagonist down-regulation in
modified antagonist protocols. Hum Reprod. 2006;21:2593–600. 17. Lossl K, Andersen AN, Loft A, Freiesleben NL, Bangsbøll S, Andersen CY. Androgen priming using
aromatase inhibitor and hCG during early-follicular-phase GnRH antagonist down-regulation in
modified antagonist protocols. Hum Reprod. 2006;21:2593–600. 18. References Bjercke S, Fedorcsak P, Abyholm T, Storeng R, Ertzeid G, Oldereid N, et al. IVF/ICSI outcome and
serum LH concentration on day 1 of ovarian stimulation with recombinant FSH under pituitary
suppression. Hum Reprod. 2005;20:2441–7. 18. Bjercke S, Fedorcsak P, Abyholm T, Storeng R, Ertzeid G, Oldereid N, et al. IVF/ICSI outcome and
serum LH concentration on day 1 of ovarian stimulation with recombinant FSH under pituitary
suppression. Hum Reprod. 2005;20:2441–7. 19. Alviggi C, Conforti A, Esteves SC, Andersen CY, Bosch E, Bühler K, et al. Recombinant luteinizing
hormone supplementation in assisted reproductive technology: a systematic review. Fertil Steril. 2018;109:644–64. 19. Alviggi C, Conforti A, Esteves SC, Andersen CY, Bosch E, Bühler K, et al. Recombinant luteinizing
hormone supplementation in assisted reproductive technology: a systematic review. Fertil Steril. 2018;109:644–64. Figures Figure 1 Page 13/14 Page 13/14 Linear relationship between serum LH concentration of HCG day and eggs retrieved
The Figure showed
that in the follicular-phase long protocol, with the increase of LHHCG, the number of retrieved eggs
decreased. Page 14/14
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https://openalex.org/W2089364433
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https://zenodo.org/records/2424858/files/article.pdf
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English
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The Work of the National Woman's Christian Temperance Union
|
The annals of the American Academy of Political and Social Science/The Annals
| 1,908
|
public-domain
| 2,176
|
BY LILLIAN M. N. STEVENS,
President. BY LILLIAN M. N. STEVENS,
President. BY LILLIAN M. N. STEVENS,
President. The Woman’s Christian Temperance Union is an organization of
Christian women banded together for the protection of the home,
the abolition of the liquor traffic and the triumph of Christ’s
Golden Rule in custom and law. It is the lineal descendant of the
Woman’s Temperance Crusade of 1873, and is now the largest
organization of women in the world. In 1873 the women of the crusade gathered in the streets to
pray and to beseech saloon keepers to give up their business, and
in two months, in upwards of 250 towns, the liquor traffic ceased to
exist. Ohio was the crusade state. Prominent among the old
crusaders were Mrs. Elizabeth J. Thompson, Hillsboro; Mother
Stewart, Springfield; Mrs. George W. Carpenter, Washington
Court House. As an outgrowth of the crusade the National Woman’s Chris-
tian Temperance Union was formed in Cleveland, 0., in 1874. The
VVoman’s Christian Temperance Union is now organized in every
state and teri-itory, the District of Columbia, and in more than 10,000
different localities. It is distinctively a total abstinence society, and
it is not only anti-saloon but anti-distillery and anti-brewery. Its
breadth is indicated by its motto, &dquo;For God and home and native
land.&dquo;
Its badge is a small bow of white ribbon and its declaration
of principles is as follows: We believe in the coming of His Kingdom, whose service is perfect free-
dom, because His laws, written in our members as well as in nature and in
grace, are perfect, converting the soul. We believe in the
l of the Golden Rule
and that each man’s habits We believe in the gospel of the Golden Rule, and that each man’s habits
of life should be an example safe and beneficent for every other man to follow. We believe that God created both
and
in His
i We believe in the gospel of the Golden Rule, and that each man s habits
of life should be an example safe and beneficent for every other man to follow. We believe that God created both man and woman in His own image,
and therefore we believe in one standard of purity for both men and women,
and in the equal right of all to hold opinions and to express the same with
equal freedom. 39 We believe in a living wage; in an eight-hour day; in courts of concilia-
tion and arbitration; in justice as opposed to greed of gain; in &dquo;peace on
earth and good-will to men.&dquo; g
We therefore formulate, and for ourselves adopt the following pledge,
asking our sisters and brothers of a common danger and a common hope,
to make common cause with us, in working its reasonable and helpful precepts
into the practice of everyday life. I hereby solemnly protnise, God helping iiie, to abstain from all distilled,
fermented and malt liquors, including wine, beer and cider/ and to employ all
~o/’~ WMM~ ~ d~fOMfog~ ~~ M.~ o/ attd ~’a~c tM ~
~ofM~. proper means to discourage the use of and this in the same. declare our
To confirm and enforce the rationale of this pledge we declare our
purpose to educate the young; to form a better public sentiment; to reform,
so far as possible, by religious, ethical and scientific means, the drinking
classes; to seek the transforming power of divine grace for, ourselves and all
for whom we work, that they and we may wilfully transcend no law of pure
and wholesome living; and finally we pledge ourselves to labor and to pray
that all these principles, founded upon the Gospel of Christ, may be worked
out into the customs of society and the laws of the land. The io,ooo local unions composing the National Woman’s
Christian Temperance Union have been the chief factors in state
campaigns for statutory prohibition and constitutional amendments,
and for securing the enactment of other reform laws, especially
those for the’ protection of girls. It began the movement for sci-
entific temperance education in the public schools, having been
instrumental in securing laws to that end in all the states, and
besides this it has secured congressional legislation, by means of
which all the territories and the District of Columbia are brought
under the same beneficent statutes. The work of the National Woman’s Christian Temperance
Union for peace and arbitration, for the children in Sunday schools,
loyal temperance legions and kindergartens; its efforts to influence
college students and to train and organize young women for a
philanthropic life; its evangelistic work for non-churchgoers, rail-
way employees, soldiers, fumbermen, miners, and especially for the
drinking men of all classes, have proved its comprehensiveness and
the tirelessness of its energy. at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from BY LILLIAN M. N. STEVENS,
President. We believe that God created both man and woman in His own image,
and therefore we believe in one standard of purity for both men and women,
and in the equal right of all to hold opinions and to express the same with
equal freedom. at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from Its efforts to reach the pauper and
the prisoner, to establish reformatories and homes for the wretched
victims of inebriety and their suffering children, and its temperance
flower mission, must appeal to every heart. 1
State and local constitutions can include the words "as a beverage," if
sired. 40 It also strives to redeem outcast women from a slavery worse
than that of chains, and by better laws to secure protection to women
and girls from the outrages of brutal and designing men. It has
been active in raising the age of consent in nearly every state in
the Union, and its influence is being strongly felt in the purification
of our literature and art. The National Woman’s Christian Temperance Union Conven-
tion is held annually in some large city. The convention of 1907
was held in Nashville, Tenn., in November. Some of the most
important features of the convention were enthusiasm over the
frequent announcement of recent prohibition victories, immense
evening audiences, gifts and pledges at one of the evening meetings
amounting to about $7,000, the presence of Confederate veterans
on the platform at demonstration night, the speech of General A. S. Daggett on the army canteen, the enthusiasm created for Y. and
L. T. L. work, the large number of life and memorial members
received, the welcome to the new prohibition states, Oklahoma and
Georgia, and the jubilee over what white ribboners and L. T. L.’s
did &dquo;to cause to come to pass.&dquo; Some of the leading efforts at the present time are to retain
the &dquo;anti-canteen&dquo; law, to secure the passage of the Littlefield or
Bacon bill, to banish the sale of liquor from all government build-
ings, to secure state laws forbidding the sale of liquor within three
or four miles of soldiers’ homes, forts, army camps, etc., to interest
all teachers and pupils, especially in normal schools and colleges, in
scientific temperance instruction, to secure at least $2.00 from every
local union, or its equivalent, from the state, for the Frances E. Willard Memorial Fund, to complete the raising of $10,000 for an
emergency fund, to increase total abstinence practice and sentiment
and to promote the prohibition of the liquor trafhc everywhere. Th
W
ld’
W
’
Ch i ti
U i The World’s Woman’s Christian Temperance Union was organ-
ized in 1883. at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from It exists in fifty-two nations with a membership of
more than half a million. Frances E. Willard, its founder, and the
first eight &dquo;around-the-world&dquo; missionaries, were instrumental in
its establishment. The officers of the World’s Woman’s Christian
Temperance Union at the present time are: The Countess of Car-
lisle, president; Mrs. L. M. N. Stevens, vice-president-at-large;
Miss Agnes E. Slack and Miss Anna A. Gordon, honorary secre-
taries ; Mrs. Mary E. Sanderson, honorary treasurer. The officers at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from 41 of the National Woman’s Christian Temperance Union at the pres-
ent time are: Mrs. Lillian M. N. Stevens, president; Miss Anna
A. Gordon, vice-president-at-large ; Mrs. Susanna M. D. Fry, corre-
sponding secretary; Mrs. Elizabeth Preston Anderson, recording
secretary; Mars. Sara H. Hoge, assistant recording secretary; Mrs. Susanna l~I. D. Fry, acting treasurer. g
The basic principles of the Woman’s Christian Temperance
Union are total abstinence and prohibition. Prohibition is the
logical associate of total abstinence, and the Woman’s Christian
Temperance Union believes that the temperance reform, in order
to go steadily and successfully forward must use as propelling forces
both these oars. The National ~’Voman’s Christian Temperance
Union teaches that alcohol is a poison; that its use breaks down the
physical nature and harmfully affects the blood, the nerves, the
heart, and makes the drinker an easy prey to disease. It also teaches
that alcohol attacks the moral nature, and its use causes an increased
need of institutions for the dependent, the delinquent and the
criminal classes. It is estimated that at least 6oo,ooo business and public Woman’s
Christian Temperance Union meetings are held in the United
States each year. The teaching and preaching at these meetings
are along the lines of prevention, education, reformation and legis-
lation. This society secures niore petitions than any other in the
world. It is estimated that not fewer_ than 20,000,000 signatures
and attestations have been secured by the Woman’s Christian Tem-
perance Union, including the polyglot petition, addressed to the
different governments of the world, asking them to do away with
the manufacture of and traffic in alcoholic liquors, opium and the
legalization of impurity. This petition has been presented to the
President of the United States, to Queen Victoria, and the Governor-
General of Canada. at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from The National Woman’s Christian Temperance Union keeps a
superintendent of legislation in Washington during the entire ses-
sion of Congress, to look after reform bills. It also maintains a
Woman’s Christian Temperance Union missionary at Ellis Island,
to meet the incoming foreigners. Fully 250,000 children are taught
in the Loyal Temperance Legion the reasons for total abstinence
and are trained as temperance workers. The Woman’s Christian
Temperance Union has created a great literature. Each of the at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from 42 forty superintendents of departments sends out large amounts, and
the several states distribute millions of pages annually. y
The &dquo;Union Signal,&dquo; the official organ of the National Woman’s
Christian Temperance Union, is owned and published by the society. It is a sixteen-page weekly and has an extensiye circulation in every
state and territory and in foreign lands. The &dquo;Crusader Monthly&dquo;
is the ofhcial organ of the Loyal Temperance Legion. It is a
sixteen-page monthly, having a large and constantly increasing cir-
culation. Forty-three state organizations publish papers devoted
entirely to Woman’s Christian Temperance Union interests. y
p
The part the Woman’s Christian Temperance Union has taken
in the recent campaigns which have brought state-wide prohibition
to Georgia, Oklahoma, Mississippi, North Carolina, Alabama and
Arkansas is well described editorially in the &dquo;Oklahoma State
Capital :&dquo; The women organized for prohibition. P
hibiti
th i
l’
d
i Prohibition was their soul’s desire. h
d
f
th
li i
l f
t They cared not for the political features so long as they in no way inter-
fered with their fight for the prohibitory clause. E
th
h
d
th
hibi
i
ith Even those who were opposed to the prohibitory measure must view with
admiration, and applause even, the steadfastness, vigor, and ability with which
thg fight was carried on by the women. The organization formed working committees all over the state, and the
marching of the children with banners through the streets of the towns and
cities had a telling influence. f
ll
d i
th t
h
i ld
i h
i fl
i After all, men must admit that the women wield a mighty influence in
the politics of State and nation. at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from A gifted Georgian has said, when speaking of the great work
of the Woman’s Christian Temperance Union in so many different
lines, that after all the most potent is &dquo;the tender mother in the
quiet seclusion of her home as she gathers her children about her
and kneels in humble prayer to the all-wise Father.&dquo;
h
W
’
Ch i ti
U i The Woman’s Christian Temperance Union was never so strong
numerically and in other ways as at the present time. The indica-
tions are that it will go on and on with its God-given work until
the victory is complete ; and it is to be completed! The day of
national prohibition &dquo;is nearer than when the Woman’s Christian
Temperance Union first believed.&dquo; at UNIV ARIZONA LIBRARY on June 9, 2015
ann.sagepub.com
Downloaded from
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The Emergence of the Bilateral Symmetry in Animals: A Review and a New Hypothesis
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Roskilde
University
The Emergence of the Bilateral Symmetry in Animals
A Review and a New Hypothesis
Toxværd, Søren
Published in:
Symmetry
DOI:
10.3390/sym13020261
Publication date:
2021
Document Version
Publisher's PDF, also known as Version of record
Citation for published version (APA):
Toxværd, S. (2021). The Emergence of the Bilateral Symmetry in Animals: A Review and a New Hypothesis.
Symmetry, 13(2), Article 261. https://doi.org/10.3390/sym13020261 Roskilde
University The Emergence of the Bilateral Symmetry in Animals
A Review and a New Hypothesis Toxværd, Søren Document Version
Publisher's PDF, also known as Version of record Citation for published version (APA):
Toxværd, S. (2021). The Emergence of the Bilateral Symmetry in Animals: A Review and a New Hypothesis. Symmetry, 13(2), Article 261. https://doi.org/10.3390/sym13020261 General rights
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• You may not further distribute the material or use it for any profit-making activity or commercial gain Citation for published version (APA):
Toxværd, S. (2021). The Emergence of the Bilateral Symmetry in Animals: A Review and a New Hypothesis.
Symmetry, 13(2), Article 261. https://doi.org/10.3390/sym13020261 General rights
C
i h
d • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • Users may download and print one copy of any publication from the public portal for the purpo You may not further distribute the material or use it for any profit making activity o
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S
S Review
The Emergence of the Bilateral Symmetry in Animals:
A Review and a New Hypothesis Søren Toxvaerd DNRF Centre “Glass and Time”, IMFUFA, Department of Sciences, Roskilde University, Postbox 260,
DK-4000 Roskilde, Denmark; st@ruc.dk Abstract: Most biological organisms exhibit different kinds of symmetry; an Animal (Metazoa), which
is our Darwinist ancestor, has bilateral symmetry, and many plants exhibit rotational symmetry. It raises some questions: I. How can the evolution from an undifferentiated cell without bilateral
symmetry to a complex biological organism with symmetry, which is based on asymmetric DNA and
enzymes, lead to the bilateral symmetry? II. Is this evolution to an organism with bilateral symmetry
obtained by other factors than DNA and enzymatic reactions? The existing literature about the
evolution of the bilateral symmetry has been reviewed, and a new hypothesis has been formulated
based on these reviews. The hypothesis is that the morphogenesis of biosystems is connected with
the metabolism and that the oscillating kinetics in the Glycolysis have played a role in the polarity of
the biological cells and in the establishment of the bilateral symmetry in Animals. Keywords: bilateral symmetry in Animals; morphogen with Turing patterns; glycolysis in Animals;
metamophosis in cell polarity https://www.mdpi.com/journal/symmetry 2. Symmetries in Animals and Plants Our first ancestor was a bacterium at least 3.5 billion years old [1,2], but the first
and simple multicellular organism with bilateral symmetry was a Bilateria or Animal
that appeared much later (almost all Animals are Bilaterians with a bilateral symmetry). The earliest fossils of Animals in the Precambrian evolution appeared in the Ediacaran
period 635–541 million years ago (Mya) [3–5], and the Bilateria was part of the Cambrian
explosion at ≈541 Mya [6]. The bilateral symmetry in Animals is probably developed
from a sea anemone (Cnidaria) with radially symmetry (Nematostella vectensis) [7–11]. In addition, many plants exhibit different kinds of symmetry, e.g., rotational symmetry,
but also bilateral symmetry. Today, most plants are flowering plants which are evolved
from the Angiospermae. They appeared, however, much later than the Animals [12,13],
at ≈140–250 Mya ago. There has been an evolution of the symmetry in Animals after the emergence of the
bilateral symmetry. On one hand, there are many examples of a tendency to develop some
asymmetry in Animals, and, on the other hand, there is a corresponding evolutionary
tendency for ensuring the bilateral symmetry. The stability of the bilateral symmetry in Animals is maintained by Darwinian natural
selections. It is in many contexts an advantage to have the Animal’s organs (ears, eyes,
limbs, etc.) placed symmetrically with respect to the symmetry plane. But beyond that,
the bilateral symmetry is also maintained by sexual preference. The bilateral symmetry
is preserved during the evolution of sexual dimorphism, which, in general, results in a
sexual differences, usually with dominating males. The success in male-male competition
reflects high quality, and it is normally assumed that the female preference for dominant
males should be widespread. But, e.g., investigations in Reference [14] show that females
of the rock lizard Lacerta monticola prefer more symmetrical males as potential mates,
by which the bilateral symmetry is preserved. The sexual preference among Humans
shows, correspondingly, that a high degree of symmetry in the human faces is attractive,
and it is found to be masculine for men and to be feminine for females [15]; bilateral
symmetry is, in general, aesthetically attractive [16]. y
y
g
y
But there is also many examples of asymmetry in Animals. Almost all Animals
are Bilaterians with bilateral embryos, but many Animals exhibit asymmetric elements. 1. Introduction A biological organism consists of stereo specific molecules.The proteins and enzymes
are polymers of L-amino acids and the carbohydrates are polymers of D-carbohydrate
units. The structures in a biological organism are obtained from an undifferentiated cell
by biochemical reactions with asymmetric enzymes. The development of the organism
depends on this quality, whereby the DNA in an undifferentiated cell ensures the complex
and unique biological order in the organism. Citation: Toxvaerd, S. The
Emergence of the Bilateral Symmetry
in Animals: A Review and a New
Hypothesis. Symmetry 2021, 13, 261. https://doi.org/10.3390/sym13020261 Nevertheless, most biological organisms exhibit different kinds of symmetry; the first
Animals (Metazoa, which our Darwinist ancestor) have bilateral symmetry (Figure 1), and
many plants exhibit rotational symmetry, and it raises some questions: Academic Editor: Markus Meringer
Received: 7 January 2021
Accepted: 27 January 2021
Published: 4 February 2021 Academic Editor: Markus Meringer
Received: 7 January 2021
Accepted: 27 January 2021
Published: 4 February 2021 Figure 1. An example of bilateral symmetry in Homo sapiens: La cueva de las manos (Argentina)
7300 BC. Copyright Wiki Commons. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the author. 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/). Figure 1. An example of bilateral symmetry in Homo sapiens: La cueva de las manos (Argentina)
7300 BC. Copyright Wiki Commons. https://www.mdpi.com/journal/symmetry Symmetry 2021, 13, 261. https://doi.org/10.3390/sym13020261 Symmetry 2021, 13, 261 2 of 10 I. How can the evolution from an undifferentiated cell without bilateral symmetry to
a complex biological organism with symmetry, which is based on asymmetric DNA and
enzymes, lead to the bilateral symmetry? y
y
y
II. Is this evolution to an organism with bilateral symmetry obtained by other factors
than DNA and enzymatic reactions? y
I shall try to answer these two questions based on the existing scientific literature,
after first describing the evolution of the bilateral symmetry in Animals. 2. Symmetries in Animals and Plants Reviews of asymmetry in Animals are given in References [17–19], with the key ques-
tion [17]: do common rules govern how direction of asymmetry is determined during
ontogeny to yield an asymmetrical individual? The answer is that both genes, environment,
and chance influence the asymmetry. There is countless examples of local asymmetry
in Animals, including asymmetry in Humans [19]. An asymmetry in Animals is, e.g.,
developed in trilobites relatively early after the emergence of the bilateral symmetry [20]. The asymmetry in a lobster with a cutter and a crusher is established during a lobsters
evolution and appears with both the crusher to the left and to the right. While the critical
period for crusher determination is genetically determined, the actual trigger is influenced
by experience [21]. A similar asymmetry is obtained for crabs [22]. Flat fish have a re-
markable asymmetry with both eyes placed on one side of the head. The asymmetry in
a flat fish is genetically determined and emerges in its late larval condition, but with the
occurrence of both asymmetries in samples of flat fish [23] (Figure 2). A similar example of
asymmetry is the Human right-/left-handed asymmetry, which is initialed at an early state
of the human embryo [24]. 3 of 10 Symmetry 2021, 13, 261 Figure 2. Two flounder caught in the Baltic Sea. They are asymmetrical Animals but exhibit together
a bilateral symmetry. A flounder has both eyes on the same side of its bilateral Animal plane. Figure 2. Two flounder caught in the Baltic Sea. They are asymmetrical Animals but exhibit together
a bilateral symmetry. A flounder has both eyes on the same side of its bilateral Animal plane. The conclusion concerning the bilateral symmetry and asymmetry in Animals is that
the bilateral symmetry first appears in the Animal at the late Precambrian evolution and
the Cambrian explosion. The asymmetries in Animals appear later and can be caused by
genetic, as well as environmental factors, and by chance [17], but with the basic bilateral
symmetry maintained in the Animals. 3.1. The Cellular Polarity and the Cell Division 3.1. The Cellular Polarity and the Cell Division There exist an extensive review literature about the developmental order and structure
in a biological system [27–35], and with the focus on the changes in cell polarity. Cell polar-
ity refers to spatial differences in shape, structure, and function within a cell. According
to Reference [27], “the origin of large-scale symmetry in biology often lies in asymmetry at a
smaller scale”; and, on a cellular level, there is already cell polarity with asymmetries in a
simple Bacteria. p
A Procariote exhibits a complex cell polarity with asymmetries [36], and, in Ref-
erence [28], the authors conclude that such a small scale asymmetry in the polarity of
cytoskeletal filaments in two bacteria (Caulobacter crescentus and Bacillus subtilis) plays a
central role in the symmetry break at the cellular level through a protrusive force genera-
tion and directional transport of molecular assemblies and organelles. In Reference [29],
the cellular polarity of RNA in bacterial cells is reviewed. Their cell division is caused by
three polarity protein determinants, MinC, MinD, and MinE, that oscillate between the cell
poles during cell division. The mechanism for the self-organization of proteins is reviewed
in Reference [30], with focus on a possible recruiting of specific proteins to the cell poles by
a self-assembling. Another review of articles concerning polarity and growth mechanism
in bacteria is given in Reference [31]. The bacterial cell growth consists of two distinct
phases: cell elongation and septum formation prior to cell division. They conclude that
temporal regulation of the peptidoglycan synthesis is a common theme. The kinetics in cell
membranes with emergence of cell polarization is reviewed in Reference [32], where the
authors conclude that the membrane kinetics is responsible for the cell polarity. The self-
organization of structures in cell with cell polarity and “mechanical forces” is reviewed
in Reference [33], with the hypothesis that it is a “ feedback interaction between polarity,
mechanics and fate” through self-organizing interactions in this tripartite relationship. The conclusion from the reviews of polarity in a Bacteria is that it is not at all “chem-
istry in a bag”, but that already a Bacterium is a highly ordered system and with a com-
plex polarity at the cell division. The polarity includes oscillating peptide locations and,
presumably, also regulation by metabolic reactions in the plasma membrane (peptidogly-
can synthesis). 3.1. The Cellular Polarity and the Cell Division The symmetry evolution from Cnidaria to Animal are reviewed in References [7–11],
and the evolution in polarity is reviewed in References [34,35]. In Reference [35], the au-
thor’s hypothesis is that the emergence of the bilateral symmetry in Animals is the optimal
response to mechanical forces, including gravity, on the multicellular biological organism. The common observation in all the reviews is that there is a polarity in the cells, the
cell-membrane, and the location of the cell proteins and genes. The explanation for the
emergence of the bilateral symmetry is that it is this structural order in the cell and the
genes which, together with an “environmental impact” on the cells, causes the bilateral
order at the evolution to an Animal. According to the reviews, the precursor could be
structural asymmetries with protrusive forces [28], self-assembling [30,33], gravity and
mechanical forces from (growth) constraints [35], mechanical forces and embryogenic cell
fate [33], and kinetics in the cell [29] and cell membrane [31,32]. But common for all the
reviews is that the genetics alone is not sufficient to ensure the evolution of symmetry, and,
if so, it answers the second question raised here. 3. The Bilateral Symmetry in Animals There is an extensive literature about the emergence of the bilateral symmetry. The Dar-
winist ancestor is as mentioned a sea anemone Cnidaria, which has rotational symmetry. Since oligomeric enzymes in fact can have rotational symmetry [25,26] despite their asym-
metric protein units, the morphogenesis of Cnidaria with rotational symmetry could alone
be caused by the rotational symmetry of oligomeric enzymes, but this subject is not to de-
bate. Here, I will focus on the evolution from a biological system with rotational symmetry
to a system with bilateral symmetry. The biological evolution is not revolutionary but characterized by gradual changes
most often over enormous times, and, despite the general accepted name: the Cambrian
explosion, this biological evolution had appeared over millions of years. An example of
religious symbols with rotational and bilateral symmetry is shown in Figure 3. The old
(and misused) religious Swastika icon has rotational symmetry but contains a cross which
also has bilateral symmetries, and the change to the Crucifix with only bilateral symmetry
can be obtained by small and continuous changes in the Swastika figure. By analogy with
this property, the change from Cnidaria to Animal can be obtained by small and continuous
evolutionary changes. As in the case of the emergence of asymmetries in Animals, one shall
expect that both genes and environment and chance influence the change of symmetry
from rotational to bilateral symmetry. 4 of 10 Figure 3. Religious symbols with symmetry. The old Indian Swastika icon with rotational symmetry
contains a cross with bilateral symmetries. The Crucifix has only a bilateral symmetry. Copyright
Wiki Commons
Figure 3. Religious symbols with symmetry. The old Indian Swastika icon with rotational symmetry
contains a cross with bilateral symmetries The Crucifix has only a bilateral symmetry Copyright Figure 3. Religious symbols with symmetry. The old Indian Swastika icon with rotational symmetry
contains a cross with bilateral symmetries. The Crucifix has only a bilateral symmetry. Copyright
Wiki Commons. A Procariote exhibits a complex cell polarity with asymmetries [36], and, in Ref-
Figure 3. Religious symbols with symmetry. The old Indian Swastika icon with rotational symmetry
contains a cross with bilateral symmetries. The Crucifix has only a bilateral symmetry. Copyright
Wiki Commons. 4 of 10 Symmetry 2021, 13, 261 4. The Kinetics in a Cell The kinetics in a cell are very complex, but the central part in the metabolism, the
Glycolysis, is common for all living organisms. The biochemical reactions with carbohy-
drates in the Glycolysis is catalyzed by enzymes, and the consecutive biochemical reactions
with stereo specific enzymes ensure the homochirality in the carbohydrates. The emergence
of homochirality in carbohydrates is not known, but it could either by obtained endoge-
nously (spontaneous Gibbs free energy favorable) [39,40] or obtained by stereo specific
enzymes [41]. The Glycolysis has a remarkable property: the concentrations of the components
in the Glycolysis oscillate. Oscillations in the concentration of NADH in a cell has been
known for a long time [42]. Figure 4 shows the oscillation in the in vivo concentration of
NADH in a yeast bacteria Saccharomyces cerevisiae [43]. In agreement with this behavior,
a comprehensive model for the consecutive metabolic reactions in the glycolysis of the yeast
bacteria exhibits oscillations of the concentrations [44]. But the experimentally determined
time-oscillating concentration in NADH in the yeast bacteria and the concentrations in the
kinetic model for the Glycolysis are mean concentrations in the cells, and the question is
whether there could be a connection between the oscillations in these concentrations of the
chemical components in the metabolism and the polarity in the cell at the cell division. Figure 4. In vivo, oscillations of NADH concentration in yeast bacteria [43]. The concentration
of NADH in arbitrary unit (a.u.) in the yeast bacteria as a function of time after the start of the
measurement. The period of the oscillations is 34.8 ± 0.2 s. Figure 4. In vivo, oscillations of NADH concentration in yeast bacteria [43]. The concentration
of NADH in arbitrary unit (a.u.) in the yeast bacteria as a function of time after the start of the
measurement. The period of the oscillations is 34.8 ± 0.2 s. The polarity and the cell growth with elongation at cell division are described in
Reference [31], with emphasis on polar growth in bacteria with cell elongation and septum
formation, and Reference [30] reviews the literature about the “self-organizing” of the genes
to the cell pole and summarizes: “In particular, the cell poles-the ends of rod-shaped cells-
constitute important platforms for cellular regulation that underlie processes as essential as
cell cycle progression, cellular differentiation, ...”. 3.2. Precursors for the Emergence of the Bilateral Symmetry in Animals 3.2. Precursors for the Emergence of the Bilateral Symmetry in Animals In searching for a precursor for the bilateral symmetry in Animals, there are only few
guide lines. There is, however, of the order 3 billion years between the emergence of the
first simple Bacteria and the Cnidaria in the Ediacaran and Cambrian period. And the
evolution of polarity in a simple Bacteria to a (Eukariote-) multicellular Cnidaria with its
cell polarity has probably evolved rather continuously in the biological systems in the
oceans over this enormous time span. It is, therefore, natural to search for an influence
on the cell, its structure, and its genes that has been present throughout this long period
of time. Gravity is not good candidate for the simple reason that a bacterium and the Symmetry 2021, 13, 261 5 of 10 5 of 10 polarity of the cells in the biological organisms did not have a constant orientation to the
gravity force. The surface tension in a cell and protrusive forces [28] is a possibility because
the cell structure with a membrane with non-uniformity in molecular structure will give
reason not only to a surface tension but also to local tensions in the membrane [37], e.g., the
ion-channels and the membrane proteins are non-uniformly distributed. Self-assembling
by self-stabilizing change is another possibility [38]. But the most obvious candidate for the
cause of the polarity in the cell and the changes in polarity at the embryonic development
is the kinetics in the cells. However, the kinetics in the cells are also determined by the
genetics of the biosystem. 4. The Kinetics in a Cell The polarity covers already for Bacteria
over a highly ordered gene structure with oscillations (change of local positions) of genes Symmetry 2021, 13, 261 6 of 10 during the cell division [29], and the question is: what kind of evolutionary kinetics could
have established this spatial organization and dynamics? during the cell division [29], and the question is: what kind of evolutionary kinetics could
have established this spatial organization and dynamics? 4.1. Early Embryonic Pattern Formation in Drosophila One of the Animals which appeared at the Cambrian explosion is the small fruit fly
Drosophila melanogaster [9], and its metamorphosis has been heavily investigated. One re-
markably feature in the evolution of Drosophila is the early embryonic pattern formation
with seven stripes with genes for regulation of the development in multicellular organisms
with cell differentiation and morphogenesis (Figure 5). The stripes contain the pair-rule
genes in the embryo preceding gastrulation (i.e., where the blastula in the embryo goes
from a single layer to a multilayer blastula) [45,46]. The stripes in the multicellular em-
bryo also contain genes for the early endosome organization for sorting of the cells in the
embryo [47] and homobox genes for the bilateral evolution [48,49]. But the figure of the
embryo and the stripes has an apparent rotational symmetry, although the genes for the
bilateral evolution may have asymmetrical positions in the stripes. The formation of the
three-dimensional bilateral structures from patterned epithelial cell sheets in Drosophila is
modeled in Reference [50]. Figure 5. Early embryonic stripe patterns (violet) of the concentration of eve RNA in a Drosophila
embryo [51]. Figure 5. Early embryonic stripe patterns (violet) of the concentration of eve RNA in a Drosophila
embryo [51]. 4.2. Models for Polarity at the Emergence of the Bilateral Evolution 4.3. The Hypothesis about the Emergence of Bilateral Symmetry in Animals 4.3. The Hypothesis about the Emergence of Bilateral Symmetry in Animals 4.3. The Hypothesis about the Emergence of Bilateral Symmetry in Animals Two questions were formulated in the Introduction: I. How can the evolution from an undifferentiated cell without bilateral symmetry to
a complex biological organism with symmetry, which is based on asymmetric DNA and
enzymes, lead to the bilateral symmetry? I. How can the evolution from an undifferentiated cell without bilateral symmetry to
a complex biological organism with symmetry, which is based on asymmetric DNA and
enzymes, lead to the bilateral symmetry? y
y
y
II. Is this evolution to an organism with bilateral symmetry obtained by other factors
than DNA and enzymatic reactions? II. Is this evolution to an organism with bilateral symmetry obtained by other factors
than DNA and enzymatic reactions? According to the present analysis, the answer to these two connected questions may
be that the evolution of the bilateral symmetry in Animals already started in a simple
Bacterium with a polarity of genes and continued in the succeeding three billion years
with biological evolution to the multicellular Cnidaria with rotational symmetry. At the
end of the Precambrian evolution and subsequent Cambrian explosion, the polarity in
the multicellular organism was polarized further and resulted in the emergence of the
Bilaterians. The polarity in the multicellular embryo of an Animal exhibits stripe patterns
of the genes in the embryo, but whether there already is a similar polarity of the gene
structure in the Cnidaria is not reported. p
The morphogenesis must be initiated by biochemical reactions, which are part of the
chemical network including the metabolism. According to the hypothesis, the evolution
of the polarities and the emergence of Bilaterians was induced by the oscillations in the
Glycolysis, which is regulated by asymmetrical enzymes. But the evolution in the polarity
is not solely controlled by the genetics. The induced Turing patterns also depend on the
cell and embryo size. 4.2. Models for Polarity at the Emergence of the Bilateral Evolution The blue color is for a relative high stationary concentration of the mor-
phogen, and yellow is for a relative small concentration. 4.2. Models for Polarity at the Emergence of the Bilateral Evolution The stripe patterns in Drosophila melanogaster can be obtained by a reaction diffusion
model with cross inhibition or activation of the kinetics for a morphogen in a cell [52]
(Figure 6), and the embryonic development during gastrulation is correspondingly obtained
by an another reaction diffusion model [53]. The patterns obtained by the reaction-diffusion
models for the embryogenesis are examples of Turing structures, and these Turing patterns
depend on the confinement [54]. In the case of oscillations of a morphogen, the spacial
stripes depends on the cross inhibition or cross-activation and on the ratio D/R2 between
the diffusion coefficient for the morphogen and the size R for the biological system [55]. For a bacteria, R is the size of the cell, and, for Drosophila, it is the size of the embryo and,
as the embryo grows, the diffusion may be regulated enzymatically in order to maintain
the ratio D/R2 and the pattern in the embryo [55]. Figures 5 and 6 show a striking resemblance, but there is no direct evidence of such a
connection between the oscillation in the Glycolysis and the formation of the stripe patterns
in the embryo. The polarity and gene patterns in Drosophila obtained in Reference [52] are
for a reaction-diffusion model of a morphogen with autocatalysis, but it is in fact also a
simple model for the oscillation in the Glycolysis [56] (see Appendix in Reference [52]). And a time-oscillating system can induce stationary patterns in another system. The sta-
tionary wavy sand patterns in a beach bottom is an example of such induced patterns
caused by the surface waves. So, the oscillations in the Glycolysis can have induced the
stripe patterns in the Drosophila. 7 of 10 Symmetry 2021, 13, 261 Figure 6. Turing concentration patterns of a morphogen in an elliptically-shaped cell with reaction-
diffusion dynamics [52]. The blue color is for a relative high stationary concentration of the mor-
phogen, and yellow is for a relative small concentration. Figure 6. Turing concentration patterns of a morphogen in an elliptically-shaped cell with reaction-
diffusion dynamics [52]. The blue color is for a relative high stationary concentration of the mor-
phogen, and yellow is for a relative small concentration. Figure 6. Turing concentration patterns of a morphogen in an elliptically-shaped cell with reaction-
diffusion dynamics [52]. 5. Summary The biological organisms evolved from Bacteria for more than 3 billion years before
the first Bilateria appeared, and a simple Bacterium is already a very complex system with
cell polarity. The biological mechanisms that caused this development are most likely also
complex. All living organisms have some characteristic features, but a given feature is
normal distributed with small deviations from the mean value. Traditionally, the evolution
is described as Darwinian with enhancements of a deviation by change and/or a ”survival
of the fittest” evolution. An example of the Darwinism survival of the fittest evolution
is illustrated in Figure 2, with two flounders that have a bilateral ancestor without the
asymmetry of the position of the eyes. There are non-genetic factors, e.g., change and self-stabilizing change [38], and cell
forces (e.g., surface tension) which also might have influenced the evolution toward the
bilateral Animals. Most likely, the evolution from a Bacterium toward a multicellular Symmetry 2021, 13, 261 8 of 10 8 of 10 Cnidarian and, finally, an Animal with bilateral symmetry has been continuous over this
long time of the evolution before the Cambrian explosion, and with small enhancements
of local symmetries, as illustrated by the religious icons in Figure 3. The success of this
evolution has been conserved in the DNA of an Animal, and, on a superficial level, it must
now seem as if the development from a undifferentiated Animal cell to its embryo, and
finally, to an Animal is only genetically determined. But, according to the above hypothesis,
this does not have to be the case either. If the above hypothesis is correct, then it is also
crucial that this development takes place first in a cell, and then in an embryo with a
compact shape, in order to obtain the Turing patterns. p
p
g p
In articles about the origin of life, one finds the view that life is more than chemistry in a
bag. But this is an unfortunate formulation because all life takes place in cells. The confined
and protected chemical environment in a cell is crucial for the stability of the biochemical
reactions [57]. The compact form of a cell is obtained by the surface tension and the osmotic
pressure, and they are examples of non-genetic factors in the evolution. 5. Summary According to the
hypothesis above, the compact form of a undifferentiated cell, and later the embryo, is also
crucial for the stability of the Turing patterns for the evolution of bilateral symmetry in
an Animal. So, the answer to the second question formulated here is the fact that all life is
obtained inside cells or collections of cells, which are also crucial for the emergence of life
and the emergence of Bilaterians. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Axel Hunding is gratefully acknowledged. This work was supported by the
VILLUM Foundation’s Matter project, grant No. 16515. Acknowledgments: Axel Hunding is gratefully acknowledged. This work was supported by the
VILLUM Foundation’s Matter project, grant No. 16515. Conflicts of Interest: The author declares no conflict of interest. Conflicts of Interest: The author declares no conflict of interest. References 1. Djokic, T.; Van Kranendonk, M.J.; Campbell, K.A.; Walter, M.R.; Ward, C.R. Earlist signs of life on land preserved in ca. 3.5 Ga hot
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Standalone inertial pocket navigation system
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Standalone Inertial Pocket Navigation System
Estefania Munoz Diaz and Ana Luz Mendiguchia Gonzalez and Fabian de Ponte M¨uller
German Aerospace Center (DLR)
Institute of Communications and Navigation
Oberpfaffenhofen, 82234 Wessling, Germany
Email: Estefania.Munoz@dlr.de Estefania Munoz Diaz and Ana Luz Mendiguchia Gonzalez and F
German Aerospace Center (DLR)
Institute of Communications and Navigation
Oberpfaffenhofen, 82234 Wessling, Germany
Email: Estefania.Munoz@dlr.de Fig. 1. The presented standalone inertial navigation system only requires
introducing the IMU in the trousers’ pocket and walking freely. Abstract—Positioning applications became more important in
recent years not only for security applications, but also for the
mass market. Having a pedestrian navigation system embedded
in a mobile phone is a realistic solution since it is equipped
with low-cost sensors and the smartphone is located in a non-
obstructive way. The location of the smartphone is important,
since the position estimation process depends on it. Therefore,
we propose to distinguish between pocket or bag, phoning,
texting and swinging. We present a standalone inertial pocket
navigation system based on an inertial measurement unit. For the
computation of the orientation, we have developed an attitude
estimator based on an unscented Kalman filter. The update stage
has two different updates based on the acceleration and the
magnetic field. Therefore, a zero acceleration detector, a magnetic
disturbances detector and a static periods detector have been
developed. The odometry in our navigation system is computed
through an extended Kalman filter. The position is predicted with
a movement model which is periodically updated through position
corrections computed by the position computer. It comprises a
step detector and a step length estimator based on the norm of
the acceleration. The performance of our attitude estimator in
comparison with the ground truth orientation is shown. The rest
of the handheld positions are also tested for orientation. Likewise,
we show pocket odometries of different users with the floor plan
superimposed. Fig. 1. The presented standalone inertial navigation system only requires
introducing the IMU in the trousers’ pocket and walking freely. assumption of a fixed orientation during the rest phases. This
is a zero angular rate update (ZARU) [2]. The previously explained methods help limiting the growth
of errors, but they require having a foot-mounted IMU. Although this is an excellent solution for pedestrian indoor
navigation, for the mass market it is more convenient to
integrate the navigation system in the mobile phone. This is
more practical and less obstructive than attaching an extra
device to the shoe, however challenging since zero velocity
corrections cannot be applied. Index Terms—Pocket, navigation, attitude estimator, magnetic
field, odometry, heading, handheld. Standalone Inertial Pocket Navigation System
Estefania Munoz Diaz and Ana Luz Mendiguchia Gonzalez and Fabian de Ponte M¨uller
German Aerospace Center (DLR)
Institute of Communications and Navigation
Oberpfaffenhofen, 82234 Wessling, Germany
Email: Estefania.Munoz@dlr.de In this paper a standalone inertial pocket navigation system
is presented. The system only requires introducing the IMU in
the trousers’ pocket and walking freely (Figure 1). The pocket
does not have to be tight. II. RELATED WORK If the IMU is attached to the body near the center of mass: There is also an initial prior work in the area of complete
attitude determination [10]–[12]. Accelerometer, gyroscope
and magnetometer measurements are combined to obtain it. Taking benefit of having two vectors, such as Earth magnetic
field and gravity, whose directions and intensities are known,
the orientation of the IMU can be extracted. • Based on a biomechanical model, where the kneeless
biped is modeled as an inverted pendulum, and scaling
the final estimation with a constant K to calibrate for
each user [16]. • Based on a biomechanical model, where the kneeless
biped is modeled as an inverted pendulum, and scaling
the final estimation with a constant K to calibrate for
each user [16]. l1 = K ·
2Lh −h2,
(3) (3) Bayesian approaches are commonly used to propagate
dynamic variables such as Euler angles. In particular, the
EKF has become the accepted basis for the major part of
orientation algorithms [10], [12], due to the non-linearities of
the equations used. However, a Madgwick filter, presented in
[13], provides a non-bayesian based orientation algorithm. In
this case, the gradient descent algorithm is used. A magnetic
distortion compensation is also incorporated on it. where l1 represents the estimated step length, h the
vertical displacement of the pelvis and L, the leg’s length. where l1 represents the estimated step length, h the
vertical displacement of the pelvis and L, the leg’s length. • Using an empirical relationship of the vertical accelera-
tion and the step length [7], [19], [25]. • Using an empirical relationship of the vertical accelera-
tion and the step length [7], [19], [25]. l2 = K ·
4amaxvi −aminvi ,
(4) (4) where l2 represents the estimated step length, K is a cal-
ibration value and amaxvi and aminvi are the maximum
and minimum values of the vertical acceleration during
step i. Respect to the sensor location, the previous work is mostly
focused on foot-mounted techniques [14], [15]. With the foot-
mounted IMU, strong corrections can be applied every step
against the accumulated errors in strapdown systems. If there are no restrictions on the IMU’s placement [20],
[21], [26]–[28], taking advantage of the relationship between
step length (l), height (h), step frequency (fstep) and some
calibration parameters (a, b, c) different for each user: For non-body-fixed sensor locations, PDR is the most
suitable solution. II. RELATED WORK There is a large amount of work in the area of pedestrian
indoor localization. Respect to the attitude/heading determina-
tion for pedestrian dead-reckoning (PDR) approaches, only the
heading or yaw angle, and not the complete attitude, seems to
merit attention in the literature. Using the complete acceleration, this technique is inde-
pendent of the sensor’s orientation. The peaks correspond
to the step occurrences because they are generated by the
vertical impact when the foot hits the ground. Since the
pattern of impact signal depends on the type of movement
and it is greatly affected by the user’s walking velocity, the
determination of the thresholds for reliable step detection
is challenging. Even considering this problem, distinguishing
steps with the norm of the vertical or complete acceleration is
also used when the sensors are handheld [19]–[22]. The combination of gyroscope and magnetic compass [6],
[7] has widely been applied to obtain the heading for such
a navigation systems. The integration of the gyroscope mea-
surements provides it directly. Due to the biases, the long
term drift in yaw needs to be corrected. The magnetometer,
having been calibrated, is used with this purpose. The short
term accuracy of the gyroscope helps detecting the short term
external disturbances of the magnetometer. Especially in [8],
[9], an interesting magnetic disturbance detector is developed. The combination of gyroscope and magnetic compass [6],
[7] has widely been applied to obtain the heading for such
a navigation systems. The integration of the gyroscope mea-
surements provides it directly. Due to the biases, the long
term drift in yaw needs to be corrected. The magnetometer,
having been calibrated, is used with this purpose. The short
term accuracy of the gyroscope helps detecting the short term
external disturbances of the magnetometer. Especially in [8],
[9], an interesting magnetic disturbance detector is developed. There is also an initial prior work in the area of complete
attitude determination [10]–[12]. Accelerometer, gyroscope
and magnetometer measurements are combined to obtain it. Taking benefit of having two vectors, such as Earth magnetic
field and gravity, whose directions and intensities are known,
the orientation of the IMU can be extracted. i
h
l
d The main current techniques to estimate the step length can
be classified depending on the IMU’s location, as specified in
[23], [24]. and and I. INTRODUCTION The market for positioning applications is rising in recent
years for both, commercial and professional users. So far,
most positioning applications are based on Global Navigation
Satellite Systems (GNSSs), such as the American Global
Positioning System (GPS) or the European Galileo. However,
due to the lack of visible satellites, the availability of GNSS
is degraded in certain scenarios such as urban canyons or
indoors. The use of inertial measurement units (IMUs) is a
viable solution to address this problem because they are able
to provide human odometries [1] and show promising potential
for reliable indoor pedestrian navigation. Additionally, IMUs
are infrastructure independent. In Section II, a brief summary of the related work is
gathered in order to offer an overview of the state of the art. The developed inertial pocket navigation system is presented
in Section III. Due to its complexity, we have dedicated a great
part of this paper, the Section IV, to describe in detail the
attitude estimator. In this section, the update equations system
is carefully explained as well as the three developed detectors,
i.e. the zero acceleration detector, the magnetic disturbances
detector and the static periods detector. The position estimator
is presented in Section V. The step detector and step length
estimator are described in this section. However, micro electromechanical systems (MEMS) based
IMUs have to face problems such as drifting sensors. Many
authors [2], [3], [4], [5] use the rest phase of the foot to limit
the growth of errors. Foxlin [3] was the first to propose an
extended Kalman filter (EKF) to estimate and subtract the
errors with the zero velocity updates (ZUPT) when the sensor
does not move. There are more suitable solutions like the Finally, the experimental results are shown in Section VI. First, we show the performance of the attitude estimator
compared to the ground truth given by a precise fiber optic
gyroscope (FOG). We offer as well a brief attitude analysis of
further handheld positions. Odometries of the described pocket
navigation system are shown with the floor plan superimposed
as a ground truth.
241 where the evaluated feature for the peaks detection is the norm
of the complete acceleration. II. RELATED WORK In this case, the computation of the user’s
position uses xstepi = xstepi−1 + l · cos(ψ)
(1)
ystepi = ystepi−1 + l · sin(ψ),
(2) xstepi = xstepi−1 + l · cos(ψ)
(1) (1) l3 = h · (a · fstep + b) + c. (5) (5) IV. ATTITUDE ESTIMATOR In this section we describe the attitude estimator. All compo-
nents such as the zero acceleration detector, the magnetic dis-
turbances detector and the static periods detector are detailed. The structure of the prediction and update stage is carefully
explained. The unscented Kalman filter (UKF) propagates mean and
covariance of the states through a linear regression between a
set of points (sigma points) drawn from the prior distribution
of the states. In the EKF the state distribution is propagated
analytically through the first-order approximation of the Taylor
series, thus the mean and covariance could be corrupted for
high non-linearities. The attitude estimator is a UKF whose states are the Euler
angles roll, pitch and yaw and the gyroscope biases. As Figure 3 shows, the attitude estimator receives di-
rectly the accelerometer, gyroscope and magnetic field sensor
measurements. The upper box in light blue represents the
prediction stage. The prediction stage for the Euler angles
consists of a simple integration of the turn rates. For clarity,
only this part has been represented in the block diagram. The
gyroscope’s biases are predicted using a random walk model. The two main tasks of the inertial pocket navigation system
are the estimation of the attitude and the estimation of the
position. In Figure 2 a block diagram is shown. For the attitude estimator filter, described in Section IV, a
UKF has been chosen due to the high non-linearities of the
equations involved. This filter has 6 states: the Euler angles,
i.e. roll, pitch and yaw and the biases of the gyroscope. It
counts with a prediction stage based on the integration of
the gyroscope for the Euler angles and on an random walk
model for the biases. For the update stage, corrections using
acceleration and magnetic field are carried out. In order to
detect periods of zero acceleration of the user, a detector
has been developed. The magnetic field measurements are
known to be distorted in certain scenarios, thus we have a
magnetic disturbances detector. We have also implemented a
static periods detector. The lower light blue box represents the update stage. For
applying the updates three specific detectors are needed. During the periods of no-user-acceleration only the gravity
acceleration is measured. Therefore, the zero acceleration
assumption (ZAA) can be only applied few milliseconds every
step and obviously when the user does not move. other blocks to help navigation. When certain constraints are hold, linearity and gaussianity,
an optimal solution is tractable, which is a Kalman filter. There are also approximations to the optimal solution when
these conditions are not fulfilled [29]. In this case, suboptimal
algorithms have been used due to the non-linearities of the
equations involved. III. INERTIAL POCKET NAVIGATION SYSTEM In this filter there are three
implemented to help applying the updates, the zero acceleration det
magnetic disturbances detector and the static periods detector. Acc,Gyro,Mag
roll,pitch,yaw
නࢍ࢟࢘
ZAdetector
MDdetector
SPdetector
Fig. 3. Attitude estimator UKF filter. The light blue boxes represent the
two stages, prediction and update. In this filter there are three detectors
implemented to help applying the updates, the zero acceleration detector, the
magnetic disturbances detector and the static periods detector. Acc,Gyro,Mag
roll,pitch,yaw
positionͲx
positionͲy
Attitude
Estimator
Position
Estimator
Fig. 2. Inertial pocket navigation system’s block diagram. It has two modules,
the attitude estimator and the position estimator, which are a UKF and a EKF
respectively. The attitude estimator has 6 states which are the Euler angles
and the gyroscope biases. The position estimator has 2 states which are the
position-x and the position-y. roll,pitch,yaw
නࢍ࢟࢘
ZAdetector
MDdetector
SPdetector Attitude
Estimator Position
Estimator Fig. 2. Inertial pocket navigation system’s block diagram. It has two modules,
the attitude estimator and the position estimator, which are a UKF and a EKF
respectively. The attitude estimator has 6 states which are the Euler angles
and the gyroscope biases. The position estimator has 2 states which are the
position-x and the position-y. Fig. 3. Attitude estimator UKF filter. The light blue boxes represent the
two stages, prediction and update. In this filter there are three detectors
implemented to help applying the updates, the zero acceleration detector, the
magnetic disturbances detector and the static periods detector. Fig. 3. Attitude estimator UKF filter. The light blue boxes represent the
two stages, prediction and update. In this filter there are three detectors
implemented to help applying the updates, the zero acceleration detector, the
magnetic disturbances detector and the static periods detector. III. INERTIAL POCKET NAVIGATION SYSTEM ystepi = ystepi−1 + l · sin(ψ),
(2) (2) The inertial pocket navigation system will be described in
this section. Firstly, we justify the choice of the filters and,
lastly, we present the two main parts of it, i.e. the attitude
estimator and the position estimator that will be described in
Section IV and Section V respectively. being xstepi the position in the x-axis at the time stamp i,
ystepi the position in the y-axis at the time stamp i, l the
estimated step length and ψ the estimated heading. For the step detection, there are different approaches de-
pending on the IMU’s location. The further the sensor is from
the foot, the more difficult is to recognize a pattern. If the
sensor is attached to the ankle [6], it is still possible to detect
steps recognising patterns on the acceleration signal. However,
for waist-/hip-mounted or pocket carried, this is not the most
ingenious solution and three approaches are mainly used. The
first one considers the peaks detection of the pelvis’ vertical
displacement [7], [16], [17]. The second method consists on
detecting foot steps finding peaks in the variance of the vertical
acceleration and introducing a threshold to avoid spurious step
detections [18]. Thresholds are also used in the third approach, The inertial pocket navigation system should track the
user’s trajectory and process the data online as it arrives. For pedestrian navigation the interesting variable is the user’s
position. Bayesian approaches are the best option to estimate
such dynamic variables. In this context, these variables are
called states. For the dynamic state estimation this algorithm
attempts to construct the posterior probability density function
of the state based on all available information, including
the received measurements. Every time a new measurement
becomes available, it is sequentially filtered. Such a filter has
two stages: prediction and update [29]. 242 Acc,Gyro,Mag
roll,pitch,yaw
positionͲx
positionͲy
Attitude
Estimator
Position
Estimator
Fig. 2. Inertial pocket navigation system’s block diagram. It has two modules,
the attitude estimator and the position estimator, which are a UKF and a EKF
respectively. The attitude estimator has 6 states which are the Euler angles
and the gyroscope biases. The position estimator has 2 states which are the
position-x and the position-y. Acc,Gyro,Mag
roll,pitch,yaw
නࢍ࢟࢘
ZAdetector
MDdetector
SPdetector
Fig. 3. Attitude estimator UKF filter. The light blue boxes repr
two stages, prediction and update. A. Zero Acceleration Detector A. Zero Acceleration Detector The zero acceleration detector is needed in this work for
different applications. This detector finds locally periods of zero or close to zero
acceleration. Firstly, for simplicity, consider the case where
the user is standing. For simplicity also consider free biases
accelerometers. If no biases are present, the measured acceler-
ation is only the gravity. The knowledge of the gravity vector
offers information of the orientation of the IMU, specifically
the roll and pitch. The orientation based on the gravity vector
is heading independent. Fig. 4. Decision tree of the magnetic disturbances detector. If no changes
on the intensity are detected, may be because the user is static. Perturbations
may be present, however the magnetic field is short-term time-invariant. In
this case there is no information to output a new state. If the user is not static
the orientation is checked. random paths in all directions and the raw data was recorded. A sphere is fitted through least-squares out from an ellipsoid
made of uncalibrated data. The biases of all 3 magnetometer
axis are set as the coordinates of the center of the sphere. The radius of the sphere is used to normalize the sensor
measurements to the previously mentioned local magnetic field
intensity. The calibration took place once in an outdoor field
disturbances free. Zero acceleration implies constant velocity. Obviously, if
the user is standing the velocity is constant and equal to zero. Additionally, during the walk, periods of zero acceleration can
be also found. The pedestrian’s step has a pattern, acceleration
and deceleration. Between those rely periods of zero accelera-
tion. Since the sensor is placed in the pocket, no zero velocity
can be found during the walk like using a foot-mounted sensor,
but zero acceleration. However, as previously mentioned, the magnetometer mea-
surements will be distorted due to the proximity of ferromag-
netic materials and electric currents [32]. That means that, even
if the magnetometer is properly calibrated, the measurements
may be distorted if the user walks, for example, through the
sidewalk close to parked cars. The formula used to find zero acceleration periods is
accel2
Xk + accel2
Yk + accel2
Zk < γ,
(6) (6) where accelik being i = X, Y, Z is the acceleration of the i-
axis measured on the time stamp k. A. Zero Acceleration Detector The parameter γ is defined
based on the observation of the accelerometers to make the
zero acceleration detector be active only when the left part of
the inequation (6) is close to the gravity value. Therefore, we have implemented a magnetic disturbances
detector. It is based on two parameters: the intensity and the
orientation of the magnetic field [33]. We have developed a magnetic disturbances detector based
on the identification of the periods where the measured mag-
netic field is constant in intensity and orientation. The fact
that the magnetic field is constant does not imply that it is
disturbances free. We have identified two cases where the
magnetic field is constant and although perturbed. The first
occurs if the sensor is static. The user may be in a building
where the magnetic field is completely distorted, however, if
the user does not move, the distortion is constant in time. This
false detection of a disturbances free magnetic field is avoided
through the static periods detector. The other false detection
arises when hard iron biases are measured by the sensor. The
hard iron errors are caused by a magnetic source which causes
a permanent field in all orientations. This is avoided in our case
through a previous magnetometer calibration. Therefore, since
these two situations are discarded, we consider our detector to
be a magnetic disturbances detector. IV. ATTITUDE ESTIMATOR In order
to correctly apply this, these no-acceleration periods have
to be accurately detected. However, applying this update is
not enough because the heading remains unobservable. The
accelerometer only allows correcting roll and pitch. The magnetic field intensity can be used for correcting the
attitude. Magnetometer measurements are always available,
however, in indoor environments and other challenging out-
door scenarios such as the vicinity of ferromagnetic materials,
the magnetic field is distorted. It is not convenient to use
directly the magnetometer measurements in these cases. In
this work, we use magnetic corrections depending on the
perturbation of the measured magnetic field. Therefore, it is
necessary to identify when the magnetic field is distorted. The
magnetic disturbances detector is based on both, changes on
the magnetic intensity and changes on the orientation of the
measured magnetic field. For the position estimator, described in Section V, a EKF
has been chosen. The states are two: position-x and position-
y. In the prediction stage the two dimensional position is
estimated through a movement model. For the position update
it has been developed a step detector and a step length
estimator. The update occurs once per user’s step. The needed
heading is taken from the attitude estimator filter. We have decided to divide the pocket navigation system in
two parts because we want a modular architecture. This will
allow us modifying separately the position estimator and the
attitude estimator. The modular architecture allows also adding The static periods detector gives an output when the user is
static, i.e. standing, sitting or lying among others. During these 243 Change
Intensity
yes
no
no
Static
Previous
State
yes
Disturbed
no
Change
Orientation
Disturbed
yes
Disturbances
Free
Fig. 4. Decision tree of the magnetic disturbances detector. If no changes
on the intensity are detected, may be because the user is static. Perturbations
may be present, however the magnetic field is short-term time-invariant. In
this case there is no information to output a new state. If the user is not static
the orientation is checked. periods, a realignment is done and the biases of the gyroscope
are updated. Change
Orientation B. Magnetic Disturbances Detector 0
100
200
300
400
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Time (s)
Norm of the total field (mGauss)
||B|| Foot-mounted
||B|| Pocket of the white noise of both, accelerometers and gyroscopes,
is known. Therefore, if the user is static, the mean of all
accelerometer and gyroscope buffers should not be greater than
this value. The static periods detector gives its first output at the
beginning of every walk, because the user is standing a couple
of seconds. This is called the alignment phase. The zero
acceleration principle is used for this phase. It is possible
during the alignment phase to compute the biases of the
gyroscope, because the sensor does not rotate. That means
the sensor measures only the biases of the gyroscopes. On
the contrary, it is not possible during the alignment phase
to compute the biases of the accelerometers, because the
knowledge of the gravity vector is not enough to distinguish
roll and pitch from biases of x- and y-axis. Therefore, during
the alignment phase, we assume the accelerometers are biases
free and everything the accelerometer measures is due to
an initial rotation of the sensor. Thus, the biases of the
accelerometer remain unobservable. Fig. 5. The green curve represents the norm of the total magnetic field for
a foot-mounted sensor and the blue curve represents the norm of the total
magnetic field for the same walk with the sensor in the pocket. The first
part of the walk was indoors, from the second 150 to the 360 the walk was
outdoors and at the end the user enters again the building. This alignment procedure is repeated every time the user
is standing, not only at the beginning, but during the walk
when they are identified by this detector. We take benefit of
these periods for the realignment of the IMU, because it may
move in the pocket during the walk. Additionally during these
periods, we update the gyroscope’s biases. is indoors, then it goes outdoors and finally indoors again to
the starting point. The difference on the starting value of both
curves can be explained because, even if both IMUs are on the
same user, the difference on the high causes much different
perturbations. Around the second 150 the user goes outdoors
and walks surrounding our office building. E. Magnetic Field Update The static periods detector and the magnetic disturbances
detector help activating the magnetic field updates. Figure 7
shows the detected disturbances free periods during the same
walk previously described in the Subsection IV-B. D. Zero Acceleration Assumption (ZAA) Update If there is a change on the intensity, the magnetic field is
disturbed. If not, may be the case that the user is static. We
detect this through the static periods detector. The magnetic
field in indoor environments is known to be constant in time
and varying in direction and intensity. Magnetic signatures are
stable over a period of several months [34] and strongly mod-
ulated in space [32]. Therefore, if the user is static (standing,
lying, sitting...) we keep the previous state, disturbances free
or disturbed. The initial hypothesis is a perturbed magnetic
field. The ZAA is an update for the Euler angles roll and pitch. As previously explained, with the knowledge of the gravity
vector the yaw remains unobservable. The update equations
are
1 φ = tan
accb
Y
accb
Z
−1
,
(8) (8) and θ = tan
⎛
⎝
−accb
X
(accb
Y )2 + (accb
Z)2
⎞
⎠
−1
,
(9) (9) If the user is not static, the change in orientation is checked. Obviously a change in orientation of the magnetic field could
be due to a change in orientation of the user. Therefore, we
verify through the gyroscope the change of the user’s heading. If the user has rotated less than the change registered by the
magnetometers, a perturbation of the magnetic field is present. We compute the magnetometer’s change in orientation through
the Equation (7) for the actual time stamp minus the same
equation for the previous time stamp. where φ represents the roll angle, θ represents the pitch
angle and accb
i for i = X, Y, Z represents the acceleration
measurement for the i-axis in the body frame. Figure 6 shows the detected periods of zero acceleration for
a 25 seconds walk. For a normal speed walk, that means not
running, theoretically a zero acceleration interval will be every
step detected. While standing, the zero acceleration is active
the whole time. After the work realized for this magnetic disturbances
detector we realized that the magnetic signatures are different
at different heights for the same (x,y) position. Therefore, they
are not only varying in x- and y-axis, but in z-axis. B. Magnetic Disturbances Detector From 160 −350
seconds the user walks close to the parking and around the
second 310 the user walks only 1 m away from a parked car. We use also this detector to help the magnetic disturbances
detector, as explained in the previous subsection. B. Magnetic Disturbances Detector The Earth’s magnetic field is modeled as a dipole. At any
location, it can be represented by a three-dimensional vector. For centuries, the Earth’s magnetic field has been used for
navigation purposes. The goal is to determine the direction of
the magnetic North. The angle between the magnetic and the true North is called
declination. Facing the magnetic North, the angle the field
makes with the horizon is called inclination. The Munich Earth
Observatory has reported an average intensity of 48219.7 nT,
an inclination angle of 64.23 degrees and a declination angle
of 2.57 degrees for January 2014 [30]. These measurements
have been taken in F¨urstenfeldbruck, 15 km away from DLR. The magnetometer measurements contain the information
of the orientation of the sensor within the Earth’s magnetic
field. The heading is extracted as follows Figure 4 represents the decision tree of the magnetic dis-
turbances detector. Firstly, the changes on the intensity of
the magnetic field are checked through the standard deviation
and compared with the standard deviation of a non-disturbed
magnetic field. The magnetic field intensity and accordingly
its standard deviation have great variations with the high of
the sensor respect to the floor. Figure 5 shows the intensity of
a foot-mounted sensor compared with the intensity registered
by a sensor in the pocket. The walk was recorded with both
IMUs at the same time. The first part of the walk the user ψ = tan
−magX
magY
−1
± D,
(7) (7) where ψ is the heading measured in radians and magi where
i = X, Y is the magnetic field intensity for the i-axis. D
represents the declination angle. Before using the magnetometer, it is recommendable to
calibrate it [31]. The sensor was manually moved describing 244 0
100
200
300
400
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Time (s)
Norm of the total field (mGauss)
||B|| Foot-mounted
||B|| Pocket
Fig. 5. The green curve represents the norm of the total magnetic field for
a foot-mounted sensor and the blue curve represents the norm of the total
magnetic field for the same walk with the sensor in the pocket. The first
part of the walk was indoors, from the second 150 to the 360 the walk was
outdoors and at the end the user enters again the building. C. Static Periods Detector The static periods detector consists on sliding window
buffers that control acceleration and gyroscope signals con-
stantly. Through the Allan variance, the standard deviation Figure 7 shows that when the user is indoors the magnetic
field is completely distorted. The two clear peaks pointing up-
wards were detected when the user goes through an automatic 245 Fig. 6. The blue curve represents the norm of the total acceleration minus
the local gravity value, measured during a walk. The first 7 seconds the user
is standing. The red circles represent the periods where the detector identifies
zero acceleration. horizontal plane, being the rotation matrix horizontal plane, being the rotation matrix ⃗
magh =
⎡
⎣
cos(θ)
sin(θ) sin(φ)
sin(θ) cos(φ)
0
cos(φ)
−sin(φ)
−sin(θ)
cos(θ) sin(φ)
cos(θ) cos(φ)
⎤
⎦
⃗
magb,
(10)
⃗
⃗ (10) where
⃗
magb and
⃗
magh are the magnetic measurements on the
body frame and projected to the horizontal plane respectively. The roll angle is represented by φ and θ represents the pitch
angle. Once the magnetic measurements are projected onto the hor-
izontal plane, Equation (7) can be used. This update presents
good results for long-term corrections, however it requires
projecting the magnetic measurements using the angles roll
and pitch. Although the magnetic measurements are supposed
to be error free because no disturbances are present, roll and
pitch angles may contain errors which are introduced in the
update of the yaw through the projection. Another source of
errors is an incorrect magnetometer calibration. Fig. 6. The blue curve represents the norm of the total acceleration minus
the local gravity value, measured during a walk. The first 7 seconds the user
is standing. The red circles represent the periods where the detector identifies
zero acceleration. However, the magnetic field has more information apart
from the heading [8]. The errors in roll, pitch and yaw
can be found through the magnetic field measurements, but
only when the magnetic field is not perturbed. Therefore, the
magnetic disturbances detector will be also used for enabling
or disabling this update. 0
100
200
300
400
100
200
300
400
500
600
700
800
900
1000
Time (s)
Norm of the total field (mGauss)
||B||
MD detections 0
100
200
300
400
100
200
300
400
500
600
700
800
900
1000
Time (s)
Norm of the total field (mGauss)
||B||
MD detections
Fig. 7. C. Static Periods Detector The blue curve represents the norm of the total magnetic field
for a sensor placed in the pocket. The red line shows the detections of the
disturbances free magnetic field. The first part of the walk was indoors, from
the second 150 to the 360 the walk was outdoors and at the end the user
enters again the building. This is the same walk represented in Figure 5. A disturbances free magnetic field implies that the measured
magnetic field does not contain errors. Given the magnetic
field at the time stamps k −1 and k, it is possible to find
the rotation between both vectors due to the user motion as
follows ⃗
magb
k = ⃗ωb ×
⃗
magb
k−1,
(11) (11) where
⃗
magb
k−1 and
⃗
magb
k is the magnetic field vector in the
body frame at time stamps k −1 and k and ⃗ωb represents the
rotation between these two vectors in the body frame. Fig. 7. The blue curve represents the norm of the total magnetic field
for a sensor placed in the pocket. The red line shows the detections of the
disturbances free magnetic field. The first part of the walk was indoors, from
the second 150 to the 360 the walk was outdoors and at the end the user
enters again the building. This is the same walk represented in Figure 5. As the magnetic field has no errors because it is not
perturbed, we consider the resulting angles of the rotation,
i.e. Δφmag, Δθmag and Δψmag, the error free rotation due
to the user motion between the time stamp k −1 and k. On the other hand, the rotation due to the user motion
between the time stamp k −1 and k can be also extracted
by integrating the turn rates of the gyroscopes to get the Euler
angles for both time stamps. Therefore, door, once for going outdoors and the last one for going into
the building again. As explained before, even though the user
was outdoors, the detected magnetic field is not completely
clean because the user was walking close to the parking and
approximately on the second 300 the user walks very close
to a parked car. This creates a perturbation on the detected
magnetic field. VI. EXPERIMENTAL RESULTS The last position is always available because it is a state,
however, this movement model needs as well two external
parameters such as the mean velocity of the user and the
heading angle. As explained before, the heading angle is
every time stamp provided by the attitude estimator. The mean
velocity of the user is computed in the step detector and step
length estimator. In order to assess the performance of the described in-
ertial pocket navigation system, we have realized a set of
experiments. In the first part of this section we focus on
the attitude estimator. We have used a FOG as a ground
truth. Then, we have decided to test our attitude estimator
created for pocket located IMUs, with the rest of the handheld
positions, i.e. swinging, calling and texting. These results are
also shown with the FOG as a ground truth. Lastly, we show
the performance of the complete inertial pocket navigation
system with a odometry. As ground truth we plot the floor
plan superimposed. V. POSITION ESTIMATOR V. POSITION ESTIMATOR V. POSITION ESTIMATOR In this section we describe the position estimator and more
in detail the structure of the prediction and update stages. The
position estimator is a EKF whose states are the 2-dimensional
position, i.e. position-x and position-y. The aim of this filter is to sequentially compute the user’s
position. Therefore, the position estimator needs the estimation
of the heading, which is sequentially provided by the attitude
estimator. In order to compute the user’s position, this filter uses
a movement model for the prediction of the position. For
the position updates and the velocity computation we have
developed a step detector and a step length estimator. A. Prediction Stage Fig. 8. The red curve represents the norm of the acceleration minus the local
gravity value for a short period of a walk. The blue curve represents the same
signal, but filtered for the same period of the walk. The green sticks represent
the detected user steps. The prediction stage of the position estimator filter consists,
as previously mentioned, on a movement model for the posi-
tion. We have chosen the following human walk model posXk = posXk−1 +
k
k−1
vel · cos(ψk),
posYk = posYk−1 +
k
k−1
vel · sin(ψk),
(13) 2) Step Length Estimator: For the step length we have de-
cided to use the empirical relationship between the maximum
and minimum of the acceleration for each step, as Equation (4)
shows. The parameter K has to be adjusted due to the low-pass
filter of the norm of the acceleration. 2) Step Length Estimator: For the step length we have de-
cided to use the empirical relationship between the maximum
and minimum of the acceleration for each step, as Equation (4)
shows. The parameter K has to be adjusted due to the low-pass
filter of the norm of the acceleration. (13) The combination of the step length and the heading gives
a new position every user step which is used to update the
prediction model. where posX and posY are the position estimates for the time
stamps k −1 and k, vel is the mean velocity of the user and
ψk is the estimated heading angle for the time stamp k. C. Static Periods Detector Δφgyr = φk −φk−1,
Δθgyr = θk −θk−1,
Δψgyr = ψk −ψk−1,
(12) (12) If the magnetic field is disturbed, no direct computation
of the yaw through Equation (7) can be used. However,
when the user is outdoors and the measured magnetic field
is disturbances free, this equation can be used to update the
yaw. being φ, θ, ψ the Euler angles roll, pitch, yaw. However, this
rotation contains errors due to the integration of the biases. The idea is to use the rotation of the magnetic vector
between the time stamp k −1 and k, which is error free,
to correct the difference of the states roll, pitch, yaw between
the time stamp k −1 and k, which contains errors due to the
integration of the biases. For applying this update, it is necessary to project the
magnetometer measurements from the body frame to the 246 Fig. 8. The red curve represents the norm of the acceleration minus the local
gravity value for a short period of a walk. The blue curve represents the same
signal, but filtered for the same period of the walk. The green sticks represent
the detected user steps. B. Update Stage Since the prediction human walk model needs the velocity
of the user, a step detector and a step length estimator have
been developed. The step length estimator provides as well
position updates every step. Two medium range IMUs, the MTx and MTw from Xsens,
have been chosen. These devices have a 3-axis accelerometer,
a 3-axis gyroscope and a 3-axis magnetometer inside. The
IMU is introduced in the pocket without a predefined ori-
entation before starting the walk. The navigation system has
been tested by 8 different users with different type of trousers
and no restriction about the tightness of the trousers has been
found. 1) Step Detector: The step detector is based on the de-
tection of consecutive minimum-maximum of the low-pass
filtered norm of the acceleration. We have decided to use the
norm of the acceleration instead of its vertical component,
which is usually used in the literature, because the norm of
the acceleration yields a detection independent of the IMU
orientation. In order to avoid false detections, some thresholds for the
amplitude and the time between detections have to be set. The
correct choice of these thresholds is one of the main difficulties
of this technique. Therefore, we have decided to low-pass filter
the acceleration. The filter smoothes the signal for its post-
processing as Figure 8 shows. In this figure, the green sticks
represent the detected user steps. In our work we define a user
step as the time between hits of the floor with every foot. A. Attitude Estimator Experiments Odometries computed with the different headings of Figure 10. 0
1
2
3
4
5
6
7
-2
-1
0
1
2
3
Distance (m)
Distance (m)
prediction
abs. yaw
mag
(b) Different update functions outdoors walk processed only with the prediction stage and
with the ZAA update. It can be clearly seen the improvement
in roll and pitch for the red curves, which have been processed
with the ZAA update. The blue curves represent the same walk
processed only with the prediction stage, without applying any
update. Figure 10 shows the heading angle for the same outdoor
walk processed with and without the magnetic updates. This
walk describes a round trip path with 16 turns of 180 degrees
that can be clearly distinguished in the figure. The final value
should be 2880 degrees, which is the sum of all turns. The blue
curve, which was processed only with the prediction stage,
reaches a final value of 2618 degrees. The green curve has
been processed only with the yaw update of Equation (7) and it
reaches a value of 2846 degrees. Finally, the red curve ends up
with 2890 degrees. This figure shows the benefit of applying
both magnetic updates. (b) Different update functions Fig. 11. Odometries computed with the different headings of Figure 10. shows have their origin in the attitude or heading estimation. This will be explained in the Subsection VI-A1. Once the different update functions have been separately
tested, the goal is to test the performance of the complete
pocket attitude estimator in comparison with a ground truth. We have chosen the DSP-1750 FOG from KVH. Before
considering this IMU as a ground truth, we have recorded
10 hours of data in the basement of our office building for
analyzing its Allan variance. The FOG IMU is two orders of
magnitude less noisy than our MEMS IMU and the biases
noise is as well more than two orders of magnitude smaller. Since it is not straight forward to analyze the difference
between the green (only applying the yaw update of Equa-
tion( 7)) and the red curve (applying both magnetic updates) of
Figure 10, we have decided to plot their odometries. Figure 11
shows the great impact of the heading for the odometries. The
first subplot shows the true trajectory, a round trip path with 6
turns of 180 degrees with a length of 3.5 meters. A. Attitude Estimator Experiments Firstly, we want to test the performance of the described
attitude estimator. We have prepared a set of indoors-outdoors
walks with different volunteers. We have chosen an outdoor walk to assess the performance
of the different update functions we have previously defined. Figure 9 shows the attitude angles roll and pitch for an 247 0
50
100
150
200
250
-50
0
50
Time (s)
Roll (degrees)
prediction
ZAA
0
50
100
150
200
250
-50
0
50
Time (s)
Pitch (degrees)
prediction
ZAA
Fig. 9. The blue curves represent the roll and pitch angles of an outdoors
walk processed with the attitude estimator without applying any update, only
with the prediction stage. The red curves represent roll and pitch angles of
the same walk process with the attitude estimator applying the ZAA update. 0
50
100
150
200
250
-3000
-2500
-2000
-1500
-1000
-500
0
500
Time (s)
Yaw (degrees)
prediction
abs. yaw
mag
Fig. 10. The blue curve represents the yaw angle for the same outdoors
walk as Figure 9 processed without applying any update, only with the
prediction stage. The green curve has been computed applying only the
absolute yaw update of Equation (7) and the red curve was computed applying
both magnetic updates. 0
50
100
150
200
250
-50
0
50
Time (s)
Roll (degrees)
prediction
ZAA 0
50
100
150
200
250
-3000
-2500
-2000
-1500
-1000
-500
0
500
Time (s)
Yaw (degrees)
prediction
abs. yaw
mag 0
50
100
150
200
250
-50
0
50
Time (s)
Pitch (degrees)
prediction
ZAA Fig. 10. The blue curve represents the yaw angle for the same outdoors
walk as Figure 9 processed without applying any update, only with the
prediction stage. The green curve has been computed applying only the
absolute yaw update of Equation (7) and the red curve was computed applying
both magnetic updates. Fig. 9. The blue curves represent the roll and pitch angles of an outdoors
walk processed with the attitude estimator without applying any update, only
with the prediction stage. The red curves represent roll and pitch angles of
the same walk process with the attitude estimator applying the ZAA update. Truetrajectory
(a) True trajectory
0
1
2
3
4
5
6
7
-2
-1
0
1
2
3
Distance (m)
Distance (m)
prediction
abs. yaw
mag
(b) Different update functions
Fig. 11. A. Attitude Estimator Experiments The
turquoise curve represents the roll angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-15
-10
-5
0
5
10
15
Time (s)
Roll (degrees)
FOG
MEMS Fig. 14. The magenta curve represents the pitch angle for an indoors
walk computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the pitch angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-600
-500
-400
-300
-200
-100
0
100
200
Time (s)
Yaw (degrees)
FOG
MEMS
Fig. 15. The magenta curve represents the yaw angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the yaw angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-600
-500
-400
-300
-200
-100
0
100
200
Time (s)
Yaw (degrees)
FOG
MEMS Fig. 13. The magenta curve represents the roll angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the roll angle for the same walk processed with
the pocket attitude estimator. walk the volunteer was asked to do 5 short stops, which can
be clearly seen in all angles as a straight line. The magenta
curves represent for every case the ground truth obtained with
the FOG IMU and the turquoise curves represent the same
walk processed by our complete attitude estimator. For the
processing of the FOG data, we have projected the raw turn
rates from the sensor frame to the body frame and then we
have applied a single integration. No correction of the biases
or updates have been applied. Fig. 15. The magenta curve represents the yaw angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the yaw angle for the same walk processed with
the pocket attitude estimator. consider this is a good starting point, the improvement of
the heading estimation is also under investigation because
of its importance later on for the odometries, as previously
explained. Figure 13 shows the roll angle for both IMUs and a
clear displacement of the turquoise curve with respect to the
magenta curve, which corresponds to the FOG IMU, can
be seen. A. Attitude Estimator Experiments This figure shows the solution we have found to attach the FOG to
our leg at the high of the pocket because it is too large to introduce it directly
in the pocket as we always do for the MEMS IMU. 0
50
100
150
200
250
-15
-10
-5
0
5
10
15
Time (s)
Roll (degrees)
FOG
MEMS
Fig. 13. The magenta curve represents the roll angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the roll angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-20
-10
0
10
20
30
40
50
Time (s)
Pitch (degrees)
FOG
MEMS
Fig. 14. The magenta curve represents the pitch angle for an indoors
walk computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the pitch angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-600
-500
-400
-300
-200
-100
0
100
200
Yaw (degrees)
FOG
MEMS Fig. 12. This figure shows the solution we have found to attach the FOG to
our leg at the high of the pocket because it is too large to introduce it directly
in the pocket as we always do for the MEMS IMU. 0
50
100
150
200
250
-20
-10
0
10
20
30
40
50
Time (s)
Pitch (degrees)
FOG
MEMS
Fig. 14. The magenta curve represents the pitch angle for an indoors
walk computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the pitch angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-20
-10
0
10
20
30
40
50
Time (s)
Pitch (degrees)
FOG
MEMS Fig. 12. This figure shows the solution we have found to attach the FOG to
our leg at the high of the pocket because it is too large to introduce it directly
in the pocket as we always do for the MEMS IMU. 0
50
100
150
200
250
-15
-10
-5
0
5
10
15
Time (s)
Roll (degrees)
FOG
MEMS
Fig. 13. The magenta curve represents the roll angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. A. Attitude Estimator Experiments The second
subplot shows the odometries computed with the different
headings of Figure 10. As previously explained, the blue curve
for the heading accumulates more than 200 degrees of drift in
a walk of approximately 200 seconds. The difference of the
green and red heading curves can be more easily evaluated
with their odometry curves. The red one keeps better the true
trajectory path. The FOG is too large to introduce it in the pocket, as
we always do with the MEMS IMU. Therefore, it has been
attached with 4 screws to a solid wood base. The piece of
wood is at the same time attached to the leg as Figure 12
shows. The optical IMU needs an external power supply. We
have introduced the battery with the rest of hardware necessary
for it to record the data in a metallic box. The MEMS IMU
for all the experiments is attached with tape to the flat face
of the FOG situated on the front. Due to the metallic box, the
screws and the rest of the required hardware, it is convenient
to recalibrate the MEMS IMU. With Figure 11 we pretend to show the great impact of
the heading in the odometry. This is not our final odometry
result because, for generating these curves, only the magnetic
updates were active. However, not all the errors the odometry In Figures 13, 14 and 15, the Euler angles roll, pitch and
yaw have been represented for an indoors walk. During the 248 0
50
100
150
200
250
-20
-10
0
10
20
30
40
50
Time (s)
Pitch (degrees)
FOG
MEMS
Fig. 14. The magenta curve represents the pitch angle for an indoors
walk computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the pitch angle for the same walk processed with
the pocket attitude estimator. 0
50
100
150
200
250
-600
-500
-400
-300
-200
-100
0
100
200
Time (s)
Yaw (degrees)
FOG
MEMS
Fig. 15. The magenta curve represents the yaw angle for an indoors walk
computed with a FOG IMU which we consider as a ground truth. The
turquoise curve represents the yaw angle for the same walk processed with
the pocket attitude estimator. Fig. 12. VII. CONCLUSIONS AND OUTLOOK Within this work we have created a standalone inertial
pocket navigation system. We have started by developing a
new attitude estimator. In contrast of the major part of the re-
lated work on PDR systems, apart of the heading, we estimate
as well the angles roll and pitch. We have obtained satisfactory
results for pocket orientations. Although it represents a good
starting point for the heading estimation, due to the great
influence of the yaw angle on the odometries, more techniques
for correcting this angle should be further investigated. Fig. 16. These curves represent a round trip walk with a 180 degrees turn
between the seconds 40−50. The magenta curves represent the ground truth,
because they have been taken from a FOG, while the cyan curves have been
taken with a MEMS IMU and processed with our attitude estimator. For the
first subplot the IMUs were held swinging, for the second one calling and for
the third one texting. g
A
B
C
D
E
Fig. 17. The red curve represents the odometry of a 200 seconds walk in
our office building. ”A” is the starting point and ”B” is the end of the walk. There is a displacement of 5 meters, because on the true trajectory this two
points coincide. We have tried our attitude estimator also for the rest of
handheld positions, i.e. texting, swinging and phoning, and the
results are optimistic. We are convinced this could be a solid
starting point for a complete navigation system embedded in
a smartphone. Fig. 17. The red curve represents the odometry of a 200 seconds walk in
our office building. ”A” is the starting point and ”B” is the end of the walk. There is a displacement of 5 meters, because on the true trajectory this two
points coincide. For the develope of our magnetic disturbances detector, we
have analyzed the magnetic field for different heights of the
sensor. We have found the results quite interesting and we
think the magnetic field should be studied as well in the
third dimension. The related work shows a magnetic signature
highly modulated indoors. This is optimal for slamming the
floor, and we think we could apply this as well at the height of
the pocket (and the rest of handheld positions). A. Attitude Estimator Experiments It is noticeable that, during the short stops where
the user was asked to remain standing, the turquoise curve
approximates to the magenta reference curve due to the ZAA
updates. It is under investigation how to improve the roll angle
results. The pitch angle represented in Figure 14 shows much
better results. On the other hand, Figure 15 shows how the
turquoise curve, which is computed with the pocket attitude
estimator, starts slowly drifting almost since the beginning,
reaching 18 degrees after 200 seconds. During the walk it can
be clearly seen 3 turns of 180 degrees, 2 single 90 degrees
turn at the beginning and at the end of the walk and 2 double
90 degrees turn in the second 60 and 160. Even though we 1) Attitude Estimator for Handheld Positions: The de-
scribed navigation system has been developed for pocket
located IMUs. However, we are also interested in the rest of
the handheld positions, i.e. swinging, calling and texting. It
has been decided to test the attitude estimator also with the
rest of handheld positions and these tests have been also done
with the FOG IMU as a reference. We have selected 3 different walks, which are represented
in the Figure 16 for swinging, calling and texting respectively. For these 3 outdoor walks the volunteers were asked to
describe a round trip path with a 180 degrees turn to go back
to the initial position. Figure 16 represents the yaw angles,
because the heading is a crucial variable for PDR systems. The magenta curves represent the ground truth extracted from 249 0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG
0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG
0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG
Fig. 16. These curves represent a round trip walk with a 180 degrees turn
between the seconds 40−50. The magenta curves represent the ground truth,
because they have been taken from a FOG, while the cyan curves have been
taken with a MEMS IMU and processed with our attitude estimator. For the
first subplot the IMUs were held swinging, for the second one calling and for
the third one texting. A
B
C
D
E
Fig. 17. VII. CONCLUSIONS AND OUTLOOK Moreover, it
would be really interesting to study the change on the magnetic
signature with the height for distinguishing the transition
between the handheld positions. the FOG IMU and the cyan curves represent the same walk
processed with our pocket attitude estimator. We consider these results satisfactory, also the roll and pitch
estimation. As an outlook, the developed attitude estimator
could be a solid starting point for the rest of handheld
positions. VIII. ACKNOWLEDGEMENTS The authors would like to thank Patrick Robertson for the
interesting attitude conversations and Francisco Javier Fuentes
S´anchez for his good ideas and support. For clarity, we have decided to show the odometry of the
walk described in the last subsection whose Euler angles roll,
pitch and yaw can be seen in Figures 13, 14 and 15 in
comparison with the ground truth. A. Attitude Estimator Experiments The red curve represents the odometry of a 200 seconds walk in
our office building. ”A” is the starting point and ”B” is the end of the walk. 0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG
0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG
0
20
40
60
80
-100
0
100
200
300
Time (s)
Yaw (degrees)
MEMS
FOG these two points are the same. The true trajectory describes the
”C” and the ”D” labels as the same point as well. Therefore,
on the left part of the odometry, for the second time the user
visits the corridor, there is a displacement of around 5 meters. Additionally, the ”E” point should be at the end of the corridor,
3 meters further. This introduces a displacement of around
3 meters on the right part of the odometry. This source of
errors can be divided in two and it is completely independent
of inaccurate heading estimation. The displacement could be
caused either by false step detection or by inaccurate step
length estimation. This result and the very similar odometries obtained for the
complete set of walks realized by 8 volunteers, invite us to
further investigate on new techniques for step detectors and
step length estimators. B. Pocket Navigation System Experiments With the odometries we have found a vast range of improve-
ments that should be further investigated in order to obtain a
better step detection and step length estimation. In order to assess the complete pocket navigation system,
the odometries of the already described set of walks realized
by 8 different volunteers have been processed. For the odom-
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and Navigation Symposium (PLANS), 2010 IEEE/ION. IEEE, 2010,
pp. 97–101. [21] D. Gusenbauer, C. Isert, and J. Krosche, “Self-contained indoor position-
ing on off-the-shelf mobile devices,” in Indoor Positioning and Indoor
Navigation (IPIN), 2010 International Conference on. IEEE, 2010, pp. 1–9. [22] M. Alzantot and M. Youssef, “Uptime: Ubiquitous pedestrian tracking
using mobile phones,” in Wireless Communications and Networking
Conference (WCNC), 2012 IEEE. IEEE, 2012, pp. 3204–3209. [23] D. Alvarez, R. C. Gonz´alez, A. L´opez, and J. C. Alvarez, “Comparison
of step length estimators from weareable accelerometer devices,” in
Engineering in Medicine and Biology Society, 2006. EMBS’06. 28th
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The Mythology of Proportionality in Judgments of the Court of Justice of the European Union on Internet and Fundamental Rights
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The mythology of proportionality in judgments of the Court of
Justice of the European Union on Internet and fundamental
rights Citation for published version:
Fontanelli, F 2016, 'The mythology of proportionality in judgments of the Court of Justice of the European
Union on Internet and fundamental rights', Oxford Journal of Legal Studies, vol. 36, no. 3, pp. 630-660. https://doi.org/10.1093/ojls/gqv037 Digital Object Identifier (DOI):
10.1093/ojls/gqv037 Link:
Link to publication record in Edinburgh Research Explorer Document Version:
Peer reviewed version Published In:
Oxford Journal of Legal Studies * filippo.fontanelli@ed.ac.uk, Lecturer in International Economic Law, University of Edinburgh. Sincere thanks to
the Journal’s editors and referees. All views are attributable to the author only. A preliminary study on the topic
was presented at the Bocconi University in Milan (October 2014).
1 Niilo Jääskinen, ‘Internet et la Cour de Justice’, in Antonio Tizzano et al. (eds), La Cour de justice de l’Union
européenne sous la présidence de Vassilios Skouris (2003-2015) (Bruylant 2015) 253-269. p
y
(
)
1 Niilo Jääskinen, ‘Internet et la Cour de Justice’, in Antonio Tizzano et al. (eds), La Cour de justice de
européenne sous la présidence de Vassilios Skouris (2003-2015) (Bruylant 2015) 253-269. Take down policy Take down policy
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investigate your claim. Download date: 24. Oct. 2024 The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. Abstract: Proportionality is the tool of choice for the EU Court of Justice’s review of measures affecting the
enjoyment of fundamental rights. The use of proportionality is normally beneficial, as it ensures that public
authorities pursue public policies without any avoidable waste of fundamental rights protection. In the field of
internet-based activities, however, certain recurrent elements make proportionality unfit for the purpose. This
article argues against the systematic recourse to the mythology of proportionality in the judgments of the Court
of Justice of the EU. Most instances of putative proportionality assessment are in fact window-dressing for
pragmatic or policy-based arguments. The claim relies on a critical reading of the recent case law of the Court in
internet-related disputes. Accordingly, it is preferable to abandon the proportionality test when certain factual
conditions – which are commonplace in the digital milieu – prevail. Keywords: proportionality, fundamental rights, internet, Court of Justice of the European Union, adjudication,
legal reasoning 1. Society evolves over time and law must apply to an ever-changing substratum. This has always been
the case and the application of EU law to internet-related matters is no exception.1 The practice of
legal interpretation and application in this field is complicated by the engagement of fundamental 1 1 rights (FRs), which also lend themselves to evolutive construction,2 because their formulation through
principles requires actualisation in particular cases.3 FR-adjudication concerning novel technologies occurs in an epistemic scene which changes
continuously. Namely, its coordinates inevitably shift along two different axes. On the one hand,
technological advancement causes social practices to reconfigure and take new shapes; on the other
hand, the flexible application of general principles to specific circumstances cannot be assessed
statically or a priori. The process of normative refinement required to regulate these activities can
take place at the legislative level and/or through legal interpretation and application, including
through the activity of judicial bodies. The nature of technological advancement makes it impossible
to rely on a backward-looking analysis of established general practice to build appropriate analogies
for the regulation of future circumstances. To be effective, refinement must instead take the form of
reformative law-making (or judicial standard-setting) rather than codification or consolidation of
practice. Whereas law cannot anticipate technological innovations, it should react to them as promptly as
possible. 2 Kanstantsin Dzehtsiarou, ‘European Consensus and the Evolutive Interpretation of the European Convention
on Human Rights’ (2011) 12 German LJ 1730, 1732; Christos L Rozakis, ‘The European Judge as Comparatist’
(2005) 80 Tul. L. Rev. 257, 260 (referring to the rudimentary nature of the provisions of the ECHR).
3 This simplification draws from the famous notion of principles in Ronald Dworkin’s Taking Rights Seriously
(HUP, 1978) 35, where he notes that rules determine the outcome of a dispute and, if they do not, they have
been disregarded. Instead, ‘[p]rinciples do not work that way; they incline a decision one way, though not
conclusively, and they survive intact when they do not prevail’. /
g
/
Limited ECR 2011, I-10269, Opinion of AG Cruz Villalón, para 31. Limited ECR 2011, I-10269, Opinion of AG Cruz Villalón, para 31. y,
y
y
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4 Joined Cases C-509/09 eDate Advertising GmbH and C-161/10 Olivier Martinez and Robert Martinez Cases C-509/09 eDate Advertising GmbH and C-161/10 Olivier Martinez and Robert Martinez v MGN
ECR 2011, I-10269, Opinion of AG Cruz Villalón, para 31. y
y
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ed Cases C-509/09 eDate Advertising GmbH and C-161/10 Olivier Martinez and Robert Martinez v MG
d ECR 2011 I 10269 O i i
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Vill ló
31 y
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4 Joined Cases C-509/09 eDate Advertising GmbH and C-161/10 Olivier Martin The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. As Advocate General Cruz Villalón noted, ‘there are currently many legal categories the
conception and scope of which require a reconsideration where they affect social and commercial
relationships occurring on the internet.’4 This article raises a warning against received thinking. Namely, it posits that the proportionality
test – as we know it – is an inadequate heuristic device to resolve legal disputes in which fundamental 2 2 rights are affected by internet activities.5 Consequently, judgments turning on a determination of
proportionality are ultimately ill-founded or seek artificial authority for conclusions based on policy
trade-offs.6 The language of proportionality has become a mythology,7 a shorthand for legitimacy. It
used to imply correctness through careful judicial evaluation, but has gradually turned into a self-
serving proclamation. Proportionality is a sign that feeds its own signified, it is a myth in which the
form draws its nourishment from an impoverished meaning.8 Constitutional (that is, FR-based) adjudication in the hands of the Court of Justice of the European
Union (the Court) is increasingly impracticable and the discipline of internet-based activities that have
FR-implications is better left to regulators or to different ways of judicial reasoning. This conclusion is
reminiscent of Balkin’s own regarding the realisation (regarding free speech) that case-law could
hardly moderate the digital brave new world. Technical and regulatory decisions are preferable to
constitutional elaboration through judicial precedents: Protecting free speech values in the digital age will be less and less a problem of constitutional
law – although these protections will remain quite important – and more and more a problem
of technology and administrative regulation.9 Protecting free speech values in the digital age will be less and less a problem of constitutional
law – although these protections will remain quite important – and more and more a problem
of technology and administrative regulation.9 of technology and administrative regulation.9 5 On the risks of basing moral and legal judgments on generic heuristic short-cuts, see Cass R. Sunstein, ‘Moral
heuristics’ (2005) 28(4) Behavioral and Brain Sciences 531. 6 Criticism of proportionality in FR-adjudication is commonplace. See Stavros Tsakyrakis, ‘Proportionality: An
assault on human rights?’ (2009) 7(3) I-Con 468; Matthias Klatt and Moritz Meister, ‘Proportionality—a benefit
to human rights? Remarks on the I·CON controversy’ (2012) 10(3) I-Con 687. For a response to this criticism, see
Kai Möller, ‘Proportionality: Challenging the critics’ (2012) 10(2) I-Con 709. p
p
y
7 Roland Barthes, Mythologies (Seuil 1957), last chapter: ‘Le mythe, aujourd’hui’. 5 On the risks of basing moral and legal judgments on generic heuristic short-cuts, see Cass R. Sunstein, ‘Moral
heuristics’ (2005) 28(4) Behavioral and Brain Sciences 531.
6 Criticism of proportionality in FR-adjudication is commonplace. See Stavros Tsakyrakis, ‘Proportionality: An
assault on human rights?’ (2009) 7(3) I-Con 468; Matthias Klatt and Moritz Meister, ‘Proportionality—a benefit
to human rights? Remarks on the I·CON controversy’ (2012) 10(3) I-Con 687. For a response to this criticism, see
Kai Möller, ‘Proportionality: Challenging the critics’ (2012) 10(2) I-Con 709. On the specific problem of
incommensurability contrast Paul-Erik N Veel, ‘Incommensurability, Proportionality, and Rational Legal Decision-
making’ (2010) 4(2) Law & Ethics of Human Rights 178 with Timothy AO Endicott, ‘Proportionality and
Incommensurability’ in Grant Huscroft, Bradley W Miller, Gregoire Webber (eds), Proportionality and the Rule
of Law (CUP 2014) 311. For a critique of the allocation of evidentiary onera within the test, see Julian Rivers,
‘The Presumption of Proportionality’ (2014) 77(3) MLR 409.
7 Roland Barthes, Mythologies (Seuil 1957), last chapter: ‘Le mythe, aujourd’hui’.
8 Ibid, (English translation, Vintage 2000) 118: ‘The meaning will be for the form like an instantaneous reserve
of history, a tamed richness, which it is possible to call and dismiss in a sort of rapid alternation’.
9 Jack M Balkin, ‘The Future of Free Expression in a Digital Age’ (2008) 36 Pepp. LR 427, 441. Balkin discusses the
continuing relevance of the Constitution’s First Amendment on free speech, arguing somewhat similarly to the
gist of this article that the technological revolution amounts to ‘a transition of enormous irony. At the very
moment that our economic and social lives are increasingly dominated by information technology and
information flows, the First Amendment seems increasingly irrelevant to the key free speech battles of the The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. On the specific problem of
incommensurability contrast Paul-Erik N Veel, ‘Incommensurability, Proportionality, and Rational Legal Decision-
making’ (2010) 4(2) Law & Ethics of Human Rights 178 with Timothy AO Endicott, ‘Proportionality and
Incommensurability’ in Grant Huscroft, Bradley W Miller, Gregoire Webber (eds), Proportionality and the Rule
of Law (CUP 2014) 311. For a critique of the allocation of evidentiary onera within the test, see Julian Rivers,
‘The Presumption of Proportionality’ (2014) 77(3) MLR 409. 8 Ibid, (English translation, Vintage 2000) 118: ‘The meaning will be for the form like an instantaneous reserve
of history, a tamed richness, which it is possible to call and dismiss in a sort of rapid alternation’. 9 Jack M Balkin, ‘The Future of Free Expression in a Digital Age’ (2008) 36 Pepp. LR 427, 441. Balkin discusses the
continuing relevance of the Constitution’s First Amendment on free speech, arguing somewhat similarly to the
gist of this article that the technological revolution amounts to ‘a transition of enormous irony. At the very
moment that our economic and social lives are increasingly dominated by information technology and
information flows, the First Amendment seems increasingly irrelevant to the key free speech battles of the 3 The assumption that the Court performs constitutional adjudication requires a clarification.10
Substantively, the Court reviews the compatibility of EU secondary legislation with the Treaties and
international law. Fundamental rights provisions are among the primary norms which secondary
legislation must respect to be valid. In the present analysis, the interpretation or validity of EU law
acts11 or national law implementing EU law turn on their compliance with fundamental rights: this kind
of judicial review is, in the substance, typically constitutional. Besides the substantive affinity, the
Court has also a formal mandate to engage in constitutional adjudication. Namely, it arbitrates the
vertical division of powers between Member States and the Union, as domestic constitutional courts
often do.12 In particular, the review of secondary legislation for compliance with Treaty law ultimately
implicates the compliance with the principle of conferral. Formally, therefore, respect of Treaty law is
a safeguard against undue encroachment of Member States’ competences. This article introduces a distinction between internet-native rules and other rules that might be
applicable in internet-related cases (section 2). future. Or, more precisely, the judge-made doctrines that I teach in my First Amendment classes seem
increasingly irrelevant’.
10 On the Court’s activity as constitutional tribunal, see Eleanor Sharpston and Geert De Baere, ‘The Court of
Justice as a Constitutional Adjudicator’ in Anthony Arnull et al. (eds), A Constitutional Order of States? (Hart
2011) 123; Bo Vesterdorf, ‘A constitutional court for the EU?’ (2006) 4(4) I-Con 607; Andreas M Donner, ‘The
Constitutional Powers of the Court of Justice of the European Communities’ (1974) 11 CMLR 127; Francis G
Jacobs, ‘Is the Court of Justice of the European Communities a Constitutional Court?’ in Deirdre Curtin et al.
(eds), Constitutional Adjudication in European Community and National Law: Essays for the Hon. Mr. Justice T.
F. O’Higgins (Butterworths 1992) 25.
11 Submitted to the Court under Articles 263 or 267 of the Treaty on the Functioning of the European Union
(TFEU).
12 Michel Rosenfeld, ‘Comparing constitutional review by the European Court of Justice and the US Supreme
Court’ (2006) 4(4) I-Con 618, 623. 12 Michel Rosenfeld, ‘Comparing constitutional review by the European Court of Justice and the US S
Court’ (2006) 4(4) I-Con 618, 623. gg
(
)
11 Submitted to the Court under Articles 263 or 267 of the Treaty on the Functioning of the Europea
(TFEU). the Court’s activity as constitutional tribunal, see Eleanor Sharpston and Geert De Baere, ‘The Court o
e as a Constitutional Adjudicator’ in Anthony Arnull et al. (eds), A Constitutional Order of States? (Ha future. Or, more precisely, the judge-made doctrines that I teach in my First Amendment classes seem
increasingly irrelevant’.
10 On the Court’s activity as constitutional tribunal, see Eleanor Sharpston and Geert De Baere, ‘The Court of
Justice as a Constitutional Adjudicator’ in Anthony Arnull et al (eds) A Constitutional Order of States? (Hart j
y
(
),
f
(
123; Bo Vesterdorf, ‘A constitutional court for the EU?’ (2006) 4(4) I-Con 607; Andreas M Donner, ‘Th e. Or, more precisely, the judge-made doctrines that I teach in my First Amendment classes see
asingly irrelevant’. The mythology of proportionality in the EU Court of Justice’s judgments on internet and
fundamental rights. The purpose of this distinction is to focus on the
constitutional component of the Court’s case-law, which is more clearly visible when it considers the
compatibility of internet-native rules with fundamental rights. Section 3 uses the Google Spain case
as an illustration of the shortcomings of proportionality in this field. The analysis develops in Section
4, which discusses other judgments to support the idea that the Court is less engaged in actual
proportionality than in a pragmatic moderation of conflicting interests. The core argument of this 4 work, synthesised in the conclusive section, is that the Court should let go of the proportionality
parlance and expose the policy-oriented thrust of its decisions on internet-related matters. This article does not purport to criticise proportionality in general, the use of balancing by the
regulator or the generic taking into account of conflicting interests in adjudication. The narrow claim
is that the three-step proportionality test is helpless to arbitrate FR-implications in internet disputes. Möller’s warning is wise: ‘[w]e should assess the value of proportionality not by asking whether there
are cases where it does not add much, but by asking whether there are cases where it does’.13
However, in the digital arena, proportionality is structurally unable to add anything, most of the times. Its damage to legal reasoning far outreaches the benefit, and should be noted. 13 Möller n 6, 727. Emphasis in the original. 14 For instance, see Directive 95/46; Directive 2000/31; Regulation 45/2001; Directive 2002/22; Directive
2002/58 (replacing Directive 97/66); Directive 2006/24. 14 For instance, see Directive 95/46; Directive 2000/31; Regulation 45/2001; Directive 2002/22; Directive
2002/58 (replacing Directive 97/66); Directive 2006/24.
15 Roger Brownsword, ‘The shaping of our on-line worlds: getting the regulatory environment right’ (2012) 20(4)
IJL&Inf Tech 249 263 265 ,
p
g
14 For instance, see Directive 95/46; Directive 2000/31; Regulation 45/2001; Directive 2002/22; D /
(
p
g
/
);
/
15 Roger Brownsword, ‘The shaping of our on-line worlds: getting the regulatory environment right’ (20
IJL&Inf. Tech. 249, 263-265. 14 For instance, see Directive 95/46; Directive 2000/31; Regulation 45/2001; Directive 200
2002/58 (replacing Directive 97/66); Directive 2006/24. g
,
IJL&Inf. Tech. 249, 263-265. 13 Möller n 6, 727. Emphasis in the original. 5 18 The FR-compliance of all acts of the EU, including normative sources, is mandated by Art. 51 of the EU Charter
of Fundamental Rights. Domestic measures implementing EU law are similarly subject to the Charter. See,
generally, Filippo Fontanelli, ‘National Measures and the Application of the EU Charter of Fundamental Rights–
Does curia.eu Know iura.eu?’ (2014) 14(2) HRLR 231-265. 16 Bert-Jaap Koops, ‘Should ICT regulation be technology-neutral?’ in Bert-Jaap Koops et al. (eds), Starting Points 2.
Regulatory connection of internet-native and non-internet norms 2. FR-adjudication inevitably entails the interpretation and application of principles. Regulation of
conduct relating to the use of internet, conversely, can be very thorough and comprise detailed rules. In fact, the EU has legislated copiously in the field.14 The process of refinement of the nexus between
EU law and internet activities has resulted in a flurry of regulation. This process has aimed to achieve,
maintain and renew ‘regulatory connection’,15 that is, the alignment between regulation and
regulated practices. New legal disciplines have emerged governing the impact that internet
technologies have on several human activities (production, distribution and consumption of
information, access to intellectual products, entertainment, marketing strategies, management of
personal data, use of intangible networks to support the activity of public entities). Resultantly, the
EU relies on a wide basis of internet-native regulation. Other EU rules (non-internet specific) antedate
internet or do not mention it specifically, but nevertheless apply to its use. When EU and domestic 5 5 judges apply rules that are not internet-specific to internet matters, regulatory connection must take
place in the courtroom, more or less ad hoc. The subject-matter of this article is the Court’s constitutional case-law in the digital milieu. The
cases are selected to gauge the process of evolutive refinement described above, whereby
adjudication secures alignment between the digital world and a subset of EU law, i.e., its fundamental
rights standards. All the disputes considered fall in a casuistic spectrum of EU applicable norms (the
application of EU law is a prerequisite for the CJEU to exercise jurisdiction). At one end, the Court
interprets or applies internet-native rules and the refinement relates to their compliance with
fundamental rights. At the other end, the Court must apply rules that are not internet-specific to an
internet-related situation; this might result from technology advancements that have not been
matched by regulatory action, or from the application of rules drafted deliberately to be ‘technology
neutral’,16 which require ‘purposive interpretation in the courts’.17 In the latter case, the regulatory connection hinges on the optimisation of existing rules to new
practices, and the constitutional duty of the Court to monitor compliance with fundamental rights is
mixed with its task to secure regulatory connection through the interpretation of non-internet rules. Quite simply, the updated application of the rule of conduct must also respect fundamental rights. 16 Bert-Jaap Koops, ‘Should ICT regulation be technology-neutral?’ in Bert-Jaap Koops et al. (eds), Starting Points
for ICT Regulation. Deconstructing Prevalent Policy One-Liners (TMC Asser 2006) 77. 19 Directive 2000/31/EC of the European Parliament and of the Council of 8 June 2000 on certain legal aspects
of information society services, in particular electronic commerce, in the Internal Market, Official Journal L 178,
17.7.2000, 1–16 ,
20 See Articles 12 to 14 of Directive 2000/31. For a commentary and a comparison with the corresponding US
regime, see Miquel Peguera, ‘The DMCA Safe Harbors and Their European Counterparts: A Comparative Analysis
of Some Common Problems’ (2009) 32 Col JL& Arts 481 2.
Regulatory connection of internet-native and non-internet norms This is a routine check that the Court must perform on all EU rules.18 Two examples illustrate this distinction, which is not clear-cut but is helpful to appreciate the
Court’s work. Two examples illustrate this distinction, which is not clear-cut but is helpful to appreciate the
Court’s work. Type 1: Consistent interpretation of internet-native rule to fundamental rights 16 Bert-Jaap Koops, ‘Should ICT regulation be technology-neutral?’ in Bert-Jaap Koops et al. (eds), Starting Points
for ICT Regulation. Deconstructing Prevalent Policy One-Liners (TMC Asser 2006) 77. 16 Bert-Jaap Koops, ‘Should ICT regulation be technology-neutral?’ in Bert-Jaap Koops et al. (eds), Starting Points
for ICT Regulation. Deconstructing Prevalent Policy One-Liners (TMC Asser 2006) 77. ,
18 The FR-compliance of all acts of the EU, including normative sources, is mandated by Art. 51 of the EU Charter
of Fundamental Rights. Domestic measures implementing EU law are similarly subject to the Charter. See,
generally, Filippo Fontanelli, ‘National Measures and the Application of the EU Charter of Fundamental Rights–
Does curia.eu Know iura.eu?’ (2014) 14(2) HRLR 231-265. 6 6 Art. 15 of Directive 2000/31 (on e-Commerce)19 provides that ‘Member States shall not impose a
general obligation on providers … to monitor the information which they transmit or store, nor a
general obligation actively to seek facts or circumstances indicating illegal activity.’ This article forms part of a set of provisions which grant a ‘safe harbour’ to selected categories of
internet service providers. These are exempted from liabilities by reason of their content-neutral
activity (caching, hosting, mere conduit).20 Article 15 clarifies that the safe harbour cannot be
frustrated by imposing filtering duties on these providers. The application of this internet-specific rule
might raise issues of compatibility with fundamental rights. For instance, certain stakeholders might
question the compatibility of this rule with their right to protection of property, insofar as this
provision spares internet service providers from a duty of monitoring and preventing a) the use of IP-
rights (see Promusicae,21 L’Oréal,22 Scarlet,23 SABAM,24 Bonnier25, PRCA,26 Papasavas27); b) access to
pictures taken and distributed illegally (see Max Mosley v Google, French28 and German29 orders); c)
the exchange of tickets for which re-sale is prohibited (see UK SC’s judgment Rugby Football Union v. Viagogo30). Type 1 cases involve balancing.31 ,
20 See Articles 12 to 14 of Directive 2000/31. 21 Case C-275/06 Productores de Música de España (Promusicae) [2008] ECR I-271. general monitoring, something which is prohibited by Article 15(1) of Directive 2000/31. In order to assess
whether that injunction is consistent with European Union law, account must also be taken of the requirements
that stem from the protection of the applicable fundamental rights’ (emphasis added). Council Regulation (EC) No 44/2001 of 22 December 2000 on jurisdiction and the recognition and enforce
judgments in civil and commercial matters, Official Journal L 012 , 16.01.2001. j
g
,
,
An extreme case is Case C-441/13 Hejduk (judgment of 22 January 2015). 2.
Regulatory connection of internet-native and non-internet norms For a commentary and a comparison with the corresponding US
regime, see Miquel Peguera, ‘The DMCA Safe Harbors and Their European Counterparts: A Comparative Analysis
of Some Common Problems’ (2009) 32 Col. JL& Arts 481. ,
20 See Articles 12 to 14 of Directive 2000/31. For a commentary and a comparison with the corresponding US
regime, see Miquel Peguera, ‘The DMCA Safe Harbors and Their European Counterparts: A Comparative Analysis
of Some Common Problems’ (2009) 32 Col. JL& Arts 481. 21 Case C-275/06 Productores de Música de España (Promusicae) [2008] ECR I-271. e C-324/09 L'Oréal and others [2011] ECR I-6011. 22 Case C-324/09 L'Oréal and others [2011] ECR I-6011. -70/10 Scarlet Extended [2011] ECR I-11959. 23 Case C-70/10 Scarlet Extended [2011] ECR I-11959. 24 Case C-360/10 SABAM (judgment of 16 February 2012). 24 Case C-360/10 SABAM (judgment of 16 February 2012). 25 Case C-461/10 Bonnier Audio and others (judgment of 19 April 2012). 26 26 Case C-360/13 Public Relations Consultants Association Ltd v The Newspaper Licensing Agency Ltd and Others
(judgment of 5 June 2014). 27 Case C-291/13 Sotiris Papasavvas v O Fileleftheros Dimosia Etaireia Ltd and Others (judgment of 11 September
2014). 28 TGI Paris, 17e ch., 6 November 2013, RG 11/07970, Max Mosley c. Google Inc et Google France, text available
at http://droitdu.net/2013/11/tgi-paris-17e-ch-6-novembre-2013-rg-1107970-max-mosley-c-google-france-et-
google-inc/. 29 Landgericht Hamburg, 24 January 2014, case 324 O 264/11, text available at http://tlmd.in/u/1456. 30 The Rugby Football Union v Consolidated Information Services Ltd [2012] UKSC 55 (21 November 2012),
available at http://www.bailii.org/uk/cases/UKSC/2012/55.html. 31 30 The Rugby Football Union v Consolidated Information Services Ltd [20
available at http://www.bailii.org/uk/cases/UKSC/2012/55.html. 31 For instance, see how the Court determined the legality of a court injunction requiring an internet provider to
monitor the users’ activity, in Scarlet n 23, 40-41: ‘that injunction would require the [provider] to carry out 7 34 For instance, consider the Court’s reflection on the application of Article 5(3) of Directive 89/104, which lists
the exclusive uses of a trademark by the holder, to the purchase from Google AdWords of words identical to a
trademark, in Cases C-236/08 to C-238/08 Google France and Google [2010] ECR I-2417, para 66: ‘an
interpretation according to which only the uses mentioned in that list are relevant would fail to have regard for
the fact that that list was drawn up before the full emergence of electronic commerce and the advertising
produced in that context. It is those electronic forms of commerce and advertising which can, by means of
computer technology, typically give rise to uses which differ from those listed’.
35 Thi
f h i
i
h
D
d M
i
4 ,
,
reme case is Case C-441/13 Hejduk (judgment of 22 January 2015). of judgments in civil and commercial matters, Official Journal L 012 , 16.01.2001.
33 An extreme case is Case C-441/13 Hejduk (judgment of 22 January 2015). of judgments in civil and commercial matters, Official Journal L 012 , 16.01.2001.
33 An extreme case is Case C-441/13 Hejduk (judgment of 22 January 2015) 36 For a distinction between subsumption and balancing, and an attempt to describe the latter process as a
neutral process (like the former), see Robert Alexy, ‘On balancing and subsumption. A structural comparison’
(2003) 16(4) Ratio Juris 433. (2003) 16(4) Ratio Juris 433.
37 Thomas M Scanlon, ‘Adjusting Rights and Balancing Values’ (2004) 74 Ford. LR 1477. Type 2: Update of non-internet specific rule Type 2: Update of non-internet specific rule Art. 5(3) of Regulation 44/200132 provides that ‘[a] person domiciled in a Member State may, in
another Member State, be sued: … in matters relating to tort, delict or quasi-delict, in the courts for
the place where the harmful event occurred or may occur.’ In this case, the interpretation of this general principle of territorial connection might prove critical
when the alleged wrongdoing and its effects (the ‘harmful event’) take place over the internet. For
web-based defamation, indeed, it is doubtful whether ‘the place’ where the event occur or might
occur is any State where the incriminated webpage is merely accessible. If so, the defendant could be
sued in any State of the world where an internet connection exists.33 From this example (and others34) it is clear that fundamental rights are somewhat ancillary to the
main legal question (i.e., whether a simple possibility to access the defamatory material online
qualifies as ‘harmful event,’ and whether the location of the internet user identifies where the
‘harmful event’ occurs for the purpose of establishing jurisdiction). In this case, fundamental rights
guarantees inform the finding of the Court only insofar as it must be ensured that the application of
Art. 5(3) of Regulation 44/2001 does not restrict disproportionately the plaintiff’s right to privacy and
the ensuing right to seek judicial protection for it.35 Type 2 cases involve primarily legal subsumption 8 (i.e., the determination of whether a norm applies to a set of facts), to which any FR-balancing – if any
– is incidental.36 9 (i.e., the determination of whether a norm applies to a set of facts), to which any FR-balancing – if any
– is incidental.36 – is incidental.36 This distinction is relevant because it allows discerning the parts of the Court’s reasoning which
use FR-argumentation as a decisive thrust towards the finding, as opposed to a simple standard of
legality applied by way of routine. To simplify, Type 1 cases have a clearer constitutional imprint, on
average, because the reasoning is free from false positives. The evolutive component in Type 2 cases,
instead, is often about updating pre-internet rules rather than about FR-based consistent
interpretation. In this sense, Type 1 cases are noise-free, because they are less concerned with
regulatory connection; the judicial reasoning on fundamental rights is at the forefront. Make no mistake: regulatory connection is critical and probes the ability of the Court to mould the
interpretation of EU law to modern needs. However, when Type 1 reasoning is deployed, the
interpretive exercise regards precisely (and exclusively) the fundamental rights standards. To simplify,
argumentation of the first category (dealing with internet-native rules) calls upon the Court to
interpret fundamental rights as living instruments, thus exalting the connection between regulation
and fundamental rights, not between technology and regulation. It forces the Court to devise the
particularisation of human rights principles for new factual predicaments and legal regulations, often
without precedents that can apply by analogy.37 Type 1 reasoning is therefore more directly
constitutional, in the limited sense explained. Its deployment is clearly visible in the application of
internet-native rules, but it can be traced also when Type 2 rules apply. Simply, there can be hybrid
cases where the updating of non-internet-specific rules occurs alongside, or even through, FR-based
arguments appealing to the balancing discretion of the Court. If the Type 1/2 distinction is relatively
clear, any case can be decoded without worrying too much about whether it is a pure example of 9 either category. Most cases are not pure specimens. Yet tracing the constitutional undercurrent of
each is easier by isolating it from the concurring elements of the Court’s reasoning. It allows to
distinguish amidst the Court’s reasoning ‘constitutional connection’ (alignment between rules and
fundamental rights) from ‘regulatory connection’. Two recent Type 2 cases (PRCA38 and Papasavvas39) illustrate how regulatory connection works
and why these cases are not ideal to observe the Court’s handling of fundamental rights. The disputes
revolved around the potential liabilities of internet service providers for infringement of intellectual
property rights and the possible application of a safe-harbour. the harmonisation of certain aspects of copyright and related rights in the information society. 41 Under Article 5(1) of Directive 2001/299/EC of the European Parliament and of the Council of 22 May n 27.
40 That is, in addition to that required to upload content on the website. 41 Under Article 5(1) of Directive 2001/299/EC of the European Parliament and of the Council of 22 May 2001 o
the harmonisation of certain aspects of copyright and related rights in the information society. n 27.
40 That is, in addition to that required to upload content on the website. (i.e., the determination of whether a norm applies to a set of facts), to which any FR-balancing – if any
– is incidental.36 They are exemplary of the potential for
litigation in circumstances of regulatory disconnection, actual or alleged. In each case, there was a far-
fetched claim. In PRCA, a licensing agency claimed that the appearance of a website’s content on a
computer screen was, in and of itself, an instance of reproduction requiring a specific clearance for
the use of any IPs contained on the website.40 The defendant objected that the appearance on the
screen of a website’s content was a ‘temporary act of reproduction’ incidental and essential to the
mere viewing of webpages, and that it was therefore exempted from the IP regime.41 Conversely, in
Papasavvas, a news company argued that the publication its newspaper online was merely hosting
and fell under the safe harbour of the e-commerce Directive (see above). Accordingly, the company
claimed immunity from an action for defamation regarding articles it published online. Obviously, the Court rejected the characterisations offered by the licensing agency in PRCA and
the newspaper company in Papasavvas. Even prima facie, on-screen projection of websites cannot
require a separate authorisation of IP use, and the for-profit operation of an online newspaper cannot
be considered a content-neutral conduit. However, attempts were made to stretch or limit the reach
of the safe harbours in the applicable Directives, in a bid to trick the Court into an adjustment of the 10 regulatory connection of norms that needed none. As preposterous as these attempts were, the Court
needed to take them seriously. It declared that IP liability exemptions ‘must allow and ensure the
development and operation of new technologies, and safeguard a fair balance between the rights and
interests of rights holders and of users of protected works who wish to avail themselves of those
technologies’.42 Whereas in the specific cases balancing was not required (the scope of the exemptions
was clear), the quote bears testimony to the occasional blending of regulatory connection and right
balancing in Type 2 reasoning.43 The suggestion, above, that precedents are of little help for FR-based adjudication in internet
matters requires a brief explanation. After all, there exists an established practice of human rights
adjudication, both at the Court and in other jurisdictions from which it can draw inspiration. 44 Variations of this metaphor are commonplace in the discussion of internet regulation. See for instance Martin
H Redish, ‘Of New Wine and Old Bottles: Personal Jurisdiction, the Internet, and the Nature of Constitutional
Evolution’ (1998) 38 Jurimetrics 575; Chris Reed, Internet law: text and materials (CUP 2004) 173 (“Old wine in
new bottles: traditional transactions in the Internet environment”); John P Barlow, ‘The Economy of Ideas’
(WIRED 2.03 1994) http://archive.wired.com/wired/archive/2.03/economy.ideas.html.
45 C-101/01 Lindqvist [2003] ECR I-12971. For a comment, see Ludovic Coudray, ‘Case C-101/01, Bodil Lindqvist’
(2004) 41(5) CMLR 1361 42 PRCA n 38, para 24. 45 C-101/01 Lindqvist [2003] ECR I-12971. For a comment, see Ludovic Coudray, ‘Case C-101/01, Bodil L
(2004) 41(5) CMLR 1361. 43 A less obvious case was the qualification of Google – in its capacity as AdWords, providing advertising
– as a neutral subject storing information under Article 14(1) of Directive 2000/31 or, instead, as a sub
IP-related liabilities. See Google France n 34, paras 106 ff. , p
43 A less obvious case was the qualification of Google – in its capacity as AdWords, providing advertising services
– as a neutral subject storing information under Article 14(1) of Directive 2000/31 or, instead, as a subject with
IP-related liabilities. See Google France n 34, paras 106 ff.
44 Variations of this metaphor are commonplace in the discussion of internet regulation. See for instance Martin
H Redish, ‘Of New Wine and Old Bottles: Personal Jurisdiction, the Internet, and the Nature of Constitutional
Evolution’ (1998) 38 Jurimetrics 575; Chris Reed, Internet law: text and materials (CUP 2004) 173 (“Old wine in
new bottles: traditional transactions in the Internet environment”); John P Barlow, ‘The Economy of Ideas’
(WIRED 2.03 1994) http://archive.wired.com/wired/archive/2.03/economy.ideas.html.
45 C-101/01 Lindqvist [2003] ECR I-12971. For a comment, see Ludovic Coudray, ‘Case C-101/01, Bodil Lindqvist’
(2004) 41(5) CMLR 1361 , p
43 A less obvious case was the qualification of Google – in its capacity as AdWords, providing advertising services
– as a neutral subject storing information under Article 14(1) of Directive 2000/31 or, instead, as a subject with 42 PRCA n 38, para 24.
43 A less obvious case was the qualification of Google
in its capacity as AdWords providing advertising – as a neutral subject storing information under Article 14(1) of D
IP-related liabilities. See Google France n 34, paras 106 ff. (i.e., the determination of whether a norm applies to a set of facts), to which any FR-balancing – if any
– is incidental.36 Yet,
internet-related activities fit hardly into the traditional models of fundamental right conflicts: we need
new bottles for the new wine.44 As a matter of legal technique, application of FR principles to digital
activities is less a question of subsumption of new facts under existing standards than it is a question
of setting new policies. The best way to illustrate the unsettling novelty of internet-based activity is
through selected cases. The discussion below remarks the aspects of these cases which are capable of
generalisation; likewise the commentaries thereon are arguably valid in general and not only ad casos. The precursor in this gallery is the Lindqvist case.45 A volunteer catechist uploaded the personal
information about some colleagues on a webpage, without their consent. From the factual
background of the main proceedings, one can appreciate Mrs Lindqvist’s good faith (she promptly 11 removed the information when things got serious). However, criminal prosecution was launched, and
Mrs Lindqvist had to endure it: this is a watershed case, symbolising internet’s loss of innocence – or
legal impunity. Among the relevant issues, the Court considered whether the mere fact that the
incriminated webpages were accessible anywhere in the world made Mrs Lindqvist liable for transfer
of the personal information to a third country, a practice restricted under Directive 95/46.46 The
reasoning of the Court is critical and exemplary: Given … the state of development of the internet at the time Directive 95/46 was drawn up …
one cannot presume that the Community legislature intended the expression transfer [of
data] to a third country’ to cover the loading, by an individual … of data onto an internet page,
even if those data are thereby made accessible to persons in third countries with the technical
means to access them. If Article 25 of Directive 95/46 were interpreted to mean that there is transfer [of data] to a
third country every time that personal data are loaded onto an internet page, that transfer
would necessarily be a transfer to all the third countries where there are the technical means
needed to access the internet. The special regime provided for by Chapter IV of the directive
would thus necessarily become a regime of general application, as regards operations on the
internet. 46 Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of
individuals with regard to the processing of personal data and on the free movement of such data, Official
Journal L 281 , 23/11/1995, 31-50, see in particular Article 25. individuals with regard to the processing of personal data and on the free movement of such data
Journal L 281 , 23/11/1995, 31-50, see in particular Article 25. 46 Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the prote Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the prote
individuals with regard to the processing of personal data and on the free movement of such data
Journal L 281 , 23/11/1995, 31-50, see in particular Article 25. 47 A history of the principle is provided in Eric Engle, ‘The General Principle of Proportionality and Aristotle’
(2013) in Liesbeth Huppens-Cluysenaer and Nuno MMS Coelho (eds), Aristotle and The Philosophy of Law:
Theory, Practice and Justice (Springer 2013) 265. y,
( p
g
)
48 Article 5 of the Treaty of the European Union; Protocol 12 on the application of the principles of subsidiarity
and proportionality. (i.e., the determination of whether a norm applies to a set of facts), to which any FR-balancing – if any
– is incidental.36 Thus, if the Commission found, pursuant to Article 25(4) of Directive 95/46, that even
one third country did not ensure adequate protection, the Member States would be obliged
to prevent any personal data being placed on the internet. On its face, this is a schoolbook example of a Type 2 question: update of a non-internet rule to a world-
cum-internet. However, it reveals the Court’s readiness to alter the balance of established principles,
if only slightly. The rationale of the applied rule is to prevent that personal data be diffused where
insufficient guarantees exist for their protection. If the rationale is valid, indeed, internet amplifies 46 Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of
individuals with regard to the processing of personal data and on the free movement of such data, Official
Journal L 281 , 23/11/1995, 31-50, see in particular Article 25. 12 this concern indefinitely; this much is unquestionable. The protection of the affected parties would
then require Member States ‘to prevent any personal data being placed on the internet.’ However,
the Court discarded this conclusion as absurd, implicitly relying on a reapportioning of the
responsibilities based on a new analysis of expected costs, that is, a covert utilitarian assessment. Since anybody uploading any personal data online would likely to incur liability, this liability is lifted,
even if it was effectively designed to protect a fundamental right. Invoking arguments relating to the original intention of the legislator (that could not foresee the
functioning of internet) and a sloppy reasoning ex absurdo (every uploader would be liable) the Court
produced a Type 1 determination in disguise, where strict proportionality determined the outcome. Specifically, it altered the established balance between the right to privacy and the right to impart
information, acknowledging that the current social and technological situation validates an
irreversible erosion of the former in favour of the latter. In a sense, this judgment has inadvertently
signalled the demotion of privacy protection to a policy objective (down from fundamental right). It
also raises the question whether proportionality is a valid tool to manage the impact of new
technologies on fundamental rights. 47 A history of the principle is provided in Eric Engle, ‘The General Principle of Proportionality and A (
)
pp
y
Theory, Practice and Justice (Springer 2013) 265. (2013) in Liesbeth Huppens-Cluysenaer and Nuno MMS Coelho (eds), Aristotle and The Philosophy
Theory, Practice and Justice (Springer 2013) 265. J 9 3.
51 Julian Rivers, ‘Proportionality and variable intensity of review’ (2006) 65(1) CLJ 174. 50 Alec Stone Sweet and Jud Mathews, ‘Proportionality balancing and global constitutionalism’ (2008) 47 Colum.
JTL 72; Mads Andenas and Stefan Zleptnig, ‘Proportionality and balancing in WTO law: a comparative
perspective’ (2006) 42 Tex. ILJ 371; T Alexander Aleinikoff, ‘Constitutional Law in the Age of Balancing’ (1987) 96
YLJ 943. Tor-Inge Harbo, ‘The Function of the Proportionality Principle in EU Law’ (2010) 16 ELJ158. 53 Yutaka Arai-Takahashi, The Margin of Appreciation Doctrine and the Principle of Proportionalit
Jurisprudence of the ECHR (Intersentia 2002). 49 On the ‘cut-and-paste’ approach of the Court of Justice, which uses judicial tests codified in its own pre 3.
Proportionality between fundamental rights in digital matters – unworkable formulae A constant of constitutional adjudication in Europe is the use of the proportionality test to balance
competing values.47 The European Union applies a homonymous principle (alongside subsidiarity) to
prevent its rules from encroaching on Member States’ competences,48 but it should be noted that
what is examined here is exclusively the use of proportionality as a judicial test, distilled from repeated 13 use.49 Other intimations of the principle and other instances of balancing lato sensu (for instance, the
balancing inherent in policy-making) are without the narrow scope of this critique. Proportionality, or the process that the test entails, is a defining element of constitutionalism
globally.50 The test of proportionality is normally used to assess the justification of restrictions to
fundamental rights caused by private or, more commonly, public measures.51 A certain proportion
must exist between the interference to a given right and the benefit that that interference brings to
another right or public interest. Proportionality has become a general principle of EU law52 and has
informed the case-law of the ECtHR,53 under the moniker of necessity. Roughly, the proportionality test used by the Court traces the one developed by the German
Federal Constitutional Court (the BvfG)54 and theorised as a prototype by Robert Alexy.55 It is a three-
step test informed by the principle of Pareto-optimisation56; each step ensures that the measure
under scrutiny is efficient, that is, there cannot be any unnecessary waste of rights’ protection. It is a
device of FR-maximation. The measure must be suitable to achieve the goal it is designed for (step 1)
and must be, among those equally suitable and reasonably available, the least encroaching on the
right restricted (step 2). The third step, usually called proportionality stricto sensu, requires weighing
the values at stake, when it is inevitable that some of them must suffer a restriction.57 Again, the
purpose is to preserve efficiency: a measure is disproportionately restrictive of a right if its see Jan Komarek, Reasoning with Previous Decisions: Beyond the Doctrine of Precedent (2013) 61 AJCL 149. 50 Alec Stone Sweet and Jud Mathews, ‘Proportionality balancing and global constitutionalism’ (2008) 47 Colum. JTL 72; Mads Andenas and Stefan Zleptnig, ‘Proportionality and balancing in WTO law: a comparative
perspective’ (2006) 42 Tex. ILJ 371; T Alexander Aleinikoff, ‘Constitutional Law in the Age of Balancing’ (1987) 96
YLJ 943. 56 Aurelien Portuese, ‘Principle of Proportionality as Principle of Economic Efficiency’ (2013) 19(5) ELJ 612. 55 Robert Alexy, ‘Constitutional rights, balancing, and rationality’ (2003) 16(2) Ratio Juris 131. p
pp
j
see Jan Komarek, ‘Reasoning with Previous Decisions: Beyond the Doctrine of Precedent’ (2013) 61 YLJ 943.
51 Julian Rivers, ‘Proportionality and variable intensity of review’ (2006) 65(1) CLJ 174. Robert Alexy, Constitutional rights, balancing, and rationality (2003) 16(2) Ratio Juris 131.
56 Aurelien Portuese, ‘Principle of Proportionality as Principle of Economic Efficiency’ (2013) 19(5) ELJ 6 p
pp
,
j
p
,
see Jan Komarek, ‘Reasoning with Previous Decisions: Beyond the Doctrine of Precedent’ (2013) 61 AJCL 149. 49 On the ‘cut-and-paste’ approach of the Court of Justice, which uses judicial tests codified in its own precedents,
see Jan Komarek, ‘Reasoning with Previous Decisions: Beyond the Doctrine of Precedent’ (2013) 61 AJCL 149.
50 Alec Stone Sweet and Jud Mathews ‘Proportionality balancing and global constitutionalism’ (2008) 47 Colum p
f
(
)
54 The seminal case is Lüth, BVerfGE 7, 198 (1958). 60 Case C-131/12 Google Spain and Google (judgment of 13 May 2014). 59 Ibid., from the Titanic judgment, BVerfGE vol. 86, 1 (1992). 58 Alexy, Constitutional rights n 55, 139. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae 14 contribution to the competing value is inferior to the restriction caused, in terms of intensity. The test,
at a closer look, invites to a comparison between states of the world (rather than values as such): one
in which the interference operates and one where it does not. This “Disproportionality Rule”58 is defined by the BvfG – convolutely but correctly – as follows: An interference with a constitutional right is disproportional if it is not justified by the fact that
the omission of this interference would give rise to an interference with another principle (or
with the same principle with respect to other persons or in other respects), provided that this
latter interference is at least as intensive as the first one.59 The proportionality test has been for decades the Court’s tool of choice to deliver judgments in hard
cases without forfeiting its legitimacy. Proportionality’s high level of proceduralisation and its
operation reminiscent of a mathematical formula facilitate the thankless task to second-guess
Member States’ preferences and review EU law’s compliance with FRs. This section discusses the use
of proportionality and FR-adjudication in selected judgments. The case of Google Spain,60 like Lindqvist, is a hybrid of Type 1 and 2. Unlike in Lindqvist, however,
the Type 1 component (the evolutionary interpretation of fundamental rights) is not disguised. The
facts warrant a synthetic account. Mr Costeja Gonzalez, a martyr of the digital age if ever there was
one, Googled his own name on one fateful day of 2009. The first results were links to the digitalised
copy of the 1998 edition of a local newspaper, reporting the notice of a public auction on real estate
properties, including his own, seised to recover social security debts. He requested Google to remove
these links from the results of a search under his name, invoking his right to privacy. More specifically, 15 he invoked a right to have certain past events not reported in widely and easily accessible documents
lacking an overriding public interest.61 First, we can observe the Type 2 component of the ruling. The Court considered a gateway
question, namely whether Google qualified as controller of personal data under Directive 95/46. 61 Two insightful commentaries are Eleni Frantziou, ‘Further Developments in the Right to be Forgotten: The
European Court of Justice's Judgment in Case C-131/12, Google Spain, SL, Google Inc v Agencia Espanola de
Proteccion de Datos’ (2014) 14 HRLR 761; John W. Kropf, ‘Google Spain SL v. Agencia Española de Protección de
Datos (AEPD). Case C-131/12’ (2014) 108(3) AJIL 502. 62 On the nature of indexing performed by search engines and the algorithms used, see Emily B Laidlaw, ‘Private
power, public interest: An examination of search engine accountability’ (2009) 17(1) IJL& Tech. 113, 128-133.
63 Google Spain n 60, Opinion of AG Jääskinen, para 29: ‘the potential scope of application of the Directive in the
modern world has become be [sic] surprisingly wide’. European Court of Justice's Judgment in Case C-131/12, Google Spain, SL, Google Inc v Agencia Espanola de
Proteccion de Datos’ (2014) 14 HRLR 761; John W. Kropf, ‘Google Spain SL v. Agencia Española de Protección de
Datos (AEPD). Case C-131/12’ (2014) 108(3) AJIL 502.
62 On the nature of indexing performed by search engines and the algorithms used, see Emily B Laidlaw, ‘Private
power, public interest: An examination of search engine accountability’ (2009) 17(1) IJL& Tech. 113, 128-133.
63 Google Spain n 60, Opinion of AG Jääskinen, para 29: ‘the potential scope of application of the Directive in the
modern world has become be [sic] surprisingly wide’.
64 Ibid, para 30.
65 Ibid, paras 89-90.
66 Contrast this characterisation with Google’s own statement that search results are a form of protected free
speech, made in US proceedings. Google’s position was frequently upheld, see for instance S. Louis Martin v.
Google Inc., case number CGC 14 539972, in the Superior Court of the State of California, County of San
Francisco, order of 13 November 2014. Google also commissioned a scientific paper that supports this notion,
see Eugene Volokh and Donald M Falk, First Amendment Protection for Search Engine Search Results, April 20,
2012 at http://www.volokh.com/wp-content/uploads/2012/05/SearchEngineFirstAmendment.pdf.
67 Google Spain n 60, paras 32-34. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae Google claimed that it did not, because it only performed a content-blind indexing of all words
uploaded online, to populate search results for its users.62 The Advocate General, after a short
reflection on how internet has taken EU law by surprise,63 advocated a ‘rule of reason’ akin to the
Lindqvist rationale.64 Drawing support from the Article 29 Working Party reports, he concluded that
holding Google responsible for the managing of personal data on the webpages listed would lead to
absurd results.65 The Advocate General’s approach is indeed reminiscent of Lindqvist: subsumption
(the application of the Directive to Google’s acts) is determined through a reasoning ex absurdo
regarding practical consequences. Regulatory connection is managed through proportionality: Type 1
and Type 2 are entangled. The Court instead rejected Google’s argument that its listing entailed an
automated processing without meaningful editorial intervention.66 Google, by indexing online data,
performs a deliberate commercial activity that can attract liability. Accordingly, Google is a controller
under the Directive (a Type 2 finding).67 under the Directive (a Type 2 finding).67 62 On the nature of indexing performed by search engines and the algorithms used, see Emily B Laidlaw, ‘Private
power, public interest: An examination of search engine accountability’ (2009) 17(1) IJL& Tech. 113, 128-133. 63 Google Spain n 60, Opinion of AG Jääskinen, para 29: ‘the potential scope of application of the Directive in the
modern world has become be [sic] surprisingly wide’. 66 Contrast this characterisation with Google’s own statement that search results are a form of protected free
speech, made in US proceedings. Google’s position was frequently upheld, see for instance S. Louis Martin v. Google Inc., case number CGC 14 539972, in the Superior Court of the State of California, County of San
Francisco, order of 13 November 2014. Google also commissioned a scientific paper that supports this notion,
see Eugene Volokh and Donald M Falk, First Amendment Protection for Search Engine Search Results, April 20,
2012 at http://www.volokh.com/wp-content/uploads/2012/05/SearchEngineFirstAmendment.pdf. 67 Google Spain n 60 paras 32-34 16 The openly constitutional reasoning, however, is deployed in the Type 1 part of the judgment. The
Court laid the groundwork for its determination noting that the outcome, seemingly framed as a
question of principle, depended on the context. Quite simply, information loaded online is too readily
available, to anyone. 68 Ibid, para 80: ‘It must be pointed out at the outset that … processing of personal data, such as that at issue
in the main proceedings, carried out by the operator of a search engine is liable to affect significantly the
fundamental rights to privacy and to the protection of personal data when the search by means of that engine
is carried out on the basis of an individual’s name, since that processing enables any internet user to obtain
through the list of results a structured overview of the information relating to that individual that can be found
on the internet — information which potentially concerns a vast number of aspects of his private life and which,
without the search engine, could not have been interconnected or could have been only with great difficulty —
and thereby to establish a more or less detailed profile of him. Furthermore, the effect of the interference with
those rights of the data subject is heightened on account of the important role played by the internet and search
engines in modern society, which render the information contained in such a list of results ubiquitous.’
69 Ibid, para 99. Ibid, para 97.
71 On the problem of commensurability between values and between interferences to values, see Niels Petersen,
‘How to Compare the Length of Lines to the Weight of Stones: Balancing and the Resolution of Value Conflicts
in Constitutional Law’ (2013) 14 GLJ 1387. For a discussion of how to represent commensurate values without
using numerical indications, see Giovanni Sartor, ‘Logic of Proportionality: Reasoning with Non-Numerical
Magnitudes’ (2013) 14 GLJ 1419, 1429 ff. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae Availability of truthful information published lawfully has become a problem –
not a matter of concern in pre-internet times.68 Now, instead, regulation is required to manage the
negative externalities of this information overload. This is partly due to how good online search
engines are: information is only as public as search engines makes it accessible. The Court concluded that the search provider is a controller which processes personal data. Therefore, if Google refuses to remove certain links from the results of a name-based search upon
request, State regulatory agencies can review the application and order the removal.69 This order can
be granted when the results of a search entail an excessive interference in the data subject’s private
life, without a concurring (and overriding) justification. Because time soothes out some of the available
justifications based on public interest, this finding was saluted as establishing a ‘right to be forgotten.’
The resulting instruction of the Court, which read into the applicable rules of the Directive a specific
duty (for the controller) and a right (for the data subject), stems from an overt use of proportionality. The passage where the reasoning of the CJEU reveals the use of proportionality stricto sensu calls
for closer analysis: The passage where the reasoning of the CJEU reveals the use of proportionality stricto sensu calls
for closer analysis: As the data subject may, in the light of his fundamental rights under Articles 7 and 8 of the
h
h
h
f
l
b
d
l bl
h
l As the data subject may, in the light of his fundamental rights under Articles 7 and 8 of the
Charter, request that the information in question no longer be made available to the general As the data subject may, in the light of his fundamental rights under Articles 7 and 8 of the As the data subject may, in the light of his fundamental rights under Articles 7 and 8 of the Charter, request that the information in question no longer be made available to the general 17 public by its inclusion in such a list of results, it should be held … that those rights override, as
a rule, not only the economic interest of the operator of the search engine but also the interest
of the general public in finding that information upon a search relating to the data subject’s
name. ,
p
y
p
g (
)
,
73 That is, we could try to analyse its collective ‘realisation-impact’ across the relevant values. Sartor n 71, 1436:
‘[t]he realisation-impact of an action α on a value v is the difference between the realisation-quantities of v
resulting from and ∅ [the status quo]’. g
(
)
,
72 Aharon Barak, ‘Proportionality and Principled Balancing’ (2010) 4 L&Ethics HR 1, 8. itudes (2013) 14 GLJ 1419, 1429 ff.
aron Barak, ‘Proportionality and Principled Balancing’ (2010) 4 L&Ethics HR 1, 8. 70 Ibid, para 97. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae However, that would not be the case if it appeared, for particular reasons, such as the
role played by the data subject in public life, that the interference with his fundamental rights
is justified by the preponderant interest of the general public in having, on account of inclusion
in the list of results, access to the information in question.70 This excerpt contrasts at least four discrete principles/values. First, the data subject’s ‘right to oblivion’
(in turn an elaboration of her rights to private life and data protection under Articles 7 and 8 of the
Charter, read through the ‘compelling reasons’ under Article 14(a) of the Directive). Second, the
operator’s economic interest, protected by Articles 15-17 of the Charter. Third, the public’s right to
impart and obtain information, protected by Article 11 of the Charter. Fourth, unspecified ‘particular
reasons’ that could tip the balance in favour of the general public’s interest at the expense of the data
subject’s own. Balancing four rights is a devilish task even if we assume, for the sake of ease, that a given measure
x can only either respect or breach each of them (that is, we disregard the degree of contribution to
the achievement of each right and the intensity of the breach thereof71). A mere head-count does not
work: the Court itself noted that the data subject’s right prevails over two competing interests (of the
public and of the economic operator). Adding the analysis of the intensity of the measure’s marginal
impact on the enjoyment of each right,72 it would perhaps be possible to determine it statically.73 For 18 instance, it could be agreed that the solution envisaged by the Court protected the rights of Mr Costeja
Gonzalez and of those like him from a substantial harm, restricting slightly the interests of the public
to know about their past, as well as the newspaper’s right to inform the public about it through online
diffusion. It also imposed a significantly burdensome restriction on the right to exercise a business
onto Google and other search engines. The Court, however, did not explain how these magnitudes
relate to, possibly off-set, each other. It is not clear whether the Court even attempted to factor into the equation the degree of
restriction of all values involved. whether we add proportionally more length to the line than we shed weight of the stone.’ g
p
, p
p
75 Sartor n 71, 1391: ‘[W]e do not compare the weight of the stone to the length of the line. Instead, we g
p
, p
p
tor n 71, 1391: ‘[W]e do not compare the weight of the stone to the length of the line. Instead, we analyz
her we add proportionally more length to the line than we shed weight of the stone.’ Google Spain n 60, para 99. Emphasis added. tor n 71, 1391: [W]e do not compare the weight of the stone to the length of the line. Instead, we analyz
her we add proportionally more length to the line than we shed weight of the stone.’ 74 Google Spain n 60, para 99. Emphasis added.
75
‘[
] d
h
h
f h
h l
h f h l
d
l 3.
Proportionality between fundamental rights in digital matters – unworkable formulae In fact, certain wording suggests instead that privacy prevails by
default over freedom of information and of conducting business: [the rights under] Articles 7 and 8 of the Charter … override, as a rule, not only the economic
interest of the operator of the search engine but also the interest of the general public in
having access to that information upon a search relating to the data subject’s name.74 It cannot be conclusively excluded that the Court performed an accurate proportionality test but, since
this does not emerge from the ruling, we have to re-create it and compare the results of the actual
decision with the hypothetical exercise. It is doubtful that the “Disproportionality Rule” could clarify
the calculus. It cannot be conclusively excluded that the Court performed an accurate proportionality test but, since
this does not emerge from the ruling, we have to re-create it and compare the results of the actual
decision with the hypothetical exercise. It is doubtful that the “Disproportionality Rule” could clarify
the calculus. Under this rule, the solution envisaged by the Court is proportionate insofar as its absence would
determine a graver breach of rights than the restriction it causes (a negative aggregate utility balance
with respect to r1, r2 … to rn).75 In other words, we should ascertain whether establishing Google’s
obligation to enforce the ‘right to oblivion’ is less restrictive of the rights under Articles 11, 15, 16 and
17 of the Charter than rejecting it would be of the rights under Articles 7 and 8. Each variable in the
calculation is adjusted for the intensity of the restriction and, presumably, for the number of people 19 actually or potentially affected. In short, although certainly it is possible to explain the outcome of the
Court along these lines, the outcome is not falsifiable because too many variables hinge on
unprincipled approximation. An outcome opposite to the one indicated by the Court, indeed, would
be just as plausible, owing to the magnitude of the burden imposed on Google and the number of
people whose access to the relevant information is restricted. Unsurprisingly, the reasoning of the
Court is very cavalier in treating the steps of the proportionality analysis, and in specifying the relative
strength of its variables.76 The proportionality test, in this field, is not a heuristic device to reach the right decision. g
g
, q
y
,
g
77 Jürgen Habermas, Between Facts and Norms (MIT 1996), 259 ff. g
,
(
78 http://en.wikipedia.org/wiki/Streisand_effect. g
g
, q
y
,
g
77 Jürgen Habermas, Between Facts and Norms (MIT 1996), 259 ff. 76 Frantziou n 61, 8: ‘while Articles 7 and 8 of the Charter suggest the creation of some obligations for EU
institutions and Member States, they do not specify what the role of private actors such as Google should be in
the enforcement of the relevant standards, or what limitations to these rights are acceptable and how they
ought to be balanced against other, equally fundamental, rights’. g
78 http://en.wikipedia.org/wiki/Streisand_effect. the enforcement of the relevant standards, or what limitations to these rights are acceptable and h
ought to be balanced against other, equally fundamental, rights’. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae Indeed,
the outcome of this contrived proportionality calculus is not falsifiable, but only opinable or
contestable. Instead of selecting the outcome of the decision, the balancing provides a malleable
template that shapes only its supporting reasoning. In the particular circumstances of this dispute,
Habermas’s critique to the notion of proportionality rings true: ‘[in the proportionality test, v]alues
must be brought into a transitive order with other values from case to case. Because there are no
rational standards for this, weighing takes place either arbitrarily or unreflectively, according to
customary standards and hierarchies’.77 The factual matrix of the Google case is, ab initio, hard to
frame as a stand-off between established fundamental rights. What bothered Mr Costeja, in whose honour the Streisand-effect78 should be re-named, was not
the existence of that piece of news in the public domain, buried as it was at p. 23 of an old magazine. Of course, the real problem was his unflattering ‘Google-identity’ or ‘Google-footprint’; consequently,
he requested that Google obey, to an extent, to his instructions on which aspects of his web-relevant
persona should pop out first through a name-search. The ‘right to be forgotten’ tag is misleading, and
so is the reference to the removal of the link from the results: what really mattered is that the 20 bothering results came on top. Had the infamous links been listed at page 23 of the Google research
results, Mr Costeja would have just not cared, exactly as he presumably did not care in 2009 about
the 1998 print publication of the very same tainting news. There is some truth in connecting one’s
rights to an apparently circumstantial fact like Google-ranking: after all, ‘to be seen is … to be highly
ranked’,79 and this author (and this Journal) would plausibly resent discovering that Google Scholar
does not host a link to the electronic version of this article, or that it is shows it at page 23 of an author-
based search.80 The idea that Google can act as arbitrary gatekeeper of all information available online,
including personal data, justifies the attempts to brace its power as a matter of policy.81 Perhaps Google v Spain announces a nascent fundamental right to the fairness, accuracy or
representativeness of our digital persona. 80 Litigation based on low search ranking was brought, without success, in the USA. See Langdon v. Google, Inc.,
474 F. Supp. 2d 622 (D. Del. 2007); Kinder-start.com, LLC v. Google, Inc., No. C 06-2057 JF (RS), 2007 WL 831806
(N.D. Cal. Mar. 16, 2007); Search King, Inc. v. Google Tech., Inc., No. CIV-02-1457-M, 2003 WE 21464568 (W.D.
Okla. May 27, 2003). y
)
81 Andrew L Shapiro, The control revolution: How the Internet is putting individuals in charge and changing the
world we know (PublicAffairs 1999) 225: ‘In a democratic society, those who control access to information have
a responsibility to support the public interest’. 83 This is one step further from the AG’s remark that ‘the universal scope of the information contributes to the
harm being potentially more acute than that suffered, for example, by means of a conventional medium’ (see
eDate n 4, para 48). 79 Laidlaw n 62, 125. p
y
pp
p
82 Laidlaw n 62, 137, referring to the notion that search engines attracts criticism ‘for being good at what they
do’. Laidlaw n 62, 125.
80 Litigation based on low search ranking was brought, without success, in the USA. See Langdon v. Google, Inc.,
474 F. Supp. 2d 622 (D. Del. 2007); Kinder-start.com, LLC v. Google, Inc., No. C 06-2057 JF (RS), 2007 WL 831806
(N.D. Cal. Mar. 16, 2007); Search King, Inc. v. Google Tech., Inc., No. CIV-02-1457-M, 2003 WE 21464568 (W.D.
Okla. May 27, 2003).
81 Andrew L Shapiro, The control revolution: How the Internet is putting individuals in charge and changing the
world we know (PublicAffairs 1999) 225: ‘In a democratic society, those who control access to information have
a responsibility to support the public interest’.
82 Laidlaw n 62, 137, referring to the notion that search engines attracts criticism ‘for being good at what they
do’.
83 This is one step further from the AG’s remark that ‘the universal scope of the information contributes to the
harm being potentially more acute than that suffered, for example, by means of a conventional medium’ (see
eDate n 4, para 48). 3.
Proportionality between fundamental rights in digital matters – unworkable formulae If so, the judgment should explain – through proportionality
– why this right trumps Google’s interest to carry out its distinctive line of business, through a lawful
collection of lawfully published content. Clearly, Google’s business performance has unintended
consequences on somebody’s life, which are perceived as unfair even if they derive from lawful facts
and acts. Google’s mind-blowingly efficient archiving and sorting activity are treated by the Court as
the kind of dangerous conduct that, although legal per se, imposes a surplus of responsibility on the
subject.82 In turn, online personality is deemed to be so vulnerable that it deserves protection also
against certain lawful acts.83 81 Andrew L Shapiro, The control revolution: How the Internet is putting individuals in charge and changing the
world we know (PublicAffairs 1999) 225: ‘In a democratic society, those who control access to information have
a responsibility to support the public interest’. p
y
pp
p
82 Laidlaw n 62, 137, referring to the notion that search engines attracts criticism ‘for being good at what they
do’. do . 83 This is one step further from the AG’s remark that ‘the universal scope of the information contributes to the
harm being potentially more acute than that suffered, for example, by means of a conventional medium’ (see
eDate n 4, para 48). 83 This is one step further from the AG’s remark that ‘the universal scope of the information contributes to the
harm being potentially more acute than that suffered, for example, by means of a conventional medium’ (see
eDate n 4, para 48). 21 In the folds of the Court’s reasoning lays the idea that, in a context of obsolescence of public
authorities, Google must operate as a quasi-public authority.84 Therefore, it has non-reciprocal and
non-contractual obligations towards its stakeholders (both the users and the persons affected by its
activity). Alternatively, Google’s duties can be framed as a regulatory brace imposed on a quasi-
monopolistic actor with predominant power over information and public opinion.85 In both
constructions, the critical element is Google’s de facto control over a public good.86 This notion,
however reasonable and legitimate, is not the result of balancing, but a regulatory choice. (
)
86 Laidlaw n 62, 126: ‘The act of framing a user’s information experience makes search engines indispensable to
access to information on the Internet thus elevating their product to public good status’, pointing to the
discussion in Lucas D Introna and Helen Nissenbaum, ‘Shaping the Web: Why the politics of search engines
matters’ (2000) 16(3) Inf. Soc. 169. (
)
( )
87 R (Quila and another) v Sec of State for the Home Dept [2011] UKSC 45, para 91 of the dissent. 85 Wolfgang Schulz, Thorsten Held and Arne Laudien, ‘Search Engines as Gatekeepers of Public Communication:
Analysis of the German Framework Applicable to Internet Search Engines Including Media Law and Anti-Trust
Law’ (2005) 6 GLJ 1418. 84 Johan Eriksson and Giampiero Giacomello, ‘Who Controls the Internet? Beyond the Obstinacy or Obso p
of the State’ (2009) 11 International Studies Review 205. p
,
y
y
of the State’ (2009) 11 International Studies Review 205. 84 Johan Eriksson and Giampiero Giacomello, ‘Who Controls the Internet? Beyond the Obstinacy or Obsolescence
of the State’ (2009) 11 International Studies Review 205 84 Johan Eriksson and Giampiero Giacomello, ‘Who Controls the Internet? Beyond the Obstinacy or Obsolescence
of the State’ (2009) 11 International Studies Review 205.
85 Wolfgang Schulz, Thorsten Held and Arne Laudien, ‘Search Engines as Gatekeepers of Public Communication:
Analysis of the German Framework Applicable to Internet Search Engines Including Media Law and Anti-Trust
Law’ (2005) 6 GLJ 1418.
86 Laidlaw n 62, 126: ‘The act of framing a user’s information experience makes search engines indispensable to
access to information on the Internet thus elevating their product to public good status’, pointing to the
discussion in Lucas D Introna and Helen Nissenbaum, ‘Shaping the Web: Why the politics of search engines
matters’ (2000) 16(3) Inf. Soc. 169.
87 R (Quila and another) v Sec of State for the Home Dept [2011] UKSC 45, para 91 of the dissent. 4.
Low-intensity actions with momentous reach. Protecting collateral victims of interne 4. Google, and in the process established Google’s unprecedented duty to arbiter individual applications
of data removal. Google, and in the process established Google’s unprecedented duty to arbiter individual applications
of data removal. In other words, there is no manifest error in the static assessment of the values at stake and of
the relative restrictions entailed by the status quo. What is unsatisfactory is the lack of reasoning
regarding the proportionality of the indicated solution (Google’s duty to remove the link upon
meritorious requests). Proportionality stricto sensu only operates – as seen above – through the
comparison between the aggregate right-implementation of the status quo and an alternative
measure. The Court found a putative disproportion in the status quo and jumped to the solution. The
better option would have been to show that the solution causes a more favourable scenario of right-
implementation. The Court failed to show the critical element of proportionality which is the putative
advantage of changing (or retaining) the status quo. Nothing suggests that it would have been easy or
even possible, but it was unavoidable if proportionality is taken seriously. The result of attributing
duties to Google is perhaps reasonable, but given the circumstances it would be preferable that the
regulator had done that88 or that the Court had appealed to the reasonableness of the decision, rather
than to its putative proportionality. 88 Shapiro n 81, 225. 3.
Proportionality between fundamental rights in digital matters – unworkable formulae Consider
Lord Brown’s dissent, concerning the UK Supreme Court’s use of proportionality to review the
requirement that visa be granted only to foreign couples when both partners are at least 21 years old
(to prevent forced marriages at an earlier age): What value … is to be attached to preventing a single forced marriage? What cost should each
disappointed couple be regarded as paying? Really these questions are questions of policy and
should be for government rather than us.87 The argument here is not that Google Spain was wrong on the merits. The crucial point is how much
novelty the Court has nonchalantly reined through legislator-like discretion, simply dangling
proportionality before the beholders. The Court combined the right to private life and the right to a
lawful use of personal data (neither of which was breached by the publication) to enforce an
unprecedented right, in unprecedented circumstances and implicating an unprecedented role for 22 88 Shapiro n 81, 225. 90 Contrast with Case C-323/09 Interflora v Mark & Spencer [2011] ECR I-8625, which also concerned the use of
a trademark by a competitor. However, the dispute was essentially between two competitors and did not involve
the interests of a wider audience. Hence, the case is only accidentally, not essentially, internet-related. 89 Joined Cases C-92/09 and C-93/09 Volker und Markus Schecke and Eifert [2010] ECR I-11063. f
[
]
90 Contrast with Case C-323/09 Interflora v Mark & Spencer [2011] ECR I-8625, which also concerne a trademark by a competitor. However, the dispute was essentially between two competitors and did
the interests of a wider audience. Hence, the case is only accidentally, not essentially, internet-rela 90 Contrast with Case C-323/09 Interflora v Mark & Spencer [2011] ECR I-8625, which also concerned the use of
a trademark by a competitor. However, the dispute was essentially between two competitors and did not involve
th i t
t
f
id
di
H
th
i
l
id
t ll
t
ti ll
i t
t
l t d 89 Joined Cases C-92/09 and C-93/09 Volker und Markus Schecke and Eifert [2010] ECR I-11063. 93 L’Oréal n 22, Opinion of AG Jääskinen, para 49. 94 Ibid, para 158. Emphasis added. 91 Specifically, Article 5(1)(a) of Directive 89/104 and Article 9(1)(a) of Regulation No 40/94. 92 As it seemingly did in Google France n 34, holding that Google’s use of certain brands to populate sponsored
ads qualified as ‘use’ under EU trademark law, and benefitted in principle from the safe harbour of Article 14 of
the e-commerce Directive.
93 measures Proportionality does not compare values, but marginal variations of value-realisation entailed by
alternative states of the world (induced or mandated by identified normative measures). However, as
argued in the previous section, internet-based activities do not lend themselves to the intuitive
generalisations that allow reviewers to attach quantitative judgments to the realisation of rights. Deprived of workable comparisons, proportionality misses its critical hinge. 23 This difficulty was prefigured in Lindqvist (where the low-intensity threat to privacy was multiplied
by the accessibility of online content, causing a formal disproportion that required a policy-oriented
ruling). A less risky assessment was necessary in Schecke.89 The applicants claimed that the publication
online of their companies’ names among the recipients of EU funding (within the Common Agricultural
Policy) was unnecessarily restrictive of the company’s privacy. The values involved were the public’s
interest in knowing the exact allocation of EU funds, the Union’s duty to transparency and the
recipients’ interest to privacy. In that occasion, however, the problem was solved through simple
Pareto-optimisation. The status quo entailing the diffusion of unnecessary information, it was
sufficient to reduce or qualify the range of published data to increase the implementation of the right
to privacy without decreasing the enjoyment of the concurring values. When Pareto-optimisation cannot occur, however, internet cases reveal their non-manageability
through balancing. A case on point is L’Oréal v eBay, which mixes the Type 1 and Type 2 reasoning in
one judgment. Apparently, the case revolved around a purely Type 2 issue and regarded fundamental
rights only tangentially. Nonetheless, the involvement of several stakeholders and several
fundamental rights makes it ideal to illustrate the elusiveness of digital litigation to balancing
parsing.90 L’Oréal sued eBay for unlawful use of its trademark. eBay pays the likes of Google to have
sponsored links appear alongside normal links as results of keyword searches. eBay’s sponsored links
advertise items sold on its auction platform, many of which branded. Therefore, the relative
trademarks are among the words that eBay selects and for which it pays Google. When private parties
sell counterfeit goods through eBay, eBay cannot be liable directly (to avoid the practice it should set
up a preventive filtering system, something it can refuse to do under the safe harbour of Art. 15 of the 24 e-Commerce Directive). 94 Ibid, para 158. Emphasis added. 93 L’Oréal n 22, Opinion of AG Jääskinen, para 49. 158. Emphasis added. measures However, L’Oréal argued that eBay, by actively buying advertising services for
the search-word ‘l’oréal’, used its brand and exploited or at least abetted the sale of counterfeit
products – an indirect breach of trademarks rights. e-Commerce Directive). However, L’Oréal argued that eBay, by actively buying advertising services for
the search-word ‘l’oréal’, used its brand and exploited or at least abetted the sale of counterfeit
products – an indirect breach of trademarks rights. The Type 2 question, in short, was whether the use of the brand L’Oréal by eBay qualified as ‘use’
under the relevant EU norms on trademark.91 If so, it could be objected by the trademark-holder, if
unlawful. Whether in 1989 the EU legislator could possibly conceive the purchase of search-embedded
advertising through keywords that can correspond to brands is beyond the point. The rationale of the
EU regime concerned the IP-holder’s power to prevent third parties from unauthorised use, hence the
regulatory connection of pre-internet rules could occur without much conceptual trouble.92 The Type 1 component of the dispute, instead, was less straightforward and did not emerge in the
judgment. It did, however, in the Opinion of the Advocate General, who reminded at the outset that
eBay listings are covered by the freedom of expression and information, under Artice 11 of the
Charter.93 Jääskinen conceded that freedom of expression cannot normally justify a breach of property
rights, including trademarks. However, the protection of trade mark proprietor’s rights in the context of electronic commerce may not
take forms that would infringe the rights of innocent users of an electronic marketplace or leave
the alleged infringer without due possibilities of opposition and defence.94 Accordingly, it would be unfairly severe to set up a system of shorthand remedies for the benefit of
IP-holders, if sellers of genuine goods were adversely affected unnecessarily. This remark reveals the
endemic problem of regulation of all internet-related conduct: any standard potentially applies to
millions of users. It is impossible to tell a priori innocent bystanders from fraudulent users, and fine- 25 tune regulation to exclude false positive. Likewise, ex post facto prosecution is unviable. The slippery
slope is around the corner every time regulation of internet behaviour purports to repose on a
seemingly neutral balancing of values, because the sheer scale of massive behaviour online makes it
impossible to strike a balance that is acceptable to all people whose fundamental rights are affected. , (
g
j
g
g p
p
y)
y
the way that the legislature or the government has done or proposes to do’. 95 Endicott n 6, (referring to judges using proportionality): ‘They assess the value of pursuing public purposes in
the way that the legislature or the government has done or proposes to do’. 95 Endicott n 6, (referring to judges using proportionality): ‘They assess the value of pursuing public pur
h
h
h l
i l
h
h
d
d ’ Endicott n 6, (referring to judges using proportionality): They assess the value of pursuing public pu
the way that the legislature or the government has done or proposes to do’. the way that the legislature or the government has done or proposes to do’. measures In short, any regulation of the activity ‘using internet’ is inevitably over-inclusive with respect to
the regulatory goal pursued. It is as if all people ‘breathing’ were required to avoid drinking, just
because all drivers are certainly ‘breathers’. Unfortunately, there seems to be no better way to
circumscribe internet regulation ratione personae or ratione materiae. As a result, proportionate
balancing is normally and demonstrably impossible to achieve; all solutions are disproportionate. This
is so since internet-regulating measures have such a massive inefficiency-creating externality (i.e.,
they create useless restrictions for users whose action is irrelevant to the regulatory purpose pursued)
that it is impossible to prove their proportionality. Hence, it is often impossible to demonstrate that
internet regulations – let alone court decisions – arbitrating between competing rights secure an
overall positive balance of right-enjoyment compared to the status quo. As a consequence, it is the
task of the regulator to deliberately assess the various trade-offs, none of which is proportionate in
the sense implicated by the proportionality test, and choose the preferable norms as a matter of
conscious policy-making. To its credit, the findings of the Court are rarely the strained result of an impracticable review of
proportionality. More commonly, they reflect indeed a policy choice,95 often dictated by the concern
of avoiding false positives and minimise the number of stakeholders whose interest is sacrificed in the
trade-off. The Court in Lindqvist spared billions of web-users from the regime applicable to those who
transfer personal information to third countries. In Google Spain, the Court operated similarly: when
the individual’s concern is plausible, Google’s duty to act upon it is the more convenient option, as 26 opposed to sacrificing her fundamental right, or asking the news outlet to retract a lawful exercise of
the freedom to impart information. This idea of (utilitarian) convenience is candidly spelled out in the
preamble of Directive 2001/2996 on copyright protection in the information society: In the digital environment … the services of intermediaries may increasingly be used by third
parties for infringing activities. In many cases such intermediaries are best placed to bring such
infringing activities to an end. Therefore, without prejudice to any other sanctions and remedies
available, rightholders should have the possibility of applying for an injunction against an
intermediary who carries a third party’s infringement of a protected work or other subject-matter
in a network. p
py g
g
y
97 Recital 59 (emphasis added). 96 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the harmonisation of
certain aspects of copyright and related rights in the information society OJ L 167, 22.6.2001, 10-19.
97 R
it l 59 (
h
i
dd d) Recital 59 (emphasis added).
98 Case C-580/13 Coty Germany (judgment of 16 July 2015) 96 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the harmoni /
/
p
y
certain aspects of copyright and related rights in the information society OJ L 167, 22.6.2001, 10-19.
97 R
it l 59 (
h
i
dd d) rtain aspects of copyright and related rights in the information society OJ L 167, 22.6.2001, 10-19.
Recital 59 (emphasis added). p
py g
97 Recital 59 (emphasis added). ve 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the harmonisation of
spects of copyright and related rights in the information society OJ L 167, 22.6.2001, 10-19. measures … The conditions and modalities relating to such injunctions should be left to the
national law of the Member States.97 There is a clear utilitarian foundation to the rightholders’ right to seek court injunctions ordering
intermediaries to remove the breach. The corresponding intermediaries’ duty does not stem from
their legal responsibility or the optimal balance between their rights and duties, but from their being
‘best placed’ to counter illegality. The intermediaries have quasi-public responsibilities reflecting their
quasi-public authority: it is easy to draw a parallel with the Google Spain decision, as constructed in
the previous section. In L’Oréal v eBay the rights of the legion of eBay sellers could not be curtailed by preventive
restrictions. eBay is held liable for infringement of IP rights only if is aware of it, for instance when it
cooperates with the user on the preparation of the listings. A comparison with the case Coty98 helps
to elucidate the special features of L’Oréal, a case that is essentially internet-related and thus,
arguably, unfit for proportionality. In Coty, the main proceedings emerged from a similar factual
background: an eBay user purchased counterfeit merchandise and sought reparation from the seller. 27 eBay revealed the identity of the seller, who denied involvement in the specific transaction. The
purchaser then required the bank with which the seller held an account (registered with eBay) to
disclose the holder’s identity. The bank refused, invoking banking secrecy. The Court was asked to
determine whether EU law allows banking secrecy to be upheld at the expense of IP protection,99 and
set out to ‘reconcile the requirements of the protection of different fundamental rights, namely the
right to an effective remedy and the right to intellectual property, on the one hand, and the right to
protection of personal data.’100 The Court held that a domestic law authorising banks to invoke
banking secrecy unconditionally can frustrate disproportionally the rights to intellectual property – by
making it impossible to pursue an effective remedy in case of breach.101 The outcome resulted from a plain application of the proportionality test. The domestic law was
considered capable of obliterating even the essence of the rights to property and to an effective
remedy, in breach of Article 52(1) of the Charter. This decision differs from L’Oréal because the
incidence of internet on the dispute was accidental. 99 Namely, under Article 8(1) and Article 8(3) of Directive 2004/48 y
, p
101 Ibid, paras 41-43. 100 Coty n 98, para 33. 99 Namely, under Article 8(1) and Article 8(3) of Directive 2004/48
100 C t
98
33 , p
102 Ibid, para 28. 100 Coty n 98, para 33. y
p
101 Ibid, paras 41-43. 99 Namely, under Article 8(1) and Article 8(3) of Directive 2004/48
100 Coty n 98, para 33. 100 Coty n 98, para 33.
101 Ibid, paras 41-43. measures The dispute was between the bank and the
purchaser, and the interests involved were just two: ‘First, the right to information and, second, the
right to protection of personal data must be complied with.’102 This case, in short, did not display some
of the common features of those disputes in which internet activities are central, and which for this
reason escape moderation through proportionality. The mythology of proportionality pervades also the cases concerning infringements of IP rights
through peer-to-peer online activities. In the main proceedings, the IP-owners (directly or through
dedicated associations) required, alternatively, that internet providers disclose their customers’
identity (Promusicae; Bonnier) or monitor their activity so as to prevent infringements (SABAM;
Scarlet). The Court, in all cases, resorted diffusely to a constitutional reasoning, claiming to consider 28 all fundamental rights involved. Generally, several rights of several subjects were at stake: the
property rights of IP-holders and their right to seek judicial protection thereof, the right to conduct
business of the internet service providers, the right to privacy and to exchange information of the
internet users (the latter arising only in the monitoring-related cases). According to the arguments presented so far, the essential matrix of these cases is sufficient to
redoubt of their compatibility with the orthodox three-step proportionality routine. A short account
confirms the doubts as meritorious. In Promusicae, the Court recalled that it is the Member States’ duty to implement a Directive
choosing measures which strike ‘a fair balance’ between the various fundamental rights and respect
proportionality.103 The Court confirmed this guideline in Bonnier, adding that an order for disclosure
of personal data is not precluded if, among other things, ‘the reasons for the measure outweigh the
nuisance or other harm which the measure may entail for the person affected by it or for some other
conflicting interest’.104 These guidelines are very vague and pass the buck to the national authorities,
which are better positioned to assess the fairness of the balance in specific cases. This implicitly
challenges the idea that there is only one balanced option, to be identified through proportionality
testing. In SABAM and Scarlet, the Court mixed subsumption and proportionality. 107 Scarlet n 23, paras 49-53; Sabam n 24, paras 47-51. 106 Scarlet n 23, para 48; Sabam n 24, para 46. , p
105 Scarlet, n 23, para 40; Sabam n 24, para 38. p
p
106 Scarlet n 23, para 48; Sabam n 24, para 46. Ibid, para 58.
105 Scarlet, n 23, para 40; Sabam n 24, para 38. Ibid, para 70.
104 Ibid, para 58. , p
104 Ibid, para 58. , p
;
, p
7 Scarlet n 23, paras 49-53; Sabam n 24, paras 47-51. 103 Ibid, para 70. 109 Case C-314/12 UPC Telekabel (judgment of 27 March 2014). 108 Scarlet n 23, para 48; Sabam n 24, para 50 (emphasis added). measures It plainly stated that the
sought-after filtering systems would require the internet service providers to carry out general
monitoring, in breach of the e-commerce Directive.105 It then stressed certain characters of the
filtering system (costly, permanent, complicated) that automatically made it unnecessarily
burdensome106 and patently unable to strike a fair balance between the rights of IP-holders and
internet providers.107 The rights of the users were mentioned only ad abundantiam. This finding is 29 impossible to square with an orthodox proportionality test. The Court simply noticed that an
obligation to monitor would impose a significant burden on internet providers, without elaborating
on the degree of benefit that it would bring to property rights. Nor did the decisions account for the
FR-balance of the preferable scenario (ample freedom of conducting business coupled by widespread
disrespect of IP rights). There is not even a hint of comparison on which to hang a proper finding of
proportionality. Likewise, the Court produced a cringeworthy non sequitur when it stated that
requiring providers to set up ‘a complicated, costly, permanent computer system … would also be
contrary to [the requirement] that measures … should not be unnecessarily complicated or costly’.108
Nothing in the Court’s reasoning supported the conclusion that the measure was stricto sensu
disproportionate, let alone unnecessary (that is, wasteful) in light of its purpose. In sum, these peer-to-peer cases show that the Court either delegated proportionality to domestic
courts or recited its elements as if it were a half-forgotten due diligence checklist. It is not
preposterous to suggest that the findings were not truly reached through proportionality, and that it
was just cited to inject legitimacy ex post deciso. In another case (UPC109), the intermediary was not eBay, and the alleged IP infringement was not
camouflaged among millions of innocent ads. The intermediary, an internet provider, was ordered by
a national court to block the users’ access to a website streaming pirated movies. In this dispute, the
possibility of a blanket shutdown did not trouble the Court: the website was clearly up to no good,
hence there was no risk of false positives being unfairly affected by the injunction. measures The balancing with
the hypothetical countervailing rights was briefly accounted as follows: … in order to prevent the fundamental rights recognised by EU law from precluding the
adoption of an injunction such as that at issue in the main proceedings, the national … in order to prevent the fundamental rights recognised by EU law from precluding the
adoption of an injunction such as that at issue in the main proceedings, the national 30 procedural rules must provide a possibility for internet users to assert their rights before the
court once the implementing measures taken by the internet service provider are known.110 procedural rules must provide a possibility for internet users to assert their rights before the
court once the implementing measures taken by the internet service provider are known.110 Even in a case like Schecke, where the outcome is based on a proper proportionality analysis (to the
point that the Court assessed the necessity of the measure challenged, and suggested a less restrictive
alternative),111 the underlying problem was more mundane. Because any information on internet
receives a disproportionate exposure, anything uploaded beyond the strict necessary breaches
somebody’s rights significantly, even when the additional disclosure is not particularly harmful in and
of itself. The fragmentation of practical responsibility for the downstream consequence of online conduct
is difficult to decipher with certainty. Resultantly, the vast disproportion that can occur (in size,
timespan, reach, effects, harmfulness) between conduct and events makes it impossible to apportion
legal responsibility according to a principled scheme, like that resulting from the proportionality test. The northern star of the Court seems the minimisation of costs, something very different from the
maximisation of rights that proportionality promises. Responsibility is regularly attributed to those
who suffer the least from bearing it. Slippery slopes are regularly shunned, a pragmatic result that
betrays the Court’s preference for sustainability over principles. There seems to be a twist, however,
which confirms the impossibility to refer to a unique method of balancing: millions of internet users
are allowed to sell goods (L’Oréal) and exchange files (SABAM) freely, even if this relatively
unregulated practice occasionally encroaches the rights of private individuals. When at stake is a
public interest, instead, the focus shifts, and the priority becomes defusing internet’s multiplying
effect of disorderly conduct. 110 Ibid, para 57. p
111 Ibid, paras 79-82. , p
111 Ibid, paras 79-82. Ibid, para 64.
115 Coty n 98, para 34: ‘EU law requires that, when transposing directives, the Member States take care to rely
on an interpretation of them which allows a fair balance to be struck between the various fundamental rights
protected by the EU legal order’; Promusicae n 21, para 68. 113 Joined cases C-293/12 and C-594/12 Digital Rights Ireland and Seitlinger and others (judgment of 8 April
2014). 112 Case C-46/08 Carmen Media Group [2010] ECR I-8149, para 105. 114 Ibid, para 64. 112 Case C-46/08 Carmen Media Group [2010] ECR I-8149, para 105. 116 That the Directive’s goal was certified as being the protection of public order and security is another point of
possible contention. The Directive, indeed, was adopted under the first pillar as a measure providing for the
harmonisation of national rules, aimed at the removal of obstacles to the realisation of a common market of
services. This economic goal, the only official one, was somewhat side-lined in the Court’s analysis favour of the
second-level purpose (the ‘material objective’) of facilitating the fight against crime, see Digital Rights Ireland n
113, para 41. measures The Court held in Carmen Media Group: a prohibition measure covering any offer of games of chance via the internet may, in principle, be
regarded as suitable for pursuing the legitimate objectives of preventing incitement to squander 31 money on gambling, combating addiction to the latter and protecting young persons, even though
the offer of such games remains authorised through more traditional channels.112 An apparent exception to this very pragmatic approach is the case Digital Rights v. Ireland,113 in which
the Court annulled the Data Retention directive, in a flamboyant exercise of proportionality testing. However, the Court maybe tried a bit too hard to sell this decision as an fearless application of
proportionality. Consider the following statement, relating to the minimum period for which
telecommunications operators must retain the data: that period is set at between a minimum of 6 months and a maximum of 24 months, but it is not
stated that the determination of the period of retention must be based on objective criteria in
order to ensure that it is limited to what is strictly necessary.114 This remark purports to prove too much: why should an EU Directive specify that its application must
comply with the principle of proportionality or respect for fundamental rights? Proportionality is a
general principle of EU law that applies, by default and not upon request, to the interpretation,
implementation and application of EU measures. Respect for fundamental rights is already a binding
principle on State acts.115 If the Directive leaves a huge margin to State implementation, Article 51(1)
of the Charter suffices to prevent the risk that implementing measures breach fundamental rights. Certainly the potential breach entailed by defective implementation must be attributed to the
Member States, not the Directive. FR-based review should hit the implementing measures, not the
implemented act, unless the latter mandates a breach of human rights, quod non in the specific case. Ibid, para 64. 115 Coty n 98, para 34: ‘EU law requires that, when transposing directives, the Member States take care to rely
on an interpretation of them which allows a fair balance to be struck between the various fundamental rights
protected by the EU legal order’; Promusicae n 21, para 68. p
117 Ibid, para 48, emphases added. 118 Ibid, para 51, emphasis added. 119 Ibid, para 49. 116 That the Directive’s goal was certified as being the protection of public order and security is another 119 Ibid, para 49. measures 32 In truth, it is plausible to concede that the Directive was probably badly drafted, and that a tighter
wording with respect to the FR-implications could help Member States to implement it appropriately,
achieving more coherence across the Union. Ultimately, the inefficient (i.e., disproportionate)
measures were the national implementing acts but the Court, perhaps knowing that the Directive
would necessarily need an overhaul in any event, applied the proportionality directly to it, in a display
of righteousness that conveyed the notion that the EU is under a rule of FR-law and the Court is ready
to enforce it unceremoniously. As for the proportionality test itself, this case was relatively simple, with essentially two interests
in tension: the privacy of the data subjects and the public security.116 The duty of telecommunication
providers to retain personal information was in fact a mere exception to the former for the benefit of
the latter, not an autonomous element for the balancing. In essence, the dispute hinged on the
regulatory limits of the exception. Using the privacy-restrictive scenario allowed (if not required) by
the Directive as the status quo baseline, the Court performed an approximate proportionality analysis. The Court employed several degree-qualifying terms, which seemed to set the scene for an
intensity-assessment. Protection of personal data plays an ‘important role’ and the Directive causes a
‘serious’ interference, hence the Court’s judicial review thereon ‘should be strict’117; moreover, the
fight against organised crime and terrorism is ‘of the utmost importance’.118 The suitability of data
retention – in general – for the purposes of fighting crime went uncontested,119 but the necessity of
its limitation under the Directive – in particular – was very much contestable. The Court even noted
that ‘derogations and limitations in relation to the protection of personal data must apply only in so 33 far as is strictly necessary’.120 This statement is presumably a reminder of how important the necessity
step is, otherwise it would indicate that limitations of certain rights other than data protection can be
justified without being strictly necessary. g
g
p
122 For instance Rotaru v. Romania, app. No. 28341/95, Judgment (Merits and Just Satisfaction), Grand C
4 May 2000, paras 57-59. 4 May 2000, paras 57 59.
123 Robert Alexy, ‘Constitutional Rights and Proportionality’ (2014) 22 Revus 51, 59. 120 Ibid, para 52, quoting Case C-473/12 IPI (judgment of 7 November 2013) para 39 , p
, q
g
(j
g
) p
121 Digital Rights Ireland n 113, para 54. gital Rights Ireland n 113, para 54.
r instance Rotaru v. Romania, app. No. 28341/95, Judgment (Merits and Just Satisfaction), Grand Chambe
y 2000, paras 57-59. g
g
, p
122 For instance Rotaru v. Romania, app. No. 28341/95, Judgment (Merits and Just Satisfaction), Grand Chamber,
4 M
2000
57 59 4 May 2000, paras 57-59. 34 pp
g
(
4 May 2000, paras 57-59. 120 Ibid, para 52, quoting Case C-473/12 IPI (judgment of 7 November 2013) para 39
121 Digital Rights Ireland n 113, para 54. g
g
p
122 For instance Rotaru v. Romania, app. No. 28341/95, Judgment (Merits and Just S 124 Digital Rights Ireland n 113, para 56. 127 Ibid, paras 63-64. 125 Ibid, paras 56-59. 126 Ibid, paras 60-62. 128 Ibid, para 65. 126 Ibid, paras 60-62. measures The Court then introduced a spurious element at the outset of the necessity test: the obligation
for the EU legislator to provide in its acts minimum safeguards against the risk of abuse of fundamental
rights.121 This unprecedented legislative duty is borrowed ‘by analogy’ from the case-law of the ECtHR
regarding vague domestic statutes, which unduly empower executive authorities to decide the scope
of FR-limitation.122 However, this parallel oddly overlooks the nature of Directives as acts which, by
definition, are not directly applicable in the Member States. The positive duty that the ECtHR bestows
on national legislators cannot be attributed inattentively by analogy to the EU legislator drafting
Directives. Directives require national implementation, so there is no analogy. Reasonably, the
required safeguards must be included precisely in the national statutes implementing the Directives,
at the hand of national authorities. The call for clauses of minimum protection is also problematic because it injects an inherent vice
in the proportionality reasoning. Alexy criticises similar instructions: A guarantee of a minimum, if not determined by balancing, would, indeed, not be the same
as optimization. It would, however, not only be different from optimization but also different
from proportionality. It would not be an alternative interpretation of proportionality. Rather,
it would be an alternative incompatible with proportionality. One who recommends the
substitution of a guarantee of a minimum for the principle of proportionality in the narrower
sense is recommending the abolishment of this principle.123 34 Even allowing that this requirement be compatible with proportionality, the notion that the Directive
should have included minimum safeguards (something it certainly did not) skewed irremediably the
necessity analysis towards a finding of breach. The Court limited itself to note that the Directive
applied to a wide and largely undifferentiated range of communications, means of communications
and users.124 Moreover, the Directive did not set a specific requirement that persons whose data ought
to be retained be suspected of a crime,125 any objective criteria to calibrate the State’s access to the
data retained,126 or any guidelines on how to restrict domestically the period of retention, down from
the over-inclusive range provided in the Directive.127 Even allowing that this requirement be compatible with proportionality, the notion that the Directive
should have included minimum safeguards (something it certainly did not) skewed irremediably the
necessity analysis towards a finding of breach. p
126 Ibid, paras 60-62. 125 Ibid, paras 56-59. p
127 Ibid, paras 63-64. , p
128 Ibid, para 65. , p
127 Ibid, paras 63-64. Ibid, paras 56 59.
126 Ibid, paras 60-62. p
128 Ibid, para 65. measures The Court limited itself to note that the Directive
applied to a wide and largely undifferentiated range of communications, means of communications
and users.124 Moreover, the Directive did not set a specific requirement that persons whose data ought
to be retained be suspected of a crime,125 any objective criteria to calibrate the State’s access to the
data retained,126 or any guidelines on how to restrict domestically the period of retention, down from
the over-inclusive range provided in the Directive.127 The Court hence concluded peremptorily that the Directive caused an interference with FRs, and
the interference was not ‘precisely circumscribed by provisions to ensure that it is actually limited to
what is strictly necessary’.128 Failure to meet the necessity requirement entailed a failure to respect
the proportionality principle and, ultimately, led to the annulment of the act. In this case, it is difficult to take the proportionality reasoning seriously, for the reasons stated
above. This judgment features, in all likelihood, in the Court’s trend of using proportionality-based
arguments for policy-based decisions. Technically, the enhanced necessity analysis, including the duty
to provide normative safeguards, made for an atypical proportionality test that cannot be traced back
to the classic model. There is an easy way to appreciate how the Court used the proportionality
narrative unorthodoxly. Even if the measure failed the necessity test, the Court did not even try to
identify alternative measures which were reasonably available and equally effective to fight crime. In
sum, the Court condemned the status quo without proving with any precision that another possible
counterfactual scenario could secure a better overall balance of FR-protection. 35 The Court held as much implicitly, noting the Directive’s over-inclusiveness. However, it is fair to
suppose that narrowing down the scope of the retention to an optimal level was a difficult task, hence
it could not be assumed that simply because the Directive balance appeared unfortunate a better
solution was readily available: perhaps it was not. Possibly, requiring that retention only occur when
the conduct of the persons involved is suspicious might delimit the investigation and undermine its
effectiveness. Likewise, the indication that the time-range for the period of retention is excessive
should have come with a cost-benefit analysis of alternative ranges, or additional criteria to narrow
the range down domestically. 129 The AG tried harder to apply proportionality. For instance, he argued that time measured ‘in years’ is
inherently disproportionate because it pertains to the ‘memory’ of the ‘historical time’ of the users, rather than
their ‘present life’ (better measured in months). Ibid, Opinion of AG Cruz Villalón para 146. He conceded that
the distinction might be unhelpful in certain cases (when criminal plans ‘are prepared well in advance’, para
149), but tried to identify a counterfactual measure yielding a better FR-outcome than the status quo. 149), but tried to identify a counterfactual measure yielding a better FR outcome than the s
130 Metro-Goldwyn-Mayer Studios v Grokster, (04-480) 545 U.S. 913 (2005) 380 F.3d 1154.
131 Ibid para 12 y
131 Ibid, para 12. p
132 Ibid, para 19. the distinction might be unhelpful in certain cases (when criminal plans ‘are prepared well in advan
149), but tried to identify a counterfactual measure yielding a better FR-outcome than the status quo. y
p
p
p
y
,
their ‘present life’ (better measured in months). Ibid, Opinion of AG Cruz Villalón para 146. He conce 133 Ibid, para 20. 129 The AG tried harder to apply proportionality. For instance, he argued that time measured ‘in inherently disproportionate because it pertains to the ‘memory’ of the ‘historical time’ of the users, rat ),
y
y
g
q
0 Metro-Goldwyn-Mayer Studios v Grokster, (04-480) 545 U.S. 913 (2005) 380 F.3d 1154.
1 Ibid, para 12. tried to identify a counterfactual measure yielding a better FR outcome than the s
-Goldwyn-Mayer Studios v Grokster, (04-480) 545 U.S. 913 (2005) 380 F.3d 1154. 134 UPC n 109, para 47: ‘it must be observed that an injunction such as that at issue in the main proceedings,
taken on the basis of Article 8(3) of Directive 2001/29, makes it necessary to strike a balance, primarily, between
(i) copyrights and related rights, which are intellectual property and are therefore protected under Article 17(2)
of the Charter, (ii) the freedom to conduct a business, which economic agents such as internet service providers
enjoy under Article 16 of the Charter, and (iii) the freedom of information of internet users, whose protection is
ensured by Article 11 of the Charter.’ See Rebecca Wong and Joseph Savirimuthu, ‘All or Nothing: This is the
Question-The Application of Article 3(2) Data Protection Directive 95/46/EC to the Internet’ (2007) 25 J. Marshall
J. Computer & Info. L. 241, 260: ‘Identity management and privacy considerations now compete with market
expectations of choice, availability, and efficiency.’ measures In short, there is no hint of real comparison in the Court’s reasoning,
which reveals that the Court itself (never mind this author) did not take proportionality seriously
either.129 In the landmark decision MGM v Gronkster, the US Supreme Court was called to address the
liability of internet service providers managing a peer-to-peer platform.130 The Supreme Court frankly
evaluated the interests at stake and the wider policies (how to prevent technology from fostering
disdain for intellectual property; how to set a liability regime that does not hinder technology
advancements). It then based its finding on an openly pragmatic remark: when infringement occurs
on such scale that individual enforcement is impossible, ‘the only practical alternative’131 is to establish
the vicarious or secondary liability of the intermediaries which induce the wrongdoing. It remarked
that the inducement rule was ‘a sensible one for copyright’132 and did not ‘compromise legitimate
commerce or discourage innovation having a lawful promise’.133 Responsible reasoning tapping on
common sense, practicality and established legal doctrines produced a well-reasoned and principled
decision. Nothing suggests that this judgment would have been more legitimate (let alone correct) if 36 it had contained an artificial calculation of the weight of the interests involved and an impressionistic
comparison of the aggregate weight corresponding to the different rules of liability discussed. Proportionality is the result of a good decision, not the other way round. it had contained an artificial calculation of the weight of the interests involved and an impressionistic
comparison of the aggregate weight corresponding to the different rules of liability discussed. Proportionality is the result of a good decision, not the other way round. ,
p
g
p
y
(
,
g
y)
137 Massimo La Torre, ‘Nine Critiques to Alexy's Theory of Fundamental Rights’ in Agustín J Menéndez 5.
Conclusions 5. The Court is couching its decisions in the familiar jargon used in the balancing of fundamental rights. This is understandable, but the recurrent use of proportionality does not evince a fil rouge of the case-
law, nor does it ensure better or more predictable decisions. Sometimes, proportionality is hardly
recognisable when its use is announced, emptied as it is of any substance. Proportionality balancing
as such would made sense only if one hoped to find a point of equilibrium, the end of a zero-sum
game, where any other alternative would be wrong (because comparatively unbalanced). This
aspiration is futile in the field of internet activities, where various recurring features advise against
using proportionality balancing as a heuristic tool. Namely, balancing often occurs between three or more groups of stakeholders and three or more
different rights;134 lawful conduct can result in harmful effects that are difficult to gauge factually and
that, by virtue of the online-multiplier, are hypothetically immense; sweeping judicial precedents are
likely to affect adversely innocent subjects; very rarely can all rights be preserved through Pareto-
optimising balancing. The challenge for the Court, in these conditions, is to let go of the comfortable
terminology/mythology on proportionality and allocate liability with policy-oriented pragmatism, as it 37 does regularly but covertly. Ultimately, however, the refinement of legal categories cannot be
entrusted to the Court alone: the incoming regulatory reform of the field is bound to provide more
guidance (and more precise rules) to judges and EU citizens alike. Regulatory connection with FR-
externalities should not be routinely entrusted courts. Regulators should translate the preferences of
society regarding technology and take responsibility for choosing a regime that will ineluctably entail
an indecipherable FR-balance sheet. Governments are allowed and required to make decisions in
circumstances of radical uncertainties, where beneficial and detrimental effects of a policy cannot be
proved.135 Difficult choices must be made, possibly outside courtrooms: …to rely on courts to rescue legislation can sometimes undermine the integrity of legal reasoning. In the interests of regulatory legitimacy and democracy, where a technology has out-run its
regulatory framework, it might be important to take time out to debate the developments that
have taken place and to determine how the regulatory framework should be adjusted. ,
q
y
y
O Eriksen (eds), Arguing Fundamental Rights (Springer 2006) 53, 61. 136 Brownsword n 15, 266. The Author also notes (ibid., 258) that different regulators might opt f
solutions, depending on the priorities they set (for instance, utilitarianism versus human dignity). 135 Endicott n 6. 137 Massimo La Torre, ‘Nine Critiques to Alexy's Theory of Fundamental Rights’ in Agustín J Menéndez and Erik
O Eriksen (eds), Arguing Fundamental Rights (Springer 2006) 53, 61. 135 Endicott n 6. 135 Endicott n 6.
136 Brownsword n 15 266 The Author also notes (ibid
258) that different regulators might opt for different ,
q
y
y
g
g
O Eriksen (eds), Arguing Fundamental Rights (Springer 2006) 53, 61. p
g
p
y
g
y
La Torre, ‘Nine Critiques to Alexy's Theory of Fundamental Rights’ in Agustín J Menéndez and Erik
ds) Arguing Fundamental Rights (Springer 2006) 53 61 5.
Conclusions Sometimes,
the social licence for a particular technology needs to be reviewed and revised.136 Granted, abandoning the FR-metrical discourse in favour of policy-oriented adjudication would have
its own risks. La Torre rightly noted that if ‘fundamental rights are seen as policies, they will … lose
their point, which is controlling and limiting State action.’137 However, limiting the exercise of public authority is hardly the most pressing problem of the
regulation of internet-related conduct, for the simple reason that public authority is already
comparatively weaker in this field. What needs urgent management are the interplay between private
rights and private interests, and the public policy inputs necessary to arbitrate or moderate between
them. The Google Spain and L’Oréal cases are, in this sense, illustrative of how the difficulty does not 38 lie in the nature of public interference into private conduct, but in the sustainability of a reciprocal
arrangement between human activities with numerous externalities. Ultimately, it is proposed here to discard the ‘mathematical’ proportionality championed by Alexy,
which has provided the Court for too long with a comfort-zone where anything goes, in terms of legal
argumentation. If the motivation of the Court’s decisions is after all policy-oriented, there is no
plausible benefit in their disguise as proportionality calculi. Google Spain is a pragmatical
apportionment of duties, so is L’Oréal v eBay and to a large extent the other IP-related cases perused
above. The outlier cases are not symptomatic: UPC spurs from the matter-of-fact consideration that
the website’s conduct was prima facie illegal; Digital Rights Ireland was really a revise-and-resubmit
note to the EU legislator with a perfunctory proportionality recitation. Certainly, more cases in the future will decide digital cases through lip-serving references to
proportionality. This knee-jerk trend is a disservice to the rule of law and obfuscates the legal
reasoning of the Court, because internet disputes are bound to fit uncomfortably in the
proportionality straitjacket. Better, the proportionality test is bound to fit too loose or too tight on
internet-based realities. Ad hoc adjustments are routinely necessary, to the point that it is not clear
anymore why courts should stick to a test that systematically needs à la carte stretching. Habermas criticised proportionality balancing for undermining the claim to correctness of
adjudication of principles. g
,
(
)
139 Matthias Klatt and Moritz Meister, The constitutional structure of proportionality (OUP 2012) 69. 138 Jürgen Habermas, Between Facts and Norms (HUP 1996) 256-259. 142 Alexy n 123, 64. Footnotes omitted. 141 Mattias Kumm, ‘The Idea of Socratic Contestation and the Right to
Proportionality Review’ (2010) 4 L&Eth. HR 141, 144. y
143 Tsakyrakis n 6, 493. 140 Paul Craig, Administrative Law (OUP 2008) 637. 141 Mattias Kumm, ‘The Idea of Socratic Contestation and the Right to Justification: The Point of Righ 141 Mattias Kumm, ‘The Idea of Socratic Contestation and the Right to Justification: The Point of Rights-Based
Proportionality Review’ (2010) 4 L&Eth. HR 141, 144. Proportionality Review’ (2010) 4 L&Eth. HR 141, 144. 143 Tsakyrakis n 6, 493. 141 Mattias Kumm, ‘The Idea of Socratic Contestation and the Right to Justification: The Point of Rights-Based
Proportionality Review’ (2010) 4 L&Eth. HR 141, 144.
142 Alexy n 123 64 Footnotes omitted 140 Paul Craig, Administrative Law (OUP 2008) 637. 142 Alexy n 123, 64. Footnotes omitted. 143 Tsakyrakis n 6, 493. 5.
Conclusions Proportionality-based reasoning, in short, would not reflect the legal
categories of right and wrong, but the policy categories of adequateness and opportunity, thus
depriving FRs of their status of legal principles.138 Alexy’s rebuttal is convincing, in general:
proportionality is better than nothing. The three-step test, in fact, envisages ‘abundant criteria to label
a proposition as correct or incorrect’139: it provides an articulate template for judicial reasoning (a 39 ‘structured form of inquiry140) in cases where free-style reasoning could be conceived as
unprincipled.141 In his words, ‘structured form of inquiry140) in cases where free-style reasoning could be conceived as
unprincipled.141 In his words, [proportionality is] an argument form of rational legal discourse. As such, it is indispensable
in order to introduce ‘order into legal thought’. It makes clear which points are decisive and
how these points are related to one another.142 This is undisputable. What makes this remark less compelling in the cases studied here is the set of
idiosyncrasies of human affairs in the digital arena. Because optimisation is not a realistic task,
proportionality cannot operate its ‘ordering’ effect and becomes a hollow formula, recited to infuse
legitimacy by way of a recurrent mythology, rather through authoritative reasoning. If the
assessments of necessity and strict proportionality are based on fuzzy and truncated reasoning
(intensity of infringements is not measured; alternative measures are not explored and compared with
the status quo; the interests of various groups are contrasted ‘impressionistically’ and not analytically;
the reasoning shifts uncontrollably and inadvertently from the interest of the dispute parties the
interest of society at large, etc); proportionality is not better than nothing, it is worse. Habermas’s warning applies to these cases. Whereas the Court’s engagement in policy-based
balancing is not to blame (what else is available?), less agreeable is its masquerading as neutral-
looking proportionality.143 If free-style judicial reasoning is the best option (if the regulator stalls, that
probably is), then indeed free judicial reasoning is preferable to reasoning constrained by formulaic
incrustations. 40
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The equation of the entropy production in a tribounit
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The equation of the entropy production in a tribounit Kharkiv National Automobile and Highway University, Kharkiv, Ukraine
*E-mail: alexhome88@gmail.com Introduction When studying the processes of the tribounit friction and wear, it is necessary to consider it as a
dynamic dissipative system in which the degradation of the energy of the macro-mechanical movement is
realized, i.e., entropy is produced. In this regard, it is quite appropriate to use the basic law of non-equilibrium
thermodynamics about the tendency to bring the entropy of a thermodynamic system to a minimum positive
value when it approaches a stationary state, which is characterized by the constancy of all parameters of the
system. As applied to the tribounit as an object of non-equilibrium thermodynamics, here we mean the friction
force and the wear rate of tribo surfaces [1]. Copyright © 2020 Ye.Ventsel, O. Shchukin, O. Orel, N. Saienko. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited. The equation of the entropy production in a tribounit
Ye.Ventsel*, O. Shchukin, O. Orel, N. Saienko
Kharkiv National Automobile and Highway University, Kharkiv, Ukraine
*E-mail: alexhome88@gmail.com The equation of the entropy production in a tribounit
Ye.Ventsel*, O. Shchukin, O. Orel, N. Saienko
Kharkiv National Automobile and Highway University, Kharkiv, Ukraine
*E-mail: alexhome88@gmail.com Abstract It is proposed to consider the tribounit as a non-equilibrium thermodynamic system in which entropy is
produced. The corresponding equation is obtained, whose analysis shows that in order to bring the tribounit to a
stationary state, in which the entropy production tends to zero, and there is a minimum of friction force and a
wear rate, it is necessary to reduce the coefficient of dynamic viscosity of the lubricating medium, the speed of
relative motion of tribo-surfaces, sizes of contaminant particles, etc. p
As a result of laboratory studies, it has been established, in particular, that the presence of contaminant
particles in the oil with the average weighted size of 10.36 μm leads to a decrease in the oil temperature, the friction
coefficient and wear of the tribo-surfaces compared to the oil in which the average weighted size of particles is 35.8 μm. This
proves the original hypothesis of the article that the tribounit is a thermodynamic system in which entropy is produced, and to
which the laws of non-equilibrium thermodynamics are applicable. Keywords: tribounit; energy; entropy; particle; dissipative system, coefficient of friction; wear. Problems of Tribology Problems of Tribology
Website: http://tribology.khnu.km.ua/index.php/ProbTrib
E-mail: tribosenator@gmail.com DOI: https://doi.org/10.31891/2079-1372-2020-96-2-12-18 Problems of Tribology, V. 25, No 2/96-2020, 12-18 Problems of Tribology, V. 25, No 2/96-2020, 12-18 The purpose of the work The purpose of the work is to establish the equation for the production of entropy in tribounits taking
into account a number of parameters characterizing them, and experimentally verify its validity. Literature review Until now, tribounits have been mainly considered as a triad consisting of three components: the friction
surface – the lubricating layer – the friction surface. This model of the tribounit is quite simple and relatively
easy to study [2 – 4]. However, due to the expansion in the number of studies of the friction and wear processes
with increasing thermal and mechanical loading of the tribounit, such a model is becoming insufficiently
informative, which in turn requires a different, more modern approach to representing the processes of friction
and wear during operation of machines. In accordance with the model of L.I. Bershadsky [5] the tribosystem includes excited volumes of
materials of friction surfaces and a lubricant layer, in which the positive entropy production is realized by any
mechanisms. The generalized equation for the production of entropy in the tribounit taking into account the properties
of the lubricating medium was obtained by A.A. Litvinov and A.Ya. Shopel [6, 7]; however, most of the
parameters included in this equation are implicit and complex functions. In this regard, an attempt was made to
theoretically establish the role of such important parameters as the speed of relative motion of tribo surfaces, the
coefficient of dynamic viscosity of the lubricating medium, the size and the number of mechanical particles as
the tribosystem approaches a stationary state. 13 Problems of Tribology 13 Presentation of research materials In accordance with the aforesaid, we analyze the evolutionary trend of various tribounit parameters when
it approaches a stationary state, using I. Prigozhin's theorem [8]. According to this theorem, the entropy production tends to a minimum value when the system approaches
a stationary state. Here we will keep in mind that it is always a positive value. As it is known, a complete change in entropy dS consists of a change in entropy produced inside the
system and entropy caused by the external environment. Considering that the entropy production is a part of the
total derivative of entropy with time: i
e
d S
d S
dS
dt
dt
dt
i
e
d S
d S
dS
dt
dt
dt
1
e
d S
dQ
dt
T
dt
is the flow of entropy. where:
1
e
d S
dQ
dt
T
dt
is the flow of entropy. dt
T
dt
pproaching a stationary state When approaching a stationary state 0
dS
dt
;
i
e
d S
d S
dt
dt
or
1
S
dQ
p
T
dt
(1) (1) accordance with the first law of thermodynamics In accordance with the first law of thermodynamics -
dQ
dU dA
. (2) (2) -
dQ
dU dA
. For a tribounit operating under the conditions of hydrodynamic lubrication, a change in the internal
energy dU is associated with a change in its temperature dT and surface energy due to the presence of particles in
the gap. That is (3) (
)
dU
c m dT
d
N
,
(3) where: C is the specific heat; m is the mass of the substance in the gap of the tribounit; is the specific
surface energy; Ω is the average surface area of an individual particle in the gap; N is the number of particles in
the gap. The work dA performed by the force of viscous friction when moving the surfaces
S of the tribounit
lue dl is equal to: dA
grad
S dl
, (4) (4) : is the coefficient of the oil dynamic viscosity. is the coefficient of the oil dynamic viscosity. where: is the coefficient of the oil dynamic viscosity. where: is the coefficient of the oil dynamic viscosity. Presentation of research materials (8) (8) ue to the fact that
(2
)
dL
dN
L
N
L
dt
dt
tends to the minimum value, the average particle size L
th t i
dL< 0 Due to the fact that
(2
)
dL
dN
L
N
L
dt
dt
tends to the minimum value, the average particle size L
decreases, that is, dL< 0. Since (8) is transformed to the inequality Due to the fact that
(2
)
dL
dN
L
N
L
dt
dt
tends to the minimum value, the average particle size L
decreases, that is, dL< 0. Due to the fact that
(2
)
dL
dN
L
N
L
dt
dt
tends to the minimum value, the average particle size L
decreases, that is, dL< 0. Since (8) is transformed to the inequality dt
dt
decreases, that is, dL< 0. Since (8) is transformed to the inequality decreases, that is, dL< 0. Since (8) is transformed to the inequality ses, that is, dL< 0. Since (8) is transformed to the inequality 2
dN
N
dL
L
, 2
dN
N
dL
L
, then taking into account the sign of dL, we have
dN
N then taking into account the sign of dL, we have
dN then taking into account the sign of dL, we have
d 2
dN
N
dL
L
. 2
dN
N
dL
L
. Whence it follows that 2
dN
N
dL
L
(9) (9) (9) As it can be seen from inequality (9), dN > 0, that is, if the tribounit approaches the stationary state, the
number of particles N in the gap increases, and their average size L decreases, and the smaller L and the larger N
are, the closer the tribounit to the stationary state is. Presentation of research materials Substituting (3) and (4) in (2) and (1), we obtain the following expression for the production of entropy: Substituting (3) and (4) in (2) and (1), we obtain the following expression for the production of entropy
1
S
d
N
dl
dT
p
grad
S
c m
T
dt
dt
dt
. (5) (5) (5) As with approaching the stationary state the production of entropy tends to some constant positive value
corresponding to its minimum, and all components in the right-hand side of expression (5) are independent of
each other, each of these components should be constantly positive. Obviously, to fulfill this condition, it is
necessary that 0
dT
dt
. (6) 0
dT
dt
. (6) Problems of Tribology 14 Problems of Tribology (
)
0
d
N
dt
(7) (
)
d
N
dt
(7) (7) Inequality (6) describes the well-known fact of a decrease in the temperature of a tribounit when it
approaches a stationary state. Inequality (6) describes the well-known fact of a decrease in the temperature of a tribounit when it
approaches a stationary state. equa ty (6) desc bes t e we
ow
act o a dec ease
t e te pe atu e o a t bou
t w e
t
approaches a stationary state. To discuss the meaning of inequality (7), we express the surface area of a particle in terms of its average
linear size L. It is obvious that To discuss the meaning of inequality (7), we express the surface area of a particle in terms of its average
linear size L. It is obvious that 2
L
, where: is the form factor, that is, the coefficient of proportionality depending on the shape of the
particle (for example, for a rod with linear dimensions L, L/2 and L/10,
1,3
) (
)
2
0
d
N
dL
dN
L
N
L
dt
dt
dt
. Problems of Tribology Let us consider the experimental data related to the effect, for example, of the average weighted size of
contaminant particles on the wear of tribo surfaces, that is, on their approaching to a stationary state. For this,
wear tests were carried out on the SMC-2 and CHSHM friction machines. On the SMC-2 friction machine, the “block – roller” test scheme (Fig. 1) was used, which provided the
contact of the samples over the surface under a mixed (hydrodynamic and boundary) lubrication mode. The use
of replaceable balls as wear elements on the ЧШМ friction machine (Fig. 2) makes it possible to obtain the
contact of the friction surfaces at a point (boundary lubrication mode). Fig. 1. Scheme of tests on the SMC-2 friction machine:
1 – cuvette with oil; 2 – roller; 3 – block
Fig. 2. Scheme of tests on the CHSHM friction machine:
1 – ball; 2 – cuvette with oil Fig. 1. Scheme of tests on the SMC-2 friction machine:
1 – cuvette with oil; 2 – roller; 3 – block Fig. 1. Scheme of tests on the SMC-2 friction machine:
1 – cuvette with oil; 2 – roller; 3 – block Fig. 2. Scheme of tests on the CHSHM friction machine:
1 – ball; 2 – cuvette with oil Fig. 2. Scheme of tests on the CHSHM friction machine:
1 – ball; 2 – cuvette with oil To carry out tests, the I-G-A-32 oil was used, which worked in the hydraulic drive of the GR-165 motor
grader for 640 machine-hours and had an average weighted size of contaminant particles of 35.8 μm. The same
oil was also tested, but after artificial dispersion of contaminant particles using a hydrodynamic dispersant [9,
10], which reduced the average size of particles to 10.36 microns. g
p
The following combinations of materials of samples were used on the SMC-2 friction machin
testing: BrAZH 9-4 bronze – 38HS steel, SHH15 steel – SHH15 steel, VCH cast iron – 18 HGT steel. The test mode on the SMC-2 friction machine was as follows: the samples were tested without load for 15
minutes, the samples were tested under load of 300 N for 2 hours, and the tests were carried out under load of
600 N for 4 hours. Presentation of research materials Given that in (5)
,
dl
dt
and also replacing
(
)
d
N
dt
in (5) in accordance with (8), we obtain: Given that in (5)
,
dl
dt
and also replacing
(
)
d
N
dt
in (5) in accordance with (8), we obtain: 1
(2
)
S
dT
dL
dN
P
grad
S
c m
L
N
L
T
dt
dt
dt
. Taking into account that dT
dt <0 and
(
)
d
N
dt
<0, the expression for the production of entropy in the final
form is as follows: Taking into account that dT
dt <0 and
(
)
d
N
dt
<0, the expression for the production of entropy in the final
form is as follows: 1
2
S
dT
dL
dN
P
grad
S
c m
L
N
L
T
dt
dt
dt
. (10) (10) As it can be seen from (10), minimizing the production of entropy, and hence the friction force and wear
of the tribounit requires a decrease in the dynamic viscosity of the lubricating medium, and therefore, a decrease
in the temperature gradient, a decrease in the speed of the relative motion of the tribo surfaces, and also the
average weighted linear size L of contaminant particles in the lubricating medium. 15 Problems of Tribology During the tests, the moment of friction was recorded using the PRS-1 potentiometer included in the
machine set, and the oil temperature was recorded by means of a thermocouple installed in the bath at a depth of
5 mm below the oil level. The wear of the pads and rollers was determined by establishing their loss of mass during the test period. Weighing was carried out with the VLA-200г-М analytical balance with an accuracy of 0.0001 g with
bringing the samples to the constant weight. g g
p
g
On the CHSHM-1 friction machine, replaceable balls with a diameter of 8 mm made of SHH15 steel with
a hardness of 62HRC were used as wear elements. 16 Problems of Tribology The test mode was as follows: the load on the balls was 80 N, the rotation speed of the upper hinge was 1
min-1, the test time was 2 hours. During the test period, the moment of friction (in standard units) was measured,
and at the end of the tests, the diameter of the wear spot was determined with the use of the MBS-9 microscope. p
p
The replication of tests of each oil with each combination of materials of friction pairs on the SMC-2 and
CHSHM-1 friction machines was chosen to be five, which, in accordance with the results of the explorary
research allowed to obtain a fractional error of not more than 0.25 with a confidence factor of 0.9. The results of testing the I-G-A-32 oil on each friction machine are shown in tables 1 and 2. Problems of Tribology Table 1
Results of oil tests on the SMC-2 friction machine
Condition of the
I-G-А-32 oil
Materials
of roller and
pad
Coefficie
nt of
friction
Wear, g
Temperature of
oil at the end
of the test
Pad
Roller
Total
Worked with a
particle size of
35.8 microns
BrAZH 9-4 –
steel 38ХС
0.0163
0.1420
0.0180
0.1600
352
Worked with a
particle size of
10.36 microns
The same
0.0135
0.0989
0.0120
0.1108
348
Worked with a
particle size of
35.8 microns
SHH-15 steel –
SHH -15 steel
SHH -15
0.0747
0.0015
0.0025
0.0040
357
Worked with a
particle size of
10.36 microns
The same
0.0692
0.0010
0,0017
0.0027
356
Worked with a
particle size of
35.8 microns
VCH cast iron –
18 HGT
steel
0.0715
0.0013
0.0035
0.0048
357
Worked with a
particle size of
10.36 microns
The same
0.0345
0.0439
0.0030
0.0469
355
Table 2
Results of oil tests on the CHSHM -1 friction machine
Condition of the I-G-A -32 oil
The moment of friction forces,
standard units
Diameter of the spot of
wear, mm
Worked with a particle size of
35.8 microns
56.6
0.402
Worked with a particle size of
10.36 microns
28.8
0.315 Table 1 Table 1
Results of oil tests on the SMC-2 friction machine Table 1
Results of oil tests on the SMC-2 friction machine
Condition of the
I-G-А-32 oil
Materials
of roller and
pad
Coefficie
nt of
friction
Wear, g
Temperature of
oil at the end
of the test
Pad
Roller
Total
Worked with a
particle size of
35.8 microns
BrAZH 9-4 –
steel 38ХС
0.0163
0.1420
0.0180
0.1600
352
Worked with a
particle size of
10.36 microns
The same
0.0135
0.0989
0.0120
0.1108
348
Worked with a
particle size of
35.8 microns
SHH-15 steel –
SHH -15 steel
SHH -15
0.0747
0.0015
0.0025
0.0040
357
Worked with a
particle size of
10.36 microns
The same
0.0692
0.0010
0,0017
0.0027
356
Worked with a
particle size of
35.8 microns
VCH cast iron –
18 HGT
steel
0.0715
0.0013
0.0035
0.0048
357
Worked with a
particle size of
10.36 microns
The same
0.0345
0.0439
0.0030
0.0469
355 Table 1
Results of oil tests on the SMC-2 friction machine Table
Results of oil tests on the CHSHM -1 friction machine
Condition of the I-G-A -32 oil
The moment of friction forces,
standard units
Diameter of the spot of
wear, mm
Worked with a particle size of
35.8 microns
56.6
0.402
Worked with a particle size of
10.36 microns
28.8
0.315 Results of oil tests on the CHSHM -1 friction machine Note: Tables 1 and 2 show the average values for the results of five tests on the oil in each of its state. Conclusions 1. The tribounit can be considered as a non-equilibrium thermodynamic system in which entropy is
produced. 2. The obtained equation for the production of entropy in the tribounit shows that in order to bring it to a
stationary state, which is characterized by the minimum of friction force and a wear rate, it is necessary to reduce
the coefficient of dynamic viscosity of the lubricating medium, the temperature gradient, the speed of the tribo
surfaces relative motion, and also the average weighted linear size L of contaminant particles in the lubricating
medium. 2. The obtained equation for the production of entropy in the tribounit shows that in order to bring it to a
stationary state, which is characterized by the minimum of friction force and a wear rate, it is necessary to reduce
the coefficient of dynamic viscosity of the lubricating medium, the temperature gradient, the speed of the tribo
surfaces relative motion, and also the average weighted linear size L of contaminant particles in the lubricating
medium. 3. The results of wear tests on the friction machines have confirmed the validity of the obtained equation
for the production of entropy, in particular, the fact that a decrease in the size of contaminant particles helps to
reduce friction and wear of tribo surfaces, as well as the temperature of the lubricating medium. 3. The results of wear tests on the friction machines have confirmed the validity of the obtained equation
for the production of entropy, in particular, the fact that a decrease in the size of contaminant particles helps to
reduce friction and wear of tribo surfaces, as well as the temperature of the lubricating medium. References 1. Ye. Ventsel, O. Orel, O. Shchukin, N. Saienko, A. Kravets', Dependence of Wear Intensity on
Parameters of Tribo Units, Tribology in Industry, Vol. 40, No. 2, pp. 195-202, 2018, DOI:
10.24874/ti.2018.40.02.03 1. Ye. Ventsel, O. Orel, O. Shchukin, N. Saienko, A. Kravets', Dependence of Wear Intensity on
Parameters of Tribo Units, Tribology in Industry, Vol. 40, No. 2, pp. 195-202, 2018, DOI:
10.24874/ti.2018.40.02.03 2. Forbes E. Antiwear and extreme pressure additives for lubricants / E.Forbes // Tribology. –2008. –
vol.3. – №3. – Pp.145-152. 2. Forbes E. Antiwear and extreme pressure additives for lubricants / E.Forbes // Tribology. –2008. –
vol.3. – №3. – Pp.145-152. 3. Akin L. An Interdiscipllinary Lubrication Theory for Gear / L.Akin // ASME Trans. – 1993. – B.36. –
№4. – Pp.1178-1195. 3. Akin L. An Interdiscipllinary Lubrication Theory for Gear / L.Akin // ASME Trans. – 1993. – B.36. –
№4. – Pp.1178-1195. Kostetskiy B.I. Trenıe, smazka i iznos v mashinah / B.I. Kostetskii. – K.: Tehnika, 1970. – 396 p. Bershadskii L.I. O vzaımosviazıah strukturnyh mehanizmov i dissipativnyh potokov pri kınetıcheskom
ovskom) trenıı i iznose // Trenie i iznos, 1989. – T.10, №2. – Pp.358-364. 6. Litvinov A.V. Obobennoe uravnenıe proizvodstva entropii v tribosıstemah s uchetom svoistv
smazochnoi sredy / A.V. Litvınov, A.Ia. Sheel // Issledovanie ekspluatatsionnyh svoistv AVIAGSM i
spetsjıdkosteı. – 1987. – Pp.139-141 6. Litvinov A.V. Obobennoe uravnenıe proizvodstva entropii v tribosıstemah s uchetom svoistv
smazochnoi sredy / A.V. Litvınov, A.Ia. Sheel // Issledovanie ekspluatatsionnyh svoistv AVIAGSM i
spetsjıdkosteı. – 1987. – Pp.139-141 p
Osıpov A.I. Termodınamıka vchera, segodnıa i zavtra. Ravnovesnaia termodinamıka / A.I. Osipov //
kıı obrazovatelnyi jurnal. – 1999. – №4, Ch.1. – Pp. 79-85. 8. Berezniakov A.I. Kompleksnaıa strukturnaia prısposoblıaemost trıbosoprıajenıı v aspekte teoremy I. Prıgojına / A. I. Bereznıakov // Trenie i ıznos. – 1993. – T.14, №1. – Pp.194–202. 8. Berezniakov A.I. Kompleksnaıa strukturnaia prısposoblıaemost trıbosoprıajenıı v aspekte teoremy I. Prıgojına / A. I. Bereznıakov // Trenie i ıznos. – 1993. – T.14, №1. – Pp.194–202. g j
,
p
9. Ventsel E.S. Gidrodınamıcheskıı dispergator dlia uluchsheniia svoistv smazochnyh masel / E.S. Ventsel // Mir tehniki i tehnologii. – 2003. – №7. – Pp. 42-43. g j
,
p
9. Ventsel E.S. Gidrodınamıcheskıı dispergator dlia uluchsheniia svoistv smazochnyh masel / E.S. Ventsel // Mir tehniki i tehnologii. – 2003. – №7. – Pp. 42-43. g
p
10. Ventsel E.S. Problems of Tribology As it can be seen from the data presented, the oil temperature, the friction coefficient (moments of force)
and wear on both friction machines with different combinations of materials of samples are less when lubricating Note: Tables 1 and 2 show the average values for the results of five tests on the oil in each of its state. Note: Tables 1 and 2 show the average values for the results of five tests on the oil in each of its state. As it can be seen from the data presented, the oil temperature, the friction coefficient (moments of force)
and wear on both friction machines with different combinations of materials of samples are less when lubricating
them with the I-G-A -32 oil with the average weighted size of particles of 10.36 μm compared to the oil, in
which the average weighted size of particles is 235.8 microns. g
g
p
In the same way, the presence of contaminant particles in the oil with the average weighted size of 10.36
μm leads to a decrease in the oil temperature and friction coefficient, which is a direct indicator that the tribounit
can be considered as a thermodynamic system, which under the certain conditions tends to an equilibrium state
(approaching zero entropy production), that is, to minimizing friction and wear. Problems of Tribology 17 Conclusions References Pіdviennia iakostі mastil, paliv і trıbovuzlіv mashin : monografiia / E.S. Ventsel, O.V. Orel, O.V. Shchukіn. – H.: FOP Brovіn O.V., 2017. – 264 p. 10. Ventsel E.S. Pіdviennia iakostі mastil, paliv і trıbovuzlіv mashin : monografiia / E.S. Ventsel, O.V. Orel, O.V. Shchukіn. – H.: FOP Brovіn O.V., 2017. – 264 p. 18 Problems of Tribology Венцель Є.С., Щукін О.В., Орел О.В., Саєнко Н.В. Рівняння виробництва ентропії в
трибосполученні Венцель Є.С., Щукін О.В., Орел О.В., Саєнко Н.В. Рівняння виробництва ентропії в
трибосполученні Запропоновано розглядати трибосполучення як нерівноважну термодинамічну систему, в якій
виробляється ентропія. Отримано відповідне рівняння, аналіз якого показує, що для наближення
трибосполучення до стаціонарного стану, при якому виробництво ентропії наближається до нуля і має
місце мінімум сили тертя і швидкості зношування, необхідно зменшення коефіцієнта динамічної
в'язкості змазуючого середовища, швидкості відносного переміщення трибосполучення, розмірів
частинок забруднень тощо. В результаті лабораторних досліджень встановлено, зокрема, що наявність в
маслі частинок забруднень з середньозваженим розміром 10,36 мкм призводить в порівнянні з маслом, в
якому середньозважений розмір частинок складає 35,8 мкм, до зменшення температури мастила,
коефіцієнта тертя і зносу трибоповерхонь. Це підтверджує вихідну гіпотезу статті про те, що
трибосполучення є термодинамічною системою, в якій виробляється ентропія і до якої застосовні закони
нерівноважної термодинаміки. Ключові слова: трибосполучення; енергія; ентропія; частка; дисипативна система, коефіцієнт
тертя; знос
|
https://openalex.org/W4294225513
|
https://www.frontiersin.org/articles/10.3389/fnbot.2022.990453/pdf
|
English
| null |
VSLAM method based on object detection in dynamic environments
|
Frontiers in neurorobotics
| 2,022
|
cc-by
| 11,315
|
VSLAM method based on object
detection in dynamic
environments KEYWORDS
dynamic target detection, VSLAM, YOLOv3, GMM, Kalman filter dynamic target detection, VSLAM, YOLOv3, GMM, Kalman filter TYPE Original Research
PUBLISHED 02 September 2022
DOI 10.3389/fnbot.2022.990453 TYPE Original Research
PUBLISHED 02 September 2022
DOI 10.3389/fnbot.2022.990453 TYPE Original Research
PUBLISHED 02 September 2022
DOI 10.3389/fnbot.2022.990453 dynamic target detection, VSLAM, YOLOv3, GMM, Kalman filter KEYWORDS
dynamic target detection, VSLAM, YOLOv3, GMM, Kalman filter VSLAM method based on object
detection in dynamic
environments OPEN ACCESS
EDITED BY
Yan Wu,
Institute for Infocomm Research
(A*STAR), Singapore
REVIEWED BY
Gongfa Li,
Wuhan University of Science and
Technology, China
Ameer Tamoor Khan,
Hong Kong Polytechnic University,
Hong Kong SAR, China
*CORRESPONDENCE
Dapeng Chen
dpchen@nuist.edu.cn
RECEIVED 10 July 2022
ACCEPTED 17 August 2022
PUBLISHED 02 September 2022
CITATION
Liu J, Gu Q, Chen D and Yan D (2022)
VSLAM method based on object
detection in dynamic environments. Front. Neurorobot. 16:990453. doi: 10.3389/fnbot.2022.990453 OPEN ACCESS
EDITED BY
Yan Wu,
Institute for Infocomm Research
(A*STAR), Singapore Jia Liu, Qiyao Gu, Dapeng Chen* and Dong Yan School of Automation, C-IMER, B-DAT, CICAEET, Nanjing University of Information Science &
Technology, Nanjing, China Augmented Reality Registration field now requires improved SLAM systems to
adapt to more complex and highly dynamic environments. The commonly
used VSLAM algorithm has problems such as excessive pose estimation
errors and easy loss of camera tracking in dynamic scenes. To solve these
problems, we propose a real-time tracking and mapping method based
on GMM combined with YOLOv3. The method utilizes the ORB-SLAM2
system framework and improves its tracking thread. It combines the afne
transformation matrix to correct the front and back frames, and employs GMM
to model the background image and segment the foreground dynamic region. Then, the obtained dynamic region is sent to the YOLO detector to find the
possible dynamic target. It uses the improved Kalman filter algorithm to predict
and track the detected dynamic objects in the tracking stage. Before building
a map, the method filters the feature points detected in the current frame and
eliminates dynamic feature points. Finally, we validate the proposed method
using the TUM dataset and conduct real-time Augmented Reality Registration
experiments in a dynamic environment. The results show that the method
proposed in this paper is more robust under dynamic datasets and can register
virtual objects stably and in real time. COPYRIGHT
© 2022 Liu, Gu, Chen and Yan. 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. 1. Introduction devices, the VSLAM system with AR (Augmented Reality)
technology needs a more excellent background update mode. Many
Augmented
Reality
Registration
methods
are
based on the front-end visual odometry of SLAM systems,
while many VSLAM systems are usually built on static
environments. However,
the
real
environment
is
much
more complicated than the ideal environment. Dynamic
objects such as people and cars are often unavoidable in
scenarios such as classrooms, hospitals, and outdoor shopping
places. Those VSLAM systems built on a static environment
have poor adaptability to dynamic and complex scenes,
leading to substantial errors in the obtained map points and
pose matrix (Cheng et al., 2019). Indirectly, it will cause
problems such as drift of virtual objects registered in the
world coordinate system. Aiming at the problems of excessive
pose estimation error and easy loss of camera tracking in
the commonly used VSLAM algorithm in high dynamic
scenes, we propose a real-time tracking and mapping method
based on GMM combined with YOLOv3. This method
can guarantee the robust registration of virtual objects in
dynamic environments. To ensure that the camera produces robust results when
moving, we combine the affine transformation matrix to correct
the continuous frame image (Sun et al., 2022). In the non-
key frame stage, we employ GMM (Gaussian Mixture Model;
Stauffer and Grimson, 1999) to model the background image,
effectively utilizes the global discontinuity of the keyframe, and
increases the GMM training time to improve the training effect
of the background model. When creating the keyframe, we
combine the image frame of the previous time series to segment
the foreground dynamic area, and provide prior knowledge
for the YOLO detector. To improve the detection accuracy
of the dynamic target of VSLAM, we employ the observation
value provided by YOLO (You Only Look Once) v3 (Redmon
and Farhadi, 2018) in the tracking thread to predict the area
of the dynamic target in real time. Our method combines
the dynamic area detected by YOLO with the dynamic area
obtained after GMM training, and uses the IOU (Intersection
Over Union) result as the probability information to obtain the
largest possible dynamic target. We choose YOLOv3 because it
is a single-stage detector that can achieve good accuracy while
meeting the real-time nature of Augmented Reality Registration. Moreover, compared with traditional methods such as frame
difference method, optical flow method, and background
removal method, YOLOv3 has better real-time performance
and robustness. 1. Introduction But the disadvantage is that YOLOv3 does not
provide prior knowledge that can identify dynamic regions. Our method is complementary to both. It uses a GMM model
to train background images, estimates motion regions when
creating new keyframes, and provides priors for YOLOv3. At
the same time, YOLOv3 meets the real-time and robustness
requirements, and can achieve dynamic target detection between
consecutive frames. y
g
y
g
g
The rest of this paper is structured as follows. The second
section describes the related work of VSLAM implementation in
a dynamic environment. The third section describes the method
of this paper in detail. The fourth section gives the experimental
results. The fifth section gives some conclusions and analysis of
the experiment. 1. Introduction Initially, SLAM (Simultaneous Localization and Mapping) was proposed to solve
the problem of robot movement in an unknown environment. After the robot observes
the environment, it immediately feeds back its posture and movement trajectory, and
constructs a map of the environment simultaneously. The early SLAM system mainly
used single-line lidar, sonar and other sensors to realize its own positioning. With the
rapid development of computer vision, the VSLAM (Visual SLAM) system with the
help of cameras has begun to become the mainstream of research by various teams due
to its convenient use and low cost. The VSLAM system has been well applied in the
fields of augmented reality (Calloway and Megherbi, 2020), driverless driving (Nguyen
et al., 2018), and robotics (Liu, 2021). Virtual objects registered with VSLAM technology
have better stability and accuracy in today’s popular augmented reality applications. To
achieve a more immersive visual experience in the dynamic environment of mobile Frontiers in Neurorobotics Frontiers in Neurorobotics 01 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 10.3389/fnbot.2022.990453 The most traditional tracking algorithm is the filtering
algorithm based on the Bayesian framework (Goan and Fookes,
2020). It utilizes prior information to make an optimal
estimation of the state of the target in the current frame to track
the target, such as the Kalman filter (Xu Y. et al., 2018) and the
Particle filter (Chakravarty et al., 2017). In actual operation, the
observed value is easily affected by factors such as the camera
itself and lighting. The traditional Kalman filter will affect the
next predicted value when an error occurs in the observed
value, leading to the accumulation of errors. We provide an
improved Kalman filter method that uses the first N groups
of observations to establish a nonlinear fitting curve to predict
the next set of observations. Then, we employ an evaluation
metric to determine whether to choose the predicted “observed
value” or the value observed by the system. After the update,
it can obtain a more accurate and practical background. We
employ this improved Kalman filter algorithm to predict and
track YOLO objects to ensure the continuity of the regional
frame. The real-time accuracy of background map construction
determines the reliability of VSLAM applications in many
directions. This method can accurately eliminate the dynamic
noise during the mapping thread, and obtain a good mapping
effect, providing a good mapping environment for Augmented
Reality Registration. The system diagram is shown in Figure 1. 2.1. Classic VSLAM The classic VSLAM system has gone through a series of
explorations and improvements and has formed an effective
execution framework. Davison et al. (2007) first proposed
MonoSLAM, a SLAM scheme based on a monocular camera. Klein and Murray (2007) proposed a keyframe mechanism in the
PTAM scheme, which realized the parallelization of tracking and
mapping, distinguished the front and back ends for the first time,
and used nonlinear optimization as the back-end optimization
scheme. The two earliest proposed VSLAM solutions have
problems such as small application scenarios and easy tracking
loss. However, these innovative framework ideas have been used
to this day. Subsequently, scholars began to improve the front-end
visual odometer. At present, the feature point method composed
of key points and descriptors is the most mainstream front-
end algorithm, such as SIFT (Lowe, 2004), SURF (Bay et al.,
2006), and ORB (Rublee et al., 2011). This kind of method Frontiers in Neurorobotics frontiersin.org 02 Liu et al. Liu et al. 10.3389/fnbot.2022.990453 FIGURE 1
System diagram. This system mainly consists of three parts: tracking thread, mapping thread, and Augmented Reality Registration thread. The
tracking thread uses GMM combined with the YOLOv3 method for dynamic object detection and uses the improved Kalman filter method for
dynamic object tracking. Next, it removes the feature points of dynamic objects in the keyframes and transfers the keyframes that retain the
static feature points to the augmented reality registration thread. In the Augmented Reality Registration thread, it registers virtual objects
through the map points and camera poses of the current frame and tracks virtual objects through matrix calculation. FIGURE 1
System diagram. This system mainly consists of three parts: tracking thread, mapping thread, and Augmented Reality Registration thread. The
tracking thread uses GMM combined with the YOLOv3 method for dynamic object detection and uses the improved Kalman filter method for
dynamic object tracking. Next, it removes the feature points of dynamic objects in the keyframes and transfers the keyframes that retain the
static feature points to the augmented reality registration thread. In the Augmented Reality Registration thread, it registers virtual objects
through the map points and camera poses of the current frame and tracks virtual objects through matrix calculation. is stable and relatively mature. Another front-end algorithm
is the direct method based on pixel brightness information. Engel et al. (2014, 2015) proposed LSD-SLAM, which uses the
direct method to build maps. 2.1. Classic VSLAM It has the advantages of fast
speed and good real-time performance, but it is very sensitive
to the camera’s internal parameters and exposure, and it is
easy to lose when the camera moves quickly. Forster et al. (2014) proposed high-speed real-time mapping using the sparse
direct method SVO. It is extremely fast, but due to abandoning
the calculation of the descriptor, its pose estimation is prone
to cumulative errors. When the camera moves quickly, the
location information is easy to lose, and it is difficult to relocate
after being lost. In the case of much noise in the dynamic
environment, the result of this method is still not satisfactory. which in turn affects the accuracy of target tracking (Huang
et al., 2022). In recent years, dynamic target detection has put forward
higher tracking accuracy and target number requirements, and
many excellent SLAM frameworks have emerged continuously
(Gehrmann et al., 2019). In the past, semantic segmentation
was used to train static objects to generate semantic maps
that increase the amount of information. For example, the
semantic map construction proposed by Goerke and Braun
(2009). When building a map in a dynamic environment, it
is necessary to segment and remove dynamic characters. The
method proposed by Wang et al. (2016) is a new method for
classifying human motion regions. It divides human activities
into categories and predicts the travel of the human body
through general movement patterns. But this method is only
suitable for fixed cameras. Riazuelo et al. (2017) proposed a
semantic SLAM method in dense portrait scenes. This method
solves the limitation of camera fixation. It completes a complete
SLAM system based on the visual odometer (Wang et al., 2007). It can detect which are dynamic objects, but it cannot detect
changes caused by static objects. Bescos et al. (2018) proposed
DynaSLAM, which employs the Mask RCNN (Ammirato and
Berg, 2019) to arrange the scene prior knowledge and estimate
the possible moving targets through the geometric view method. This method removes the feature points of the moving target
through a mask to maintain the algorithm’s accuracy. After
removing the dynamic target, the previously observed static
information is used to repair the area. However, when repairing
the occluded background of the current frame, using the pixel
area corresponding to the last frame will cause the accumulation
of errors. Zhong et al. (2018) proposed Detect-SLAM. 2.1. Classic VSLAM It
combines the single shot multibox detector (Liu et al., 2016) Mur-Artal et al. (2015) proposed a monocular ORB-SLAM
system. ORB-SLAM utilizes unified ORB features in each
link of tracking, mapping, relocation and loop detection. It
has high computational efficiency, good rotation and scaling
invariance (Mur-Artal and Tardós, 2017; Campos et al., 2021),
and its performance in a dynamic environment can be further
improved. Many SLAM systems improved through dynamic
target detection and deep learning are also implemented under
the ORB-SLAM’s framework. Frontiers in Neurorobotics 2.2. Dynamic VSLAM scheme of deep
learning and geometric view In terms of dynamic target detection, traditional methods
are greatly affected by scene brightness changes, noise, etc., and
there will be false detections and missed detections in the target
detection process. This also leads to drift during target tracking, Frontiers in Neurorobotics frontiersin.org 03 Liu et al. 10.3389/fnbot.2022.990453 of precise detection and segmentation of moving targets, and
it is not outstanding in real-time performance. Cheng et al. (2019) inspired by deep neural networks, proposed SMR-
SLAM, which employs the Bayesian formula to solve the
probability distribution of the feature point area of the geometric
view. Small-probability events are eliminated to help SLAM
distinguish dynamic regions as much as possible. It can learn
and perform well in scenes with low dynamics, but the error is
more evident in highly dynamic scenes or excessive complexity. Liu et al. (2022) optimized the sparse point cloud map through
the YOLOv4 framework to enhance the interactivity of the
robot. Gao et al. (2020) proposed a feature map fusion one-shot
multi-box detector, which has higher detection accuracy and
real-time performance compared to SSD and DSSD methods. The occlusion of the hand is also one of the reasons for the
failure of virtual object registration, and the detection of the
hand is also very necessary (Gao et al., 2019). YOLOv3 has
better real-time and accuracy in hand detection, and can detect
hand occlusion in real time, helping to complete better virtual
object registration. on the basis of ORB-SLAM, and uses semantic information to
eliminate the influence of dynamic targets in SLAM. In addition,
it also contains a method to propagate the dynamic possibilities
of each feature point in real time, which solves the problem
of delay in the transmission of semantic information. Yu et al. (2018) proposed DS-SLAM, which utilizes the optical flow
method to track feature points and employs RANSAC (Raguram
et al., 2012) to eliminate outliers and calculate the basic matrix. The dynamic and static points are judged based on the distance
from the feature point to the epipolar line. Then, SegNet
(Badrinarayanan et al., 2017) is used to divide the dynamic area
and eliminate the feature points of the dynamic area. However,
because the semantic information is not comprehensive enough
and the semantics are untargeted, there are problems in dynamic
filtering in some aspects, such as gesture occlusion. Xiao et al. (2019) proposed Dynamic-SLAM. 2.2. Dynamic VSLAM scheme of deep
learning and geometric view Based on the same work as
DynaSLAM (Bescos et al., 2018), it reduces the dynamic error
and builds a better map (Fan et al., 2020). These SLAM systems for dynamic scenes generally use
semantic information, either using geometric information or a
simple combination of methods for dynamic object detection. Cui and Ma (2019) proposed SOF-SLAM, which combines
semantics and optical flow methods. It fully utilizes the dynamic
characteristics of features hidden in semantic and geometric
information. Cui and Ma (2020) proposed SDF-SLAM, utilizes
a depth filter to describe each map point’s inverse depth,
updates the inverse depth of the 3D map points in the Bayesian
framework, and divides the 3D map points into active or inactive
points. However, the problem of using a semantic combination
of VSLAM is still undeniable. They all rely heavily on the
training effect of the network model. The prestage workload is
enormous, but it can only be divided and cannot be tracked well. For the classic network model of target detection, the candidate
area method proposed by RCNN is very time-consuming and
cannot be run in real time. YOLO innovatively proposed
merging the candidate area and recognition process in RCNN
to increase computing speed significantly (Redmon and Farhadi,
2018). We provide the method of GMM combined with YOLOv3. Our method uses the ORB-SLAM2 framework and improves
its tracking thread to analyze dynamic targets. The method
provided in this paper detects and tracks dynamic targets,
eliminates dynamic points in real time, and optimizes the update
mode of the background to ensure the accuracy of pose solving
and map creation. 3.1.1. Moving target detection algorithm based
on GMM GMM is a method to accurately quantify things with a
Gaussian probability density function and decompose them into
several models based on a Gaussian probability density function. The GMM application in background elimination establishes
a Gaussian mixture model for each pixel in the video frame. If the pixel model has a significant weight, it is indicated as
a background pixel; otherwise, it is a foreground image. Since
background pixels often occupy high weights, the generated data
is more trustworthy on the background pixels so that GMM can
distinguish the foreground and the background in the long-term
observation sequence generated by the video. Inspired by deep learning, improved view geometry methods
are also constantly advancing, and new system models appear. Sun et al. (2017) proposed a motion removal method based
on RGB-D cameras. Since this method relies on the maximum
posterior scheme to determine the foreground, the segmentation
results are limited (Sun et al., 2019). Xu X. et al. (2018) proposed
a multi-view spectral clustering framework that combines
multiple models together, integrating the affine, tomography,
and basic matrix. Sun et al. (2018) proposed MR-SLAM, which
improved their previous method (Sun et al., 2017) to model
prospects in different classes, so the number of moving objects
was not limited during segmentation. This method adds online
learning capabilities, allowing it to update the foreground model
incrementally. Although, MR-SLAM can effectively deal with
dynamic factors, it consumes too much time in the process Our method employs the ORB feature extraction and
matching to extract feature points, calculates the affine matrix
M by matching the front and back frames, corrects the current
frame image through the affine matrix M. Then, the method
employs GMM to learn background pixels and segment the
foreground and background images by finding the pixel group Frontiers in Neurorobotics 04 frontiersin.org Liu et al. Liu et al. 10.3389/fnbot.2022.990453 FIGURE 3
Flow chart of dynamic target detection combining GMM and
afne matrix. FIGURE 4
Schematic diagram of bounding box regression. FIGURE 2
GMM dynamic solution. FIGURE 3
Flow chart of dynamic target detection combining GMM and
afne matrix. FIGURE 2
GMM dynamic solution. closest to the background. The dynamic target detection process
is as follows: a. In the initialization phase, the method completes the initial
setting of the parameters of the GMM model. b. 3.1.1. Moving target detection algorithm based
on GMM In the non-key frame stage, the feature points are extracted
from the VSLAM to match the continuous frame images. Calculate the affine transformation matrix M by this method,
utilize the M matrix to make affine changes, and set the
threshold (the threshold is 20 in this paper) to correct the
current frame. FIGURE 3
Flow chart of dynamic target detection combining GMM and
afne matrix. Flow chart of dynamic target detection combining GMM and
afne matrix. FIGURE 4
Schematic diagram of bounding box regression. FIGURE 4
Schematic diagram of bounding box regression. c. At the same time, to reduce the image shift caused by the
affine matrix error, the method uses the mean filter to process
the images before and after the transformation. d. Our method trains the corrected image on the GMM
Gaussian mixture model. It combines the image frame of the
previous time series to determine the foreground dynamic
area when the keyframe is created. The
principle
diagram
of
dynamic
target
detection
combining VSLAM and GMM is shown in Figure 2, and the
specific flow chart is shown in Figure 3. FIGURE 4
Schematic diagram of bounding box regression. Frontiers in Neurorobotics 3.1.2. Target detection algorithm based on
YOLOv3 Due to the
instability of the feature points, the calculated affine matrix has
errors, so when the dynamic target is moving, the real-time
calculation result using the frame difference method is often
not satisfactory. Our method in this paper establishes dynamic
candidate areas through keyframes. At the same time, it
employs the YOLOv3 algorithm to receive each candidate area
FIGURE 6
Schematic diagram of dynamic area detection. detection fails [such as 1⃝], discard other static targets obtained
by YOLOv3 [such as 4⃝], and detect the area (solid box) such as
2⃝3⃝. FIGURE 5
YOLOv3 detection process. FIGURE 5
YOLOv3 detection process. bw = pwetw
(3)
bh = pheth
(4) bw = pwetw
(3)
bh = pheth
(4) FIGURE 6
Schematic diagram of dynamic area detection. (3) FIGURE 6
Schematic diagram of dynamic area detection. (4) where tx, ty, tw, and th represent the offset of x coordinate,
y coordinate, width, and height offset, respectively. bx, by, bw,
and bh represent the result of the final goal box. σ (x) represents
the Sigmoid function. The result of x is normalized to speed
up network convergence, where pw and ph are the width and
height of the bounding box, respectively. The overall YOLOv3
detection process is shown in Figure 5. In
VSLAM,
to
ensure
the
accuracy
of
map
point
construction, it is necessary to eliminate all possible dynamic
targets in the keyframe by comparing the information of
the last frame when generating the keyframe. Due to the
instability of the feature points, the calculated affine matrix has
errors, so when the dynamic target is moving, the real-time
calculation result using the frame difference method is often
not satisfactory. Our method in this paper establishes dynamic
candidate areas through keyframes. At the same time, it
employs the YOLOv3 algorithm to receive each candidate area
and discard candidate areas that cannot be identified. The
method employs the GMM model to train the background
image, estimates the motion area when creating new keyframes,
provides prior knowledge for YOLOv3, and exploits the fast
and robust advantages of YOLOv3 to achieve dynamic target
detection between consecutive frames. With the advantage
of discontinuous VSLAM keyframes in time series, each time
a keyframe is established, this method analyzes the dynamic
area to increase or decrease the dynamic tracking frame. 3.1.2. Target detection algorithm based on
YOLOv3 This
method can ensure the real-time performance of VSLAM and
avoid the problem of local map tracking failure caused by too
few map points due to multiple additions and reductions of
candidate areas. The schematic diagram is shown in Figure 6. FIGURE 6
Schematic diagram of dynamic area detection. detection fails [such as 1⃝], discard other static targets obtained
by YOLOv3 [such as 4⃝], and detect the area (solid box) such as
2⃝3⃝. Frontiers in Neurorobotics 3.1.2. Target detection algorithm based on
YOLOv3 converted into a regression problem, and the 4 coordinates
tx, ty, tw, th (as shown in formulas 1–4) of each bounding box
are predicted. For the problem of bounding box regression, for
the 13×13 feature scale map, we utilize three bounding boxes
of 10×13, 16×30, and 33×23 pixels; for the 26×26 feature
scale map, we utilize three bounding boxes of 30×61, 62×45,
59×119 pixels; for the 52×52 feature scale map, we utilize
three bounding boxes of 116×90, 156×198, 373×326 pixels. The
regression diagram of the bounding box is shown in Figure 4. The moving target detection algorithm based on the GMM
extracts the keyframes and then performs the difference. Between keyframes, we employ the YOLOv3 algorithm to detect
objects that may need to be tracked. YOLOv3 is a single-stage
detector that can meet real-time performance for Augmented
Reality Registration while maintaining accuracy compared to
methods via R-CNN. The method divides the input image into
a 13×13 table and then lets each cell detect the target. The
bounding box and the discrimination probability value through
each grid are obtained to judge whether the target object and
the position information and probability information of the
target area in the grid. The dimensional clustering method on
the bounding box is chosen to select 3 scales and nine types
of bounding boxes, the bounding box detection problem is The definition formula for the bounding box is as follows bx = σ (tx) + cx
(1)
by = σ
ty
+ cy
(2) (1) bx = σ (tx) + cx by = σ
ty
+ cy
(2) (2) frontiersin.org Frontiers in Neurorobotics 05 frontiersin.org Liu et al. Liu et al. 10.3389/fnbot.2022.990453 FIGURE 5
YOLOv3 detection process. bw = pwetw
(3)
bh = pheth
(4)
where tx, ty, tw, and th represent the offset of x coordinate,
y coordinate, width, and height offset, respectively. bx, by, bw,
and bh represent the result of the final goal box. σ (x) represents
the Sigmoid function. The result of x is normalized to speed
up network convergence, where pw and ph are the width and
height of the bounding box, respectively. The overall YOLOv3
detection process is shown in Figure 5. In
VSLAM,
to
ensure
the
accuracy
of
map
point
construction, it is necessary to eliminate all possible dynamic
targets in the keyframe by comparing the information of
the last frame when generating the keyframe. 3.2. Dynamic target tracking based on an
improved Kalman filter Because of the special relationship between
the observation equation and the state equation in this paper, Hk
is a 4×8 matrix, where the observation equation is only related
to the first four dimensions of the current state vector, that is, the
displacement point, so take: necessarily accurate, whether due to the influence of the camera
or lighting effects. To solve such problems, we propose an
improved Kalman filter method. We exploit the improved
Kalman filter to predict the maximum probability position and
length and width information of the next frame. It uses its error
covariance to calculate the predicted value of the state variable,
find the observed value by combining the detection algorithm,
correct the predicted value with Kalman gain, and finally obtain
the optimal value of the variable. The improved Kalman filtering algorithm exploits the first
N groups of observations to establish a nonlinear fitting curve
to predict the next group of observations. The algorithm uses an
evaluation index to determine the selected predicted “observed
value” or the value observed by the system. Since the feature
points are affected by environmental factors or camera shake
factors, linear fitting is performed according to the absolute
values of the errors of the previous N-1 groups of predictions
and observations. While ensuring the real-time performance
of the algorithm, it can distinguish whether the target is
moving fast or instantaneously due to observation errors. The
improvement principle is shown in Figure 7 ( ˙Xn are fitted
observations, and Xn is an actual observation. P1, P2... Pn−1
represent the error covariance). 3.2. Dynamic target tracking based on an
improved Kalman filter Multi-target detection algorithms are easily affected by
factors such as illumination, occlusion, and pixel blur when
moving (Li and Shi, 2019). The dynamic area will not disappear
irregularly, so we build a tracking model to achieve multi-
target tracking between two keyframes to ensure the continuity
of the bounding box detected by YOLO. The Kalman filter
algorithm itself is a linear system. Since the value observed in
this paper is the state value, it is easy to estimate the value
from the previous state to the next state by using the state
transition matrix of the Kalman filter algorithm. The Kalman
filter algorithm only considers the relationship between the
upper and lower frames to a certain extent, so the Kalman filter
algorithm needs a very accurate observation effect. However,
in the actual operation process, the observed values are not The dotted box represents the dynamic candidate area
provided by GMM, and the solid box represents all targets
detected by YOLOv3. Then we use the IOU result as the
probability information to get the largest possible dynamic
target [such as at 2⃝], discard the area where GMM dynamic 06 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 10.3389/fnbot.2022.990453 Liu et al. state vector values represent the center pixel position x and
y, the aspect ratio and height of the bounding box, and
their corresponding velocity values. Expressed by the equation
of motion xk = Akxk−1 + Bkµk + wk, due to the lack of a
control vector, Bk is set to a 0 vector, which satisfies the state
transition matrix: FIGURE 7
Improved observation value selection principle diagram. A =
1 0 0 0 1 0 0 0
0 1 0 0 0 1 0 0
0 0 1 0 0 0 1 0
0 0 0 1 0 0 0 1
0 0 0 0 1 0 0 0
0 0 0 0 0 1 0 0
0 0 0 0 0 0 1 0
0 0 0 0 0 0 0 1
(5) This formula expresses the displacement of the previous
state plus the unit velocity to represent the displacement
of the current state, and considers the system error and
the observation error, wk ∼N
0, Qk
, vk ∼N
0, Rk
. The
observation equation is expressed as zk = Hkxk + vk according
to the Kalman filter. Frontiers in Neurorobotics 3.2. Dynamic target tracking based on an
improved Kalman filter Hk =
1 0 0 0 0 0 0 0
0 1 0 0 0 0 0 0
0 0 1 0 0 0 0 0
0 0 0 1 0 0 0 0
(6) (6) To satisfy the system’s optimal estimation of the state
equation, we modify the state value during the observation phase
and introduce the covariance matrix to update: Pk|k−1 = A · Pk−1|k−1 · AT + Q
(7) (7) where Pk|k−1 represents the covariance matrix of the
predicted state value and obtains the optimal estimation of the
current state through the prediction result of the current system
and the measurement of the current state: The observation values selected in this paper are the central
pixels
1
2
P2
2i x2i, 1
2
P3
2i+1 y2i+1
of the four boundary corners
of the target image to input to the Kalman filter system to obtain
the predicted value. Then, take the predicted point as the center,
use the value of max
xj −xi
obtained in the last frame as the
width of the rectangle, and the value of max
yj −yi
as the
length of the rectangle, and then crop a new area. The camera
pose is detected and calculated in this area to obtain a new set
of measured values, and the rectangular area size and area of the
next frame are updated from the measured values and the new
boundary corner points. xk|k = xk|k−1 + Kk
zk −H · xk|k−1
(8) (8) where Kk represents the current Kalman gain coefficient,
which is represented by the covariance matrix P and the
measurement matrix H: where Kk represents the current Kalman gain coefficient,
which is represented by the covariance matrix P and the
measurement matrix H: Kk = Pk|k−1 ·
HT
H · Pk|k−1 · HT + R
(9) (9) We bring the Kalman gain at this time into the optimal
estimation solution and exploit this gain to calculate the required
covariance matrix value at the next moment: First, we establish an 8-dimensional state vector and
a 4-dimensional observation vector according to the linear
condition satisfied by the Kalman filter. The 8-dimensional Pk|k =
1 −Kk · H
Pk|k−1
(10) (10) frontiersin.org 07 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 We can completely predict the center point’s position at the
next moment through observations. 3.2. Dynamic target tracking based on an
improved Kalman filter However, the selection of
observations affects the stability of the entire system. In the case
of minimal noise, if there is a significant error in the observed
value, the predicted value will also be inaccurate. We improve
the performance of the entire system by improving the selection
of observations, and the method is as follows: FIGURE 8
Improved Kalman forecast update flow chart. 3.3. Incremental model The method provided in this paper performs the above
dynamic target detection and tracking on the image sequence
between every two keyframes. When VSLAM constructs a
keyframe, it rejudges whether a new target area needs to be
constructed. Therefore, the following incremental model is
added during the keyframe construction to ensure that the
dynamic increment can be tracked stably in the tracking thread
or use the incremental model to determine whether to cancel
tracking the lost target information. The incremental model is
shown in Figure 9, where FLast represents the last frame of the
keyframe, FCur represents the current frame that can also be
understood as a keyframe, and Tracker is the tracker designed
in this paper. (3) Judgment stage Our method has obtained two sets of observation points:
the observed points and the predicted “observation points.”
Of course, it is hoped that the actual observation points are
accurate, but regardless of the presence of noise or the influence
of light factors, the observed data may always be wrong. This
paper introduces third-party evaluation indicators to determine
which value is more accurate. We assume that the previous observation data are accurate
(or the observation data has been corrected), and there are also
errors between the predicted value of the Kalman filter and
the observation of the next frame, and the error may be small. Our method builds a set of fitting data by the absolute value of
the error between the observation value of the next frame and (1) Initialization phase First, we establish a non-linear loss function model. Our
method sets the model as: f (x) −exp
a · x2 + b · x + c
(11) (11) the predicted value of Kalman filter. At the same time, we fit
the linear equations with the previous N-1 sets of data, predict
the “observed value” of the Nth set of data, and calculate the
absolute value of the error between it and the Kalman predicted
value. Finally, we judge which observation value is more reliable
according to the error growth rate. The calculation function of
the judgment is as follows: Secondly, we define N groups of observation data (N is set
to 20 in this chapter), and establish the least square function
through the observation data: again
X
x
||f (x) −exp
a · x2 + b · x + c
||2
(12) (12) At the same time, we assign values to the initial values of
the first N groups. If all the first N groups are assigned a value
of 0, the finally obtained parameters are easy to fall into the
local optimal solution, and the parameters to be sought are
solved incorrectly in the initialization stage. Therefore, we add
Gaussian disturbance to the value of f(x) and x to make them in
a fluctuating state. zk = min
zk
3 ×
b
zk −pk−1
−gk
,
zk −pk−1
−gk zk = min
zk
3 ×
b
zk −pk−1
−gk
,
zk −pk−1
−gk
(1 (13) where gk is the predicted value of the error, b
zk is the
predicted “observed value,” zk is the observation value of the
system, and pk−1 is the predicted value of the last frame. In order
to ensure the reliability of system prediction, we assign weight
to both of them to avoid local optimization. Finally, the closer
“observation point” is selected as the new observation point. The
flow chart of the algorithm is shown in Figure 8. (2) Solving stage To ensure that the data of a given fitting does not increase
over time, the problem of incorrect fitting parameters and a
significant increase in the number of calculations does not
occur. Our method accepts new data while removing the old
data to maintain it at the value of N. Within the parameter
range. Our method uses the L-M method to iterate, and finally
finds the solution of the unit at the next time through the
known parameters, which is the “observation point” for solving
the prediction. Frontiers in Neurorobotics Data sets distribution and the higher the detection accuracy. In order to
reflect the superiority of the detection algorithm proposed in this
paper, this experiment employs the traditional frame difference
method, optical flow method and other algorithms that are often
used in dynamic target detection to compare. Table 1 shows
the results (calculate the average IOU value for each frame
detected under each data set). For multiple dynamic targets in an
image frame, calculate the average value of IOU in the current
frame and then map it to the global data set. The algorithm’s
performance in this paper on the data set fr2/desk with person is
shown in Figure 10. of the tracking algorithm based on the improved Kalman filter. Finally, the algorithm is integrated into the VSLAM to eliminate
the dynamic target. We verify the effectiveness of the algorithm
proposed in this paper by two metrics: ATE (absolute trajectory
error) and RPE (relative pose error). The test platform for this
experiment is Ubuntu 16.04, the primary language for building
the platform is C++, and the Python environment is applied for
ATE and RPE analysis. It can be seen from the data in Table 1 that the target
detection algorithm used in this paper effectively improves the
detection accuracy of the dynamic region. 4. Experiments This experiment utilizes the dynamic objects dataset in the
TUM dataset for dynamic target detection and the verification 08 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 FIGURE 9
Incremental model. TABLE 1 Dynamic target detection results. Data sets
Temporal
difference method
Optical
flow method
Ours
fr2/desk with person
0.2514
0.4613
0.5756
fr3/sitting static
0.1011
0.2167
0.4783
fr3/sitting xyz
0.2331
0.5098
0.5933
fr3/sitting halfsphere
0.4060
0.4200
0.5749
fr3/sitting rpy
0.1991
0.2340
0.6764
fr3/warking static
0.4788
0.6993
0.6032
fr3/warking xyz
0.5423
0.5745
0.7220
fr3/warking halfsphere
0.4421
0.6421
0.6854
fr3/warking rpy
0.5322
0.3210
0.6010
Bold values mean the best result among the methods. of the tracking algorithm based on the improved Kalman filter. Finally, the algorithm is integrated into the VSLAM to eliminate
the dynamic target. We verify the effectiveness of the algorithm
proposed in this paper by two metrics: ATE (absolute trajectory
error) and RPE (relative pose error). The test platform for this
experiment is Ubuntu 16.04, the primary language for building
the platform is C++, and the Python environment is applied for
ATE and RPE analysis. FIGURE 10
Dynamic detection performance efect of the data set. distribution and the higher the detection accuracy. In order to
reflect the superiority of the detection algorithm proposed in this
paper, this experiment employs the traditional frame difference
method, optical flow method and other algorithms that are often
used in dynamic target detection to compare. Table 1 shows
the results (calculate the average IOU value for each frame
detected under each data set). For multiple dynamic targets in an
image frame, calculate the average value of IOU in the current
frame and then map it to the global data set. The algorithm’s
performance in this paper on the data set fr2/desk with person is
shown in Figure 10. It can be seen from the data in Table 1 that the target
d
l
h
d
h
ff
l
h FIGURE 9
Incremental model. TABLE 1 Dynamic target detection results. Data sets
Temporal
difference method
Optical
flow method
Ours
fr2/desk with person
0.2514
0.4613
0.5756
fr3/sitting static
0.1011
0.2167
0.4783
fr3/sitting xyz
0.2331
0.5098
0.5933
fr3/sitting halfsphere
0.4060
0.4200
0.5749
fr3/sitting rpy
0.1991
0.2340
0.6764
fr3/warking static
0.4788
0.6993
0.6032
fr3/warking xyz
0.5423
0.5745
0.7220
f 3/
ki
h lf
h
0 4421
0 6421
0 6854
FIGURE 10
Dynamic detection performance efect of the data set. FIGURE 9
Incremental model. Frontiers in Neurorobotics 4. Experiments FIGURE 10
Dynamic detection performance efect of the data set. FIGURE 10
Dynamic detection performance efect of the data set. TABLE 1 Dynamic target detection results. Data sets
Temporal
difference method
Optical
flow method
Ours
fr2/desk with person
0.2514
0.4613
0.5756
fr3/sitting static
0.1011
0.2167
0.4783
fr3/sitting xyz
0.2331
0.5098
0.5933
fr3/sitting halfsphere
0.4060
0.4200
0.5749
fr3/sitting rpy
0.1991
0.2340
0.6764
fr3/warking static
0.4788
0.6993
0.6032
fr3/warking xyz
0.5423
0.5745
0.7220
fr3/warking halfsphere
0.4421
0.6421
0.6854
fr3/warking rpy
0.5322
0.3210
0.6010
Bold values mean the best result among the methods. 4.3. Analysis of experimental results
based on the VSLAM dynamic
environment error for each camera pose are represented as the black,
blue, and red segments, respectively. The algorithm proposed
in this paper is compared and analyzed with ORB-SLAM2
(Mur-Artal and Tardós, 2017) and SMR-SLAM (Cheng et al.,
2019). Figure 13 shows the analysis results of the performance
comparison between the proposed algorithm and ORB-SLAM2
under the conditions of three dynamic data sets fr3/w half,
fr3/w rpy, and fr3/w xyz. Figure 14 shows the analysis results of
the performance comparison between the proposed algorithm
and SMR-SLAM under the conditions of three dynamic data sets
fr3/w half, fr3/w xyz, and fr2/desk with person. The segmentation idea we adopt is that under the target area
frame, the proportion of target pixels is always the larger one, so
we perform a sliding window search according to the depth value
of the depth image to search for the pixel area with the largest
proportion (we divide the depth image pixels into 16. There are
16-pixel areas per copy to ensure that each pixel value from 0
to 255 can be searched). In the augmented reality technology,
the reason for the deviation of the virtual object in the map is
often the calculation error of the posture point. Therefore, we
use two indicators, ATE and RPE, to verify the algorithm in this
paper. At the same time, in order to ensure that the method
can be effectively applied to the augmented reality environment,
we exploit the TUM data set fr3/w xyz combined with the
Augmented Reality Registration algorithm for verification. The
feature collection effect of our method under the TUM data set
fr3/w xyz is shown in Figure 12. The binary image on the left is
the result of dynamic target segmentation, and the image on the
right is the feature points detected by VSLAM. Through the analysis of Figures 13, 14, it can be seen
from the results of absolute trajectory error analysis that
the algorithm proposed in this paper has more significant
advantages in dynamic scenes and still maintains good results
in low-dynamic scenes. To reflect that the algorithm in this paper can maintain
stable and superior performance under different data sets,
we employ the official ATE and RPE test files provided by
TUM to test the fr2 and fr3 series of data sets and obtain
the data results shown in Tables 2–4. 4.2. Analysis of long-term tracking results
based on YOLOv3 and improved Kalman
filter At present, there is no clear data set for dynamic target
detection in a dynamic environment. In order to verify the
robustness of the dynamic target detection algorithm proposed
in this paper, we search for dynamic targets in the Dynamic
Object dataset in the TUM dataset. First, we employ YOLOv3
to set prior knowledge to label dynamic targets artificially. Next, we find the IOU value of the target detected by
YOLOv3 and the result of dynamic target detection. The larger
the experimental result, the more concentrated the detection This experiment utilizes the Dynamic Object dataset to
verify the effectiveness of target tracking, and utilizes the MOT16
dataset to verify the robustness of the multi-target tracking
algorithm used in this paper. This experiment utilizes YOLOv3
to detect pedestrians, and utilizes an improved Kalman filter
algorithm to track the observation results provided by YOLOv3. Frontiers in Neurorobotics 09 frontiersin.org Liu et al. Liu et al. 10.3389/fnbot.2022.990453 FIGURE 11
Operation efect of the tracking algorithm MOT16 in this paper. FIGURE 11
Operation efect of the tracking algorithm MOT16 in this paper. We employ the Hungarian algorithm to find the match between
the previous and next frames in terms of data association. The
effect of running on MOT16 is shown in Figure 11. FIGURE 12
The feature collection efect of our method under the TUM data
set fr3/w xyz. The experimental results show that in a highly dynamic
environment, the detection and tracking algorithm can better
assign weights and find the best prediction results. It assigns
the maximum possible motion trajectory to the target through
cascade matching, avoiding the problem of target loss caused
by occlusion. FIGURE 12
The feature collection efect of our method under the TUM data
set fr3/w xyz. Frontiers in Neurorobotics 4.3. Analysis of experimental results
based on the VSLAM dynamic
environment RMSE is the mean root
mean square error, and SD is the standard deviation, using
ORB-SLAM2 (RGB-D) (Mur-Artal and Tardós, 2017), MR-
SLAM (Sun et al., 2018), and SMR-SLAM (Cheng et al., 2019)
as comparisons. We analyze the results of multiple dynamic data sets in
the TUM data set, and employ the absolute trajectory error
graph ATE to verify the algorithm in this paper. It directly
measures the point difference between the real trajectory and
the estimated trajectory. The longer the red segment, the larger
the estimation error and the lower the positioning accuracy. The
ground truth, the estimated camera motion, and the localization It can be seen from Tables 2–4 that the performance results
of the VSLAM method proposed in this paper on the dynamic 10 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 FIGURE 13
The (top) row is the trajectory error graph of ORB-SLAM2, and the (bottom) row is the trajectory error graph of ours. The ground truth, the
estimated camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. FIGURE 14
The (top) row is the trajectory error graph of SMR, and the (bottom) row is the trajectory error graph of ours. The ground truth, the estimated
camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. data set are much better than ORB-SLAM2. Compared with
the more advanced VSLAM systems, MR-SLAM and SMR-
SLAM, currently proposed, it also has an advantage. Although
the performance on the low-dynamic dataset is slightly inferior
advantage than ORB-SLAM2. This result is consistent with the
results in Figures 13, 14. Our method occupies an absolute
advantage in evaluating rotation drift, which can effectively
avoid errors caused by dynamic target interference in many FIGURE 13
The (top) row is the trajectory error graph of ORB-SLAM2, and the (bottom) row is the trajectory error graph of ours. The ground truth, the
estimated camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. FIGURE 13
The (top) row is the trajectory error graph of ORB-SLAM2, and the (bottom) row is the trajectory error graph of ours. The ground truth, the
estimated camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. Frontiers in Neurorobotics 4.3. Analysis of experimental results
based on the VSLAM dynamic
environment FIGURE 14
The (top) row is the trajectory error graph of SMR, and the (bottom) row is the trajectory error graph of ours. The ground truth, the estimated
camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. FIGURE 14 The (top) row is the trajectory error graph of SMR, and the (bottom) row is the trajectory error graph of ours. The ground truth, the estimated
camera motion, and the localization error for each camera pose are represented as the black, blue, and red segments, respectively. advantage than ORB-SLAM2. This result is consistent with the
results in Figures 13, 14. Our method occupies an absolute
advantage in evaluating rotation drift, which can effectively
avoid errors caused by dynamic target interference in many
applications such as AR. data set are much better than ORB-SLAM2. Compared with
the more advanced VSLAM systems, MR-SLAM and SMR-
SLAM, currently proposed, it also has an advantage. Although
the performance on the low-dynamic dataset is slightly inferior
to that of the SMR-SLAM algorithm, it still maintains a better Frontiers in Neurorobotics 11 frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 TABLE 2 Ate in meters for the experiments using ORB-SLAM2, MR-SLAM, SMR-SLAM, and Ours. TABLE 2 Ate in meters for the experiments using ORB-SLAM2, MR-SLAM, SMR-SLAM, and Ours. Data sets
ORB-SLAM2
MR-SLAM
SMR-SLAM
Ours
RMSE
S.D. RMSE
S.D. RMSE
S.D. RMSE
S.D. w halfsphere
0.2668
0.1429
0.0668
0.0266
0.0352
0.0207
0.0342
0.0206
w xyz
0.2774
0.1230
01230
0.0657
0.0186
0.0098
0.0331
0.0176
w rpy
0.1677
0.0958
0.0729
0.0335
0.0436
0.0253
0.0347
0.0160
w static
0.0250
0.0147
0.0334
0.0207
0.0238
0.0113
0.0142
0.0071
s halfsphere
0.0219
0.0133
0.0664
0.0386
0.0210
0.0127
0.0438
0.0305
s xyz
0.0089
0.0046
0.0514
0.0280
0.0138
0.0076
0.0255
0.0113
desk person
0.0056
0.0030
0.0759
0.0313
0.0068
0.0031
0.0728
0.0207
Bold values mean the best result among the methods. ational drift (RPE) in m/s for the experiments using ORB-SLAM2, MR-SLAM, SMR-SLAM, and Ours. Data sets
ORB-SLAM2
MR-SLAM
SMR-SLAM
Ours
RMSE
S.D. RMSE
S.D. RMSE
S.D. RMSE
S.D. 4.4. Experiments with augmented reality
registration method are shown in Figure 15. The experimental results of our
proposed method are shown in Figure 16. We choose to insert a virtual square in the 20th frame of the
data set fr3/w xyz. From frame 100 to frame 500, we sampled the
result six times. In these six images, there are objects entering,
a single object moving slowly, a single object moving quickly,
multiple objects moving, the lens moving up and down, the
lens moving left and right, and the lens rotating. Figures 15,
16 show the AR implementation effects of the ORB-SLAM2
method and the method in this paper, respectively. It can be seen
from Figure 15 that under the influence of camera motion and Frontiers in Neurorobotics 4.3. Analysis of experimental results
based on the VSLAM dynamic
environment w halfsphere
0.8078
0.4958
0.0611
0.0268
0.0816
0.0419
0.0539
0.0301
w xyz
0.6181
0.3778
0.0668
0.0369
0.0337
0.0162
0.0470
0.0227
w rpy
1.5083
0.9031
0.0968
0.0510
0.0337
0.0162
0.0214
0.0134
w static
0.5436
0.3783
0.0307
0.0205
0.0829
0.0479
0.0276
0.0165
s halfsphere
0.0326
0.0198
0.0547
0.0318
0.0307
0.0183
0.0654
0.0429
s xyz
0.0132
0.0063
0.0357
0.0225
0.0242
0.0106
0.0363
0.0167
desk person
0.0383
0.0228
0.0213
0.0151
0.0369
0.0213
0.0121
0.0646
Bold values mean the best result among the methods. Data sets Data sets
ORB-SLAM2
MR-SLAM
SMR-SLAM
Ours
RMSE
S.D. RMSE
S.D. RMSE
S.D. RMSE
S.D. w halfsphere
17.7267
10.2391
1.9004
0.7629
1.1556
0.5359
1.0076
0.4283
w xyz
10.9428
7.1977
1.5950
0.8236
0.7473
0.4333
0.7427
0.4266
w rpy
28.0287
17.3043
2.5936
1.3210
1.6024
0.9284
1.0777
0.5112
w static
9.9384
6.9106
0.8998
0.6470
1.1366
0.6269
0.4823
0.2975
s halfsphere
0.8217
0.3594
2.2677
1.3861
0.8038
0.3495
1.0254
0.4454
s xyz
0.5775
0.3016
1.0362
0.5304
0.6905
0.3474
0.6601
0.2998
desk person
1.4668
0.6857
0.7744
0.4767
1.3784
0.6742
1.4410
0.6932
Bold values mean the best result among the methods. 4.4.1. Robustness experiments The above experiment is the result analysis of the VSLAM
algorithm we proposed under the dynamic data set. At the same
time, in order to verify the robustness of our proposed method in
the Augmented Reality system, we utilize the fr3/w xyz data set
to test, select the appropriate Kth frame, insert a virtual object,
and observe the dynamic performance of the virtual object
during operation. The experimental results of the ORB-SLAM2 12 Frontiers in Neurorobotics frontiersin.org Liu et al. Liu et al. 10.3389/fnbot.2022.990453 200th, 350th, and 500th frames after initialization, calculate
the response time, and compare with our method through
several classical algorithms such as SURF+KLT, ORB-SLAM2, video portrait motion, ORB-SLAM2 cannot accurately analyze
the plane, and the error situation shown in Figure 15 often
occurs. In terms of long-term attitude tracking, the ORB-SLAM2
method has attitude offset, which will also cause the inserted
virtual object to not be in the original position. It can be seen
from the results in Figure 16 that the VSLAM method proposed
in this paper can accurately fit and create a virtual object, which
greatly improves the registration of augmented reality and the
tracking of virtual objects. TABLE 5 Real-time analysis (ms). Frame
SURF+KLT
VINS-Mono
ORB-SLAM2
Ours
50
42.5
26.3
22.3
23.9
100
44.5
29.5
22.5
23.1
200
49.0
44.9
23.5
24.5
350
50.2
69.9
23.2
24.9
500
50.5
108.6
23.4
24.8
Bold values mean the best result among the methods. Frame
SURF+KLT
VINS-Mono
ORB-SLAM2
Ours 4.4.2. Real-time experiment We
conduct
real-time
comparison
experiments
of
Augmented Reality Registration in a dynamic laboratory
environment. We register virtual objects at the 50th, 100th, FIGURE 15
The application efect of ORB-SLAM2 method in augmented reality experiment. FIGURE 16
The application efect of our method in the augmented reality experiment. FIGURE 15
The application efect of ORB-SLAM2 method in augmented reality experiment. FIGURE 15
The application efect of ORB-SLAM2 method in augmented reality experiment. FIGURE 16
The application efect of our method in the augmented reality experiment. FIGURE 16
The application efect of our method in the augmented reality experiment. 13 13 Frontiers in Neurorobotics frontiersin.org Liu et al. 10.3389/fnbot.2022.990453 FIGURE 17
Application of VINS and our method in augmented reality experiment. FIGURE 17
Application of VINS and our method in augmented reality experiment. small number of dynamic points in the map will significantly
affect the registration effect. The dynamic target detection and
tracking algorithm proposed in this paper can effectively help
the stable operation of the Augmented Reality Registration
technology in a dynamic environment. The stable operation
of YOLOv3 can effectively help eliminate the feature points of
small dynamic targets. Considering that the offset of augmented
reality in the map is always the calculation error of the pose
point is too large, this paper uses the ATE and RPE indicators
to verify the algorithm of this paper. The final result analysis
shows that the algorithm proposed in this paper has an excellent
performance in each target detection stage and long-term
tracking. The results of the ATE and RPE indicators indicate
that the algorithm proposed in this paper performs well in both
small and large dynamic scenarios and can be well applied in
augmented reality technology. When we integrated the object
detection method into the SLAM system, we did not choose
the more efficient YOLOv4 due to the problem of computing
power. Therefore, we use the prior data provided by GMM
to compensate for the accuracy problem, which can use less
computing power while maintaining the accuracy and real-
time required for Augmented Reality Registration. There are
better solutions now, like YOLOv5 and the recently released
YOLOv7, and we’re working on it. And, we need to optimize the
computational cost in the next work so that Augmented Reality
Registration requires less computational power and has better
real-time performance. and VINS-Mono (Mur-Artal and Tardós, 2017). The data
are shown in Table 5. 4.4.3. Comparison experiment with VISLAM VISLAM is the most commonly used registration method
for AR today. Although, the use of IMU provides good
assistance for camera pose, it does not perform so well when
tracking for long periods of time in dynamic environments. As shown in the VINS-Mono data in Table 5, after 350
frames, it shows a great drift and the registration time is
also much longer. In the laboratory dynamic environment,
we carried out many experiment of dragging the chair
to move. After initialization, insert a virtual object, and
verify by dragging the chair to move together. We select
one of the experimental results for comparison, as shown
in Figure 17. It can be seen from the results that the
virtual objects registered by the VINS-Mono method are not
very robust in dynamic environments. However, the virtual
objects registered by our method remain stable in long-term
dynamic environments. 4.4.2. Real-time experiment The experimental results show that
the registration real-time performance of our method is
better than the traditional SURF+KLT method at different
time stages. Although, the computational cost of detection
causes our method to consume slightly more time than the
ORB-SLAM2 method for Augmented Reality Registration, this
method provides better robustness while the registration latency
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Environments for Measurement Systems and Applications (CIVEMSA) (Tunis). doi: 10.1109/CIVEMSA48639.2020.9132969 Gehrmann, S., Strobelt, H., Krüger, R., Pfister, H., and Rush, A. M. (2019). Visual
interaction with deep learning models through collaborative semantic inference. IEEE Trans. Vis. Comput. Graph. 26, 884–894. doi: 10.1109/TVCG.2019.2934595 Campos, C., Elvira, R., Rodríguez, J. J. G., Montiel, J. M., and Tardós,
J. D. (2021). ORB-SLAM3:
an
accurate
open-source
library
for
visual,
visual-inertial,
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multimap
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Trans. Robot. 37,
1874–1890. doi: 10.1109/TRO.2021.3075644 Goan,
E.,
and
Fookes,
C. (2020). 5. Conclusion The original contributions presented in the study are
included in the article/supplementary material, further inquiries
can be directed to the corresponding author/s. In recent years, augmented reality technology is prevalent,
and it is often applied in small map scenarios. Therefore, a Frontiers in Neurorobotics 14 frontiersin.org Liu et al. Liu et al. 10.3389/fnbot.2022.990453 Author contributions under Grant JSSCBS202030576, and the Natural Science
Research Project of Jiangsu Higher Education Institutions
under Grant 20KJB520029. JL and DC provided research ideas and plans. JL and QG
improved the algorithm. QG and DY wrote the programs and
conducted the experiments. DC and QG were responsible for
collecting data. QG wrote the manuscript with the help of JL
and DC. DC revised the manuscript and approved the final
submission. All authors contributed to the article and approved
the submitted version. Publisher’s note This work was partially supported by the Key R&D
Program
of
Jiangsu
Province
(Industry
Prospects
and
Key Core Technologies) under Grant BE2020006-2, the
National Natural Science Foundation of China under Grants
61773219 and 62003169, the Natural Science Foundation
of Jiangsu Province under Grant BK20200823, the Jiangsu
Innovation and Entrepreneurship Talent Program Project All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. Conflict of interest The authors declare that the research was conducted in the
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Generating Factoid Questions With Recurrent Neural Networks: The 30M Factoid Question-Answer Corpus
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* First authors.
◦Email: {iulian.vlad.serban,caglar.gulcehre,
sungjin.ahn,sarath.chandar.anbil.parthipan,
aaron.courville,yoshua.bengio}@umontreal.ca
⋄Email: alberto.garcia-duran@utc.fr
† CIFAR Senior Fellow Generating Factoid Questions With Recurrent Neural Networks:
The 30M Factoid Question-Answer Corpus Generating Factoid Questions With Recurrent Neural Networks:
The 30M Factoid Question-Answer Corpus Iulian Vlad Serban∗◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada Alberto Garc´ıa-Dur´an∗⋄
Universit´e de Technologie de Compi`egne CNRS
Rue du Dr Schweitzer,
Compigne, France Sungjin Ahn◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada Sarath Chandar◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada Caglar Gulcehre◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada Aaron Courville◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada Yoshua Bengio†◦
University of Montreal
2920 chemin de la Tour,
Montr´eal, QC, Canada 1Freebase is now a part of WikiData. Proceedings of the 54th Annual Meeting of the Association for Computational Linguistics, pages 588–598,
Berlin, Germany, August 7-12, 2016. c⃝2016 Association for Computational Linguistics Abstract A major obstacle for training question-answering
(QA) systems has been due to the lack of labeled
data. The question answering field has focused
on building QA systems based on traditional in-
formation retrieval procedures (Lopez et al., 2011;
Dumais et al., 2002; Voorhees and Tice, 2000). More recently, researchers have started to utilize
large-scale knowledge bases (KBs) (Lopez et al.,
2011), such as Freebase (Bollacker et al., 2008),
WikiData (Vrandeˇci´c and Kr¨otzsch, 2014) and
Cyc (Lenat and Guha, 1989).1 Bootstrapping QA
systems with such structured knowledge is clearly
beneficial, but it is unlikely alone to overcome the
lack of labeled data. To take into account the rich
and complex nature of human language, such as
paraphrases and ambiguity, it would appear that
labeled question and answer pairs are necessary. The need for such labeled pairs is even more criti-
cal for training neural network-based QA systems,
where researchers until now have relied mainly on
hand-crafted rules and heuristics to synthesize ar-
tificial QA corpora (Bordes et al., 2014; Bordes et
al., 2015). Over the past decade, large-scale super-
vised learning corpora have enabled ma-
chine learning researchers to make sub-
stantial advances. However,
to this
date, there are no large-scale question-
answer corpora available. In this paper
we present the 30M Factoid Question-
Answer Corpus, an enormous question-
answer pair corpus produced by apply-
ing a novel neural network architecture
on the knowledge base Freebase to trans-
duce facts into natural language ques-
tions. The produced question-answer pairs
are evaluated both by human evaluators
and using automatic evaluation metrics,
including well-established machine trans-
lation and sentence similarity metrics. Across all evaluation criteria the question-
generation model outperforms the compet-
ing template-based baseline. Furthermore,
when presented to human evaluators, the
generated questions appear to be compa-
rable in quality to real human-generated
questions. Motivated by these recent developments, in this
paper we focus on generating questions based on
the Freebase KB. We frame question generation as
a transduction problem starting from a Freebase
fact, represented by a triple consisting of a sub-
ject, a relationship and an object, which is trans- 588 588 duced into a question about the subject, where
the object is the correct answer (Bordes et al.,
2015). We propose several models, largely in-
spired by recent neural machine translation mod-
els (Cho et al., 2014; Sutskever et al., 2014; Bah-
danau et al., 2015), and we use an approach sim-
ilar to Luong et al. (2015) for dealing with the
problem of rare-words. We evaluate the produced
questions in a human-based experiment as well as
with respect to automatic evaluation metrics, in-
cluding the well-established machine translation
metrics BLEU and METEOR and a sentence simi-
larity metric. We find that the question-generation
model outperforms the competing template-based
baseline, and, when presented to untrained human
evaluators, the produced questions appear to be in-
distinguishable from real human-generated ques-
tions. This suggests that the produced question-
answer pairs are of high quality and therefore that
they will be useful for training QA systems. Fi-
nally, we use the best performing model to con-
struct a new factoid question-answer corpus – The
30M Factoid Question-Answer Corpus – which is
made freely available to the research community.2 following the analysis of Graesser et al. (1992),
who classify questions according to a taxonomy
consisting of 18 categories. The works discussed so far propose ways to
map unstructured text to questions. This implies
a two-step process: first, transform a text into a
symbolic representation (e.g. a syntactic represen-
tation of the sentence), and second, transform the
symbolic representation of the text into the ques-
tion (Yao et al., 2012). On the other hand, go-
ing from a symbolic representation (structured in-
formation) to a question, as we will describe in
the next section, only involves the second step. Closer to our approach is the work by Olney et
al. (2012). They take triples as input, where the
edge relation defines the question template and the
head of the triple replaces the placeholder token in
the selected question template. In the same spirit,
Duma et al. (2013) generate short descriptions
from triples by using templates defined by the rela-
tionship and replacing accordingly the placeholder
tokens for the subject and object. Our baseline is similar to that of Olney et al. (2012), where a set of relationship-specific tem-
plates are defined. These templates include place-
holders to replace the string of the subject. 588 The
main difference with respect to their work is that
our baseline does not explicitly define these tem-
plates. Instead, each relationship has as many
templates as there are different ways of framing a
question with that relationship in the training set. This yields more diverse and semantically richer
questions by effectively taking advantage of the
fact-question pairs, which Olney et al. did not have
access to in their experiments. 2www.agarciaduran.org 4It is not necessary for the object to be the only answer,
but it is required to be one of the possible answers. 2
Related Work Question generation has attracted interest in recent
years with notable work by Rus et al. (2010), fol-
lowed by the increasing interest from the Natural
Language Generation (NLG) community. A sim-
ple rule-based approach was proposed in different
studies as wh-fronting or wh-inversion (Kalady et
al., 2010; Ali et al., 2010). This comes at the
disadvantage of not making use of the semantic
content of words apart from their syntactic role. The problem of determining the question type (e.g. that a Where-question should be triggered for loca-
tions), which requires knowledge of the category
type of the elements involved in the sentence, has
been addressed in two different ways: by using
named entity recognizers (Mannem et al., 2010;
Yao and Zhang, 2010) or semantic role labelers
(Chen et al., 2009). In Curto et al. (2012) ques-
tions are split into classes according to their syn-
tactic structure, prefix of the question and the cat-
egory of the answer, and then a pattern is learned
to generate questions for that class of questions. After the identification of key points, Chen et
al. (2009) apply handcrafted-templates to generate
questions framed in the right target expression by Unlike the work by Berant and Liang (2014),
which addresses the problem of deterministically
generating a set of candidate logical forms with a
canonical realization in natural language for each,
our work addresses the inverse problem: given a
logical form (fact) it outputs the associated ques-
tion. It should also be noted that recent work in ques-
tion answering have used simpler rule-based and
template-based approaches to generate synthetic
questions to address the lack of question-answer
pairs to train their models (Bordes et al., 2014;
Bordes et al., 2015). 589 Questions
Entities
Relationships
Words
108,442
131,684
1,837
∼77k
Table 1: Statistics of SimpleQuestions 3.2
Transducing Facts to Questions We aim to transduce a fact into a question, such
that: 1. The question is concerned with the subject
and relationship of the fact, and 4
Model 2. The object of the fact represents a valid an-
swer to the generated question. We propose to attack the problem with the models
inspired by the recent success of neural machine
translation models (Sutskever et al., 2014; Bah-
danau et al., 2015). Intuitively, one can think of
the transduction task as a “lossy translation” from
structured knowledge (facts) to human language
(questions in natural language), where certain as-
pects of the structured knowledge is intentionally
left out (e.g. the name of the object). These models
typically consist of two components: an encoder,
which encodes the source phrase into one or sev-
eral fixed-size vectors, and a decoder, which de-
codes the target phrase based on the results of the
encoder. We model this in a probabilistic framework as a
directed graphical model: P(Q|F) =
N
Y
n=1
P(wn|w<n, F),
(1) (1) where F = (subject, relationship, object) rep-
resents the fact, Q = (w1, . . . , wN) represents the
question as a sequence of tokens w1, . . . , wN, and
w<n represents all the tokens generated before to-
ken wn. In particular, wN represents the question
mark symbol ’?’. 3.1
Knowledge Bases In general, a KB can be viewed as a multi-
relational graph, which consists of a set of
nodes (entities) and a set of edges (relation-
ships) linking nodes together. In Freebase (Bol-
lacker et al., 2008) these relationships are di-
rected and always connect exactly two enti-
ties. For example, in Freebase the two enti-
ties fires creek and nantahala national forest are
linked together by the relationship contained by. Since the triple {fires creek, contained by, nan-
tahala national forest} represents a complete and
self-contained piece of information, it is also
called a fact where fires creek is the subject (head
of the edge), contained by is the relationship and
nantahala national forest is the object (tail of the
edge). at a time and they were asked to phrase a ques-
tion such that the object of the presented fact be-
comes the answer of the question.4 Consequently,
both the subject and the relationship are explic-
itly given in each question. But indirectly char-
acteristics of the object may also be given since
the humans have an access to it as well. Often
when phrasing a question the annotators tend to be
more informative about the target object by giving
specific information about it in the question pro-
duced. For example, in the question What city is
the American actress X from? the city name given
in the object informs the human participant that it
was in America - information, which was not pro-
vided by either the subject or relationship of the
fact. We have also observed that the questions are
often ambiguous: that is, one can easily come up
with several possible answers that may fit the spec-
ifications of the question. Table 1 shows statistics
of the dataset. 3www.mturk.com 4.1
Encoder We use the SimpleQuestions dataset (Bordes et al.,
2015) in order to train our models. This is by far
the largest dataset of question-answer pairs created
by humans based on a KB. It contains over 100K
question-answer pairs created by users on Amazon
Mechanical Turk3 in English based on the Free-
base KB. In order to create the questions, human
participants were shown one whole Freebase fact In contrast to the neural machine translation
framework, our source language is not a proper
language but instead a sequence of three vari-
ables making up a fact. We propose an encoder
sub-model, which encodes each atom of the fact
into an embedding. Each atom {s, r, o}, may 590 stand for subject, relationship and object, respec-
tively, of a fact F = (s, r, o) is represented as
a 1-of-K vector xatom, whose embedding is ob-
tained as eatom = Einxatom, where Ein ∈RDEnc×K
is the embedding matrix of the input vocabulary
and K is the size of that vocabulary. The en-
coder transforms this embedding into Enc(F)atom
∈RHDec as Enc(F)atom = WEnceatom, where WEnc
∈RHDec×DEnc. This embedding matrix, Ein, could be another
parameter of the model to be learned, however, as
discussed later (see Section 4.3), we have learned
it separately and beforehand with TransE (Bordes
et al., 2013), a model aimed at modeling this kind
of multi-relational data. We fix it and do not allow
the encoder to tune it during training. We call fact embedding Enc(F) ∈R3HDec the
concatenation [Enc(F)s, Enc(F)r, Enc(F)o] of
the atom embeddings, which is the input for the
next module. Figure 1:
The computational graph of the
question-generation model, where Enc(F) is the
fact embedding produced by the encoder model,
and c(F, hn−1) for n = 1, . . . , N is the fact rep-
resentation weighed according to the attention-
mechanism, which depends on both the fact F
and the previous hidden state of the decoder RNN
hn−1 . For the sake of simplicity, the attention-
mechanism is not shown explicitly. 4.2
Decoder For the decoder, we use a GRU recurrent neu-
ral network (RNN) (Cho et al., 2014) with an
attention-mechanism (Bahdanau et al., 2015) on
the encoder representation to generate the associ-
ated question Q to that fact F. Recently, it has
been shown that the GRU RNN performs equally
well across a range of tasks compared to other
RNN architectures, such as the LSTM RNN (Gr-
eff et al., 2015). The hidden state of the decoder
RNN is computed at each time step n as: rameters of the GRU and c(F, hn−1) is the con-
text vector (defined below Eq. 6). The vector gr
is called the reset gate, gu as the update gate
and ˜h the candidate activation. By adjusting gr
and gu appropriately, the model is able to cre-
ate linear skip-connections between distant hid-
den states, which in turn makes the credit as-
signment problem easier and the gradient signal
stronger to earlier hidden states. Then, at each
time step n the set of probabilities over word to-
kens is given by applying a softmax layer over
Votanh(Vhhn + VwEoutwn−1 + Vcc(F, hn−1)),
where Vo ∈RV ×HDec, Vh, Vc ∈RHDec×HDec and
Vw ∈RHDec×DDec. Lastly, the function c(F, hn−1)
is computed using an attention-mechanism: gr
n = σ(WrEoutwn−1 + Crc(F, hn−1) + Urhn−1)
(2)
gu
n = σ(WuEoutwn−1 + Cuc(F, hn−1) + Uuhn−1)
(3)
˜h = tanh(WEoutwn−1 + Cc(F, hn−1)
(4)
+ U(gr
n ◦hn−1))
hn = gu
n ◦hn−1 + (1 −gu
n) ◦˜h,
(5) (5) where σ is the sigmoid function, s.t. σ(x) ∈[0, 1],
and the circle, ◦, represents element-wise mul-
tiplication. The initial state h0 of this RNN is
given by the output of a feedforward neural net-
work fed with the fact embedding. The product
Eoutwn ∈RDDec is the decoder embedding vec-
tor corresponding to the word wn (coded as a 1-
of-V vector, with V being the size of the output
vocabulary), the variables Ur, Uu, U, Cr, Cu, C ∈
RHDec×HDec, Wr, Wu, W ∈RHDec×DDec are the pa- where σ is the sigmoid function, s.t. σ(x) ∈[0, 1], where σ is the sigmoid function, s.t. σ(x) ∈[0, 1],
and the circle, ◦, represents element-wise mul-
tiplication. The initial state h0 of this RNN is
given by the output of a feedforward neural net-
work fed with the fact embedding. 4.3
Modeling the Source Language A particular problem with the model presented
above is related to the embeddings for the enti-
ties, relationships and tokens, which all have to
be learned in one way or another. If we learn
these naively on the SimpleQuestions training set,
the model will perform poorly when it encoun-
ters previously unseen entities, relationships or to-
kens. Furthermore, the multi-relational graph de-
fined by the facts in SimpleQuestions is extremely
sparse, i.e. each node has very few edges to other
nodes, as can be expected due to high ratio of
unique entities over number of examples. There-
fore, even for many of the entities in SimpleQues-
tions, the model may perform poorly if the embed-
ding is learned solely based on the SimpleQues-
tions dataset alone. We need a coarse representation for the entities
contained in SimpleQuestions, capturing the ba-
sic information, like the profession or nationality,
the annotators tend to use when phrasing the ques-
tions, and accordingly we have ensured the em-
beddings contain this information by taking triples
coming from the Freebase graph5 regarding: 1. Category
information:
given
by
the
type/instance
relationship,
this
ensures
that all the entities of the same semantic
category are close to each other. Although
one might think that the expected category of
the subject/object could be inferred directly
from the relationship, there are fine-grained
differences in the expected types that be
extracted only directly by observing this
category information. On the source side, we can resolve this is-
sue by initializing the subject, relationship and
object embeddings to those learned by apply-
ing multi-relational embedding-based models to
the knowledge base. Multi-relational embedding-
based models (Bordes et al., 2011) have recently
become popular to learn distributed vector embed-
dings for knowledge bases, and have shown to
scale well and yield good performance. Due to
its simplicity and good performance, we choose
to use TransE (Bordes et al., 2013) to learn such
embeddings. TransE is a translation-based model,
whose energy function is trained to output low val-
ues when the fact expresses true information, i.e. a
fact which exists in the knowledge base, and other-
wise high values. Formally, the energy function is
defined as f(s, r, o) = ||es + er −eo||2, where es,
er and eo are the real-valued embedding vectors
for the subject, relationship and object of a fact. Further details are given by Bordes et al. (2013). 2. 5Extracted from one of the latest Freebase dumps (down-
loaded in mid-August 2015) https://developers.
google.com/freebase/data 4.2
Decoder The product
Eoutwn ∈RDDec is the decoder embedding vec-
tor corresponding to the word wn (coded as a 1-
of-V vector, with V being the size of the output
vocabulary), the variables Ur, Uu, U, Cr, Cu, C ∈
RHDec×HDec, Wr, Wu, W ∈RHDec×DDec are the pa- is computed using an attention-mechanism: c(F, hn−1) = αs,n−1Enc(F)s + αr,n−1Enc(F)r
+ αo,n−1Enc(F)o,
(6) (6) where
αs,n−1, αr,n−1, αr,n−1
are
real-valued
scalars, which weigh the contribution of the
subject, relationship and object representations. 591 Embeddings for entities with few connections
are easy to learn, yet the quality of these embed-
dings depends on how inter-connected they are. In
the extreme case where the subject and object of a
triple only appears once in the dataset, the learned
embeddings of the subject and object will be se-
mantically meaningless. This happens very often
in SimpleQuestions, since only around 5% of the
entities have more than 2 connections in the graph. Thus, by applying TransE directly over this set of
triples, we would eventually end up with a lay-
out of entities that does not contain clusters of se-
mantically close concepts. In order to guarantee
an effective semantic representation of the embed-
dings, we have to learn them together with addi-
tional triples extracted from the whole Freebase
graph to complement the SimpleQuestions graph
with relevant information for this task. They correspond to the attention of the model,
and are computed by applying a one-layer neural
network with tanh-activation function on the
encoder representations of the fact, Enc(F), and
the previous hidden state of the RNN, hn−1,
followed by the sigmoid function to restrict the
attention values to be between zero and one. The
need for the attention-mechanism is motivated by
the intuition that the model needs to attend to the
subject only once during the generation process
while attending to the relationship at all other
times during the generation process. The model is
illustrated in Figure 1. 4.3
Modeling the Source Language Geographical
information:
sometimes
the annotators have included information
about
nationality
(e.g. Which
French
president. . . ?)
or location (e.g. Where
in Germany. . . ?)
of the subject and/or
object. This information is given by
the
relationships
person/nationality
and
location/contained by. By including these
facts in the learning, we ensure the existence
of a fine-grained layout of the embeddings
regarding this information within a same
category. 592 Closest neighbors to
Warner Bros. Entertainment
Manchester
hindi language
SQ
Billy Gibbons
Ricky Anane
nepali indian
Jenny Lewis
Lee Dixon
Naseeb
Lies of Love
Jerri Bryne
Ghar Ek Mandir
Swordfish
Greg Wood
standard chinese
SQ + FB
Paramount Pictures
Oxford
dutch language
Sony Pictures Entertainment
Sale
italian language
Electronic Arts
Liverpool
danish language
CBS
Guildford
bengali language
Table 2: Examples of differences in the local structure of the vector space embeddings when adding more
FB facts Table 2: Examples of differences in the local structure of the vector space embeddings when adding more
FB facts Table 2: Examples of differences in the local structure of the vector space embeddings when adding more
FB facts 3. Gender:
similarly, sometimes annotators
have included information about gender (e.g. Which male audio engineer. . . ?). This in-
formation is given by the relationship per-
son/gender. 3. Gender:
similarly, sometimes annotators
have included information about gender (e.g. Which male audio engineer. . . ?). This in-
formation is given by the relationship per-
son/gender. cause then the entities and relationships in the in-
put would not be able to transmit semantic (e.g. topical) information to the decoder. If we had in-
cluded placeholder tokens in the input language,
the model would not be able to generate informa-
tive words regarding the subject in the question
(e.g. it would be impossible for the model to learn
that the subject Paris may be accompanied by the
words French city when generating a question, be-
cause it would not see Paris but only a placeholder
token). To this end, we have included more than
300, 000 facts from Freebase in addition to the
facts in SimpleQuestions for training. Table 2
shows the differences in the embeddings before
and after adding additional facts for training the
TransE representations. A single placeholder token for all question types
could unnecessarily limit the model. 4.4
Generating Questions To resolve the problem of data sparsity and previ-
ously unseen words on the target side, we draw in-
spiration from the placeholders proposed for han-
dling rare words in neural machine translation by
Luong et al. (2015). For every question and an-
swer pair, we search for words in the question
which overlap with words in the subject string of
the fact.6 We heuristically estimate the sequence
of most likely words in the question, which cor-
respond to the subject string. These words are
then replaced by the placeholder token <place-
holder>. For example, given the fact {fires creek,
contained by, nantahala national forest} the orig-
inal question Which forest is Fires Creek in? is transformed into the question Which forest is
<placeholder>in?. The model is trained on these
modified questions, which means that model only
has to learn decoder embeddings for tokens which
are not in the subject string. At test time, after
outputting a question, all placeholder tokens are
replaced by the subject string and then the outputs
are evaluated. We call this the Single-Placeholder
(SP) model. The main difference with respect
to that of Luong et al. (2015) is that we do not
use placeholder tokens in the input language, be- 6We use the tool difflib: https://docs.python.
org/2/library/difflib.html. 4.3
Modeling the Source Language We there-
fore also experiment with another model, called
the Multi-Placeholder (MP) model, which uses 60
different placeholder tokens such that the place-
holder for a given question is chosen based on
the subject category extracted from the relation-
ship (e.g. contained by is classified in the category
location, and so the transformed question would
be Which forest is <location placeholder> in?). This could make it easier for the model to learn
to phrase questions about a diverse set of entities,
but it also introduces additional parameters, since
there are now 60 placeholder embeddings to be
learned, and therefore the model may suffer from
overfitting. This way of addressing the sparsity in
the output reduces the vocabulary size to less than
7000 words. 5.1
Training Procedure All neural network models were implemented in
Theano (Theano Development Team, 2016). To
train the neural network models, we optimized the
log-likelihood using the first-order gradient-based
optimization algorithm Adam (Kingma and Ba,
2015). To decide when to stop training we used
early stopping with patience (Bengio, 2012) on
the METEOR score obtained for the validation set. In all experiments, we use the default split of the
SimpleQuestions dataset into training, validation
and test sets. The results are shown in Table 3. Exam-
ple questions produced by the model with mul-
tiple placeholders are shown in Table 4. The
neural network models outperform the template-
based baseline by a clear margin across all met-
rics. The template-based baseline is already a rel-
atively strong model, because it makes use of a
separate template for each relationship. Qualita-
tively the neural networks outperform the base-
line model in cases where they are able to levage
additional knowledge about the entities (see first,
third and fifth example in Table 4). On the other
hand, for rare relationships the baseline model ap-
pears to perform better, because it is able to pro-
duce a reasonable question if only a single exam-
ple with the same relationship exists in the train-
ing set (see eighth example in Table 4). Given
enough training data this suggests that neural net-
works are generally better at the question genera-
tion task compared to hand-crafted template-based
procedures, and therefore that they may be useful
for generating question answering corpora. Fur-
thermore, it appears that the best performing mod-
els are the models where TransE are trained on the
largest set of triples (TransE++). This set con-
tains, apart from the supporting triples described
in Section 4.3, triples involving entities which are
highly connected to the entities found in the Sim-
pleQuestions facts. In total, around 30 millions of
facts, which have been used to generate the 30M
Factoid Question-Answer Corpus. Lastly, it is not
clear whether the model with a single placeholder
or the model with multiple placeholders performs
best. This motivates the following human study. We trained TransE embeddings with embedding
dimensionality 200 for each subject, relationship
and object. Based on preliminary experiments, for
all neural network models we fixed the learning
rate to 0.00025 and clipped parameter gradients
with norms larger than 0.1. 7We use the Word2Vec embeddings pretrained on the
Google News Corpus: https://code.google.com/
p/word2vec/. 4.5
Template-based Baseline To compare our neural network models, we pro-
pose a (non-parametric) template-based baseline
model, which makes use of the entire training set
when generating a question. The baseline oper-
ates on questions modified with the placeholder as
in the preceding section. Given a fact F as in-
put, the baseline picks a candidate fact Fc in the
training set at uniformly random, where Fc has the
same relationship as F. Then the baseline consid-
ers the questions corresponding to Fc and as in the 593 SP model, in the final step the placeholder token
in the question is replaced by the subject string of
the fact F. Although we believe that METEOR and BLEU
are reasonable evaluation metrics, they may have
not recognize certain paraphrases, in particular
paraphrases of entities. We therefore also make
use of a sentence similarity metric, as proposed
by Rus and Lintean (2012), which we will denote
Embedding Greedy (Emb. Greedy). The metric
makes use of a word similarity score, which in
our experiments is the cosine similarity between
two Word2Vec word embeddings (Mikolov et al.,
2013).7 The metric finds a (non-exclusive) align-
ment between words in the two questions, which
maximizes the similarity between aligned words,
and computes the sentence similarity as the mean
over the word similarities between aligned words. 5.2
Evaluation To investigate the performance of our models, we
make use of both automatic evaluation metrics and
human evaluators. 5.1
Training Procedure We further fixed the
embedding dimensionality of words to be 200, and
the hidden state of the decoder RNN to have di-
mensionality 600. 5.2.1
Automatic Evaluation Metrics BLEU
(Papineni
et
al.,
2002)
and
ME-
TEOR (Banerjee and Lavie,
2005) are two
widely used evaluation metrics in statistical
machine translation and automatic image-caption
generation (Chen et al., 2015). Similar to sta-
tistical machine translation, where a phrase in
the source language is mapped to a phrase in the
target language, in this task a KB fact is mapped to
a natural language question. Both tasks are highly
constrained, e.g. the set of valid outputs is limited. This is true in particular for short phrases, such
as one sentence questions. Furthermore, in both
tasks, the majority of valid outputs are paraphrases
of each other, which BLEU and METEOR have
been designed to capture. We therefore believe
that BLEU and METEOR constitute reasonable
performance metrics for evaluating the generated
questions. 594 Model
BLEU
METEOR
Emb. Greedy
Baseline
31.36
33.12
74.02
SP Triples
33.27
35.07
76.72
MP Triples
32.76
34.97
76.70
SP Triples TransE++
33.32
35.38
76.78
MP Triples TransE++
33.28
35.29
77.01 Table 3: Test performance for all models w.r.t. BLEU, METEOR and Emb. Greedy performance met-
rics, where SP indicates models with a single placeholder and MP models with multiple placeholders. TransE++ indicates models where the TransE embeddings have been pretrained on a larger set of triples. The best performance on each metric is marked in bold font. Fact
Human
Baseline
MP Triples TransE++
bayuvi dupki
– contained by –
europe
where is bayuvi dupki? what state is the city
of bayuvi dupki located
in? what continent is bayuvi
dupki in? illinois
– contains –
ludlow township
what is in illinois? what
is
a
tributary
found in illinois? what is the name of a place
within illinois? neo contra
– publisher –
konami
who published
neo contra? which company pub-
lished the game neo
contra? who is the publisher for the
computer videogame neo
contra? fumihiko maki
– structures designed –
makuhari messe
fumihiko
maki
de-
signed what structure? what park did fumihiko
maki help design? what’s a structure designed
by fumihiko maki? cheryl hickey
– profession –
actor
what is cheryl hickey’s
profession? what is cheryl hickey? what is cheryl hickey’s pro-
fession in the entertainment
industry? cherry
– drugs with this flavor –
tussin expectorant for adults
100 syrup
name a cherry flavored
drug? what is a cherry fla-
vored drug? what’s a drug that cherry
shaped like? 5.2.1
Automatic Evaluation Metrics pop music
– artists –
nikki flores
what artist is known for
pop music? An example of pop music is
what artist? who’s
an
american
singer that plays pop
music? Table 4: Test examples and corresponding questions. Table 4: Test examples and corresponding questions. 8The probabilities for the “comparable” class in Table 5
can be computed in each row as 100 minus the third and
fourth column in the table. 6
Conclusion We propose new neural network models for map-
ping knowledge base facts into corresponding nat-
ural language questions. The neural networks
combine ideas from recent neural network ar-
chitectures for statistical machine translation, as
well as multi-relational knowledge base embed-
dings for overcoming sparsity issues and place-
holder techniques for handling rare words. The
produced question and answer pairs are evalu-
ated using automatic evaluation metrics, includ-
ing BLEU, METEOR and sentence similarity, and
are found to outperform a template-based base-
line model. When evaluated by untrained human
subjects, the question and answer pairs produced
by our best performing neural network appears to
be comparable in quality to real human-generated
questions. Finally, we use our best performing
neural network model to generate a corpus of 30M
question and answer pairs, which we hope will en-
able future researchers to improve their question
answering systems. The preference of each example is defined as
the question which is preferred by the majority of
the evaluators. Examples where neither of the two
questions are preferred by the majority of the eval-
uators, i.e. when there is an equal number of eval-
uators who prefer each question, are assigned to a
separate preference class called “comparable”.8 The results are shown in Table 5. In total,
3, 810 preferences were recorded by 63 indepen-
dent human evaluators. The questions produced
by each model model pair were evaluated in 5
batches (HITs). Each human evaluated 44-75 ex-
amples (facts and corresponding question pairs) in
each batch and each example was evaluated by
3-5 evaluators. In agreement with the automatic
evaluation metrics, the human evaluators strongly
prefer either the human or the neural network
model over the template-based baseline. Further-
more, it appears that humans cannot distinguish
between the human-generated questions and the
neural network questions, on average showing a
preference towards the later over the former ones. We hypothesize this is because our model penal-
izes uncommon and unnatural ways to frame ques-
tionsand sometimes, includes specific information
about the target object that the humans do not (see
last example in Table 4). This confirms our earlier 5.2.2
Human Evaluation Study An asterisk next to the preferred
model indicates a statistically significance likelihood-ratio test, which shows that the model is preferred
in at least half of the presented examples with 95% confidence. The name MP Triples TransE++ indi-
cates the model with multiple placeholders and TransE embeddings pretrained on a larger set of triples. The last column shows the Fleiss’ kappa averaged across batches (HITs) with different evaluators and
questions. assertion, that the neural network questions can be
used for building question answering systems. responding pair of questions one by one. To avoid
presentation bias, we randomly shuffle the order
of the examples and the order in which questions
are shown by each model. During each experi-
ment, we also show four check facts and corre-
sponding check questions at random, which any
attentive human annotator should be able to an-
swer easily. We discard responses of human eval-
uators who fail any of these four checks. 5.2.2
Human Evaluation Study though less so compared to the human generated
questions. In the next experiment, we therefore
measure the holistic quality of the questions. We carry out pairwise preference experiments on
Amazon Mechanical Turk. We setup experiments comparing:
Human-
Baseline (human and baseline questions), Human-
MP (human and MP Triples TransE++ ques-
tions) and Baseline-MP (baseline and MP Triples
TransE++ questions). We show human evaluators
a fact along with two questions, one question from
each model for the corresponding fact, and ask the
them to choose the question which is most relevant
to the fact and most natural. The human evaluator
also has the option of not choosing either question. This is important if both questions are equally
good or if neither of the questions make sense. At
the beginning of each experiment, we show the hu-
man evaluators two examples of statements and a
corresponding pair of questions, where we briefly
explain the form of the statements and how ques-
tions relate to those statements. Following the in-
troductory examples, we present the facts and cor- Initially, we considered carrying out separate
experiments for measuring relevancy and fluency
respectively, since this is common practice in ma-
chine translation. However, the relevancy of a
question is determined solely by a single factor,
i.e. the relationship, since by construction the sub-
ject is always in the question. Measuring rel-
evancy is therefore not very useful in our task. To verify this we carried out an internal pairwise
preference experiment with human subjects, who
were repeatedly shown a fact and two questions
and asked to select the most relevant question. We found that 93% of the questions generated
by the MP Triples TransE++ model were either
judged better or at least as good as the human gen-
erated questions w.r.t. relevancy. The remaining
7% questions of the MP Triples TransE++ model
questions were also judged relevant questions, al- 595 Model A
Model B
Model A Preference (%)
Model B Preference (%)
Fleiss’ kappa
Human
Baseline
∗56.329 ± 5.469
34.177 ± 5.230
0.242
Baseline
MP Triples TransE++
32.484 ± 5.180
∗60.828 ± 5.399
0.234
Human
MP Triples TransE++
38.652 ± 5.684
51.418 ± 5.833
0.182 Table 5: Pairwise human evaluation preferences computed across evaluators with 95% confidence inter-
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Traffic injury as a medical and sanitary consequence of an emergency of man-made nature in Ukraine. Report three: analysis and characterization of victims depending on the sign of participation in the traffic
|
Medicina neotložnyh sostoânij
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| 8,573
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tation: Emergency Medicine (Ukraine). 2024;20(1):35-42. doi: 10.22141/2224-0586.20.1.2024.1656 Abstract. Background. Road traffic injury remains a significant medical and social problem for all humanity at
any time. The aim of the research: to determine the characteristics of the victims of traffic accidents depending on
the sign of their participation in traffic and living conditions. Materials and methods. This article is a continuation of
previous publications in the journal “Emergency medicine (Ukraine)”, that is, this component of the comprehensive
study was conducted using a single factual material. The actual material of the study is 1,696 cases of traffic injuries,
which were selected by the method of irreversible randomization. Model 1 of the real-life type was chosen to be
a metropolis with a volume of actual material of 1,139 victims with traffic injuries, model 2 (rural area) — 315
cases, model 3 (regional city) — 242 cases. The study of the actual material was carried out using the methods of
parametric and non-parametric statistics, the law of formal logic and fractal analysis. Results. The study found that
the properties of the research models have a significant impact on the occurrence of injuries depending on the sign of
participation in traffic. The largest specific weight of drivers was recorded in the metropolis (40.65 %), the smallest —
in rural areas (16.19 %). The specific weight of passengers was the highest in the regional city (36.36 %), rural areas
(23.49 %), and the lowest in the metropolis (22.43 %). The highest specific weight of pedestrians was observed in
rural areas (60.32 %), in the metropolis this figure is 36.88 %, and the lowest specific weight is in the regional city
(27.28 %). In addition, the influence of the characteristics of the models on the formation of the research array was
found depending on the sign of participation in traffic and the victim’s gender. Attention is drawn to the very low
share of female drivers (4.55 %) in rural areas, while in a regional city, this figure is 24.39 %. The influence of the
properties of the models and signs of participation in traffic on the occurrence of a negative outcome of the traumatic
process in the victims of traffic accidents has also been proven. Original Researches UDC 614.8:616-08-039.71:656.089
DOI: https://doi.org/10.22141/2224-0586.20.1.2024.1656
S.O. Guryev1 , V.A. Kushnir2 , O.S. Soloviov1 , N.I. Iskra3
1State Institution “Ukrainian Scientific and Practical Center for Emergency Medical Care and Disaster Medicine
of the Ministry of Health of Ukraine”, Kyiv, Ukraine
2Institute of Public Administration and Research in Civil Protection, Kyiv, Ukraine
3Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine UDC 614.8:616-08-039.71:656.089
DOI: https://doi.org/10.22141/2224-0586.20.1.2024.1656
S.O. Guryev1 , V.A. Kushnir2 , O.S. Soloviov1 , N.I. Iskra3
1State Institution “Ukrainian Scientific and Practical Center for Emergency Medical Care and Disaster Medicine
of the Ministry of Health of Ukraine”, Kyiv, Ukraine
2Institute of Public Administration and Research in Civil Protection, Kyiv, Ukraine
3Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine DOI: https://doi.org/10.22141/2224-0586.20.1.2024.1656 S.O. Guryev1 , V.A. Kushnir2 , O.S. Soloviov1 , N.I. Iskra3
1State Institution “Ukrainian Scientific and Practical Center for Emergency Medica
of the Ministry of Health of Ukraine”, Kyiv, Ukraine
2Institute of Public Administration and Research in Civil Protection, Kyiv, Ukraine
3Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine Оригінальні дослідження © 2024. The Authors. This is an open access article under the terms of the Creative Commons Attribution 4.0 International License, CC BY, which allows others to freely distribute the
published article, with the obligatory reference to the authors of original works and original publication in this journal. ір Віталій Андрійович, кандидат медичних наук, старший науковий співробітник, завідувач кафедри домедичної підготовки, Інститут державного управління та
вільного захисту, вул. Вишгородська, 21, м. Київ, 02000, Україна; e-mail: kv78@i.ua; тел.: +380(97)5485138 наукових досліджень з цивільного захисту, вул. Вишгородська, 21, м. Київ, 02000, Україна; e-mail: kv78@i.ua; тел.: +380(97)5485138
For correspondence: Vitalii Kushnir, PhD, Senior Research Fellow, Head of the Department of Pre-medical Training, Institute of Public Administration and Research in Civil Protection, Vyshhorodska st.,
21, Kyiv, 02000, Ukraine; e-mail: kv78@i.ua; phone: +380(97)5485138
F ll li
f
h
’i f
i
i
il bl
h
d f h
i l tation: Emergency Medicine (Ukraine). 2024;20(1):35-42. doi: 10.22141/2224-0586.20.1.2024.1656 As a result of the analysis, the impact of research
models on the emergence and formation of the array of victims with a negative outcome of the traumatic process was
determined, depending on their age and participation in the traffic. Conclusions. Probable and direct influence of
living conditions and signs of participation in the traffic on the formation of an array of victims of traffic accidents
has been found. Gender has a significant impact in all research models, and it is most pronounced in the conditions
of a metropolis in male victims. The sign of participation in traffic has a significant effect on the outcome of the
traumatic process in victims of traffic accidents. This effect is especially pronounced in those who were injured in
rural areas. The highest fatality rate among all road users is observed among pedestrians in rural areas (37.37 %),
and the lowest among drivers in metropolitan areas (3.02 %). Keywords: traffic accident; traffic injury; participation in the traffic; victims; comparative analysis © 2024. The Authors. This is an open access article under the terms of the Creative Commons Attribution 4.0 International License, CC BY, which allows others to freely distribute the
published article, with the obligatory reference to the authors of original works and original publication in this journal. у у
р
р
Kushnir, PhD, Senior Research Fellow, Head of the Department of Pre-medical Training, Institute of Public Administration and Research in Civil Protection, Vyshhorodska st.,
mail: kv78@i.ua; phone: +380(97)5485138 Для кореспонденції: Кушнір Віталій Андрійович, кандидат медичних наук, старший науковий співробітник, завідувач кафедри домедичної підготовки, Інститут д
наукових досліджень з цивільного захисту, вул. Вишгородська, 21, м. Київ, 02000, Україна; e-mail: kv78@i.ua; тел.: +380(97)5485138 Для кореспонденції: Кушнір Віталій Андрійович, кандидат медичних наук, старший науковий співробітник, завідувач кафедри домедичної підготовки, Інститут державного управління та
наукових досліджень з цивільного захисту, вул. Вишгородська, 21, м. Київ, 02000, Україна; e-mail: kv78@i.ua; тел.: +380(97)5485138
For correspondence: Vitalii Kushnir, PhD, Senior Research Fellow, Head of the Department of Pre-medical Training, Institute of Public Administration and Research in Civil Protection, Vyshhorodska st.,
21, Kyiv, 02000, Ukraine; e-mail: kv78@i.ua; phone: +380(97)5485138
Full list of authors’information is available at the end of the article y
p
Full list of authors’ information is available at the end of the article. 35 https://emergency.zaslavsky.com.ua Vol. 20, No. 1, 2024 Оригінальні дослідження / Original Researches Introduction epidemiological, clinical-nosological characteristics, clinical
features of the course of the traumatic process, principles of
providing medical care)”, approved by the Scientific Council
of the Ukrainian Scientific and Practical Center for Emer
gency Medical Care and Disaster Medicine of the Ministry
of Health of Ukraine (protocol of the meeting of the Scien
tific Council No. 6 of December 15, 2020) in compliance
with the terms of the Declaration of Helsinki and by the bio
ethics commission of the Ukrainian Scientific and Practical
Center for Emergency Medical Care and Disaster Medicine
of the Ministry of Health of Ukraine (protocol No. 10 of
December 8, 2020). Road traffic injury remains a significant medical and
social problem for all mankind at any time [1–5]. How
ever, the study of the problem of road traffic injuries as a
medical and social consequence of an emergency situation
of man-made nature remains insufficient, especially this
concerns the origin of medical and sanitary consequences
of road traffic accidents [6, 7]. It is also important to study
such consequences in different medical-geographical and
socio-economic conditions, which is especially relevant
for Ukraine. This is due to the variety of natural condi
tions, in particular the country’s landscape and infrastruc
ture [8]. An important component is the sign of participa
tion in the traffic of the victims, which was pointed out by
many researchers [9–14], but such comprehensive studies
were not conducted on the territory of Ukraine, especially
in a comparative aspect depending on the medical and
geographical conditions. Object of the research The sign of participation is one of the most important
clinical and epidemiological signs that characterizes a traffic
collision as a phenomenon as a whole. In order to study the
structure of accidents based on traffic participation and the
influence of the properties of the research model on such
distribution, an analysis of specific traffic participants in
the research models was carried out. Its results are shown
in Table 1. This article is a continuation of previous publications
in the journal “Emergency medicine (Ukraine)”, i.e. this
component of the comprehensive study was conducted using
a single factual material [15]. The actual material of the study
is 1,696 cases of traffic injuries, which were selected by the
method of irreversible randomization from the total array of
21,000 victims using the methodology of random numbers. The material was selected in the period of 2019–2020. The
resulting array was distributed according to the conditions
of an epidemiological experiment on a natural type model,
formed in accordance with the requirements and criteria of
the law of large numbers. The volume of the study array for
each model exceeded the necessary and sufficient, and this
made it possible to conduct a full-fledged and high-quality
analysis of the actual material and obtain reliable results. Model 1 of the natural type was chosen to be a metropolis
with an actual material of 1,139 victims with traffic injuries,
model 2 — rural area (315 cases), model 3 — regional city
(242 victims). As a result of data analysis in Table 1, it was found that
the drivers of model 1 (metropolis) are in the first place —
40.65 %, and the drivers of model 2 (rural area) are in the
last place — 16.19 %. The ratio of the maximum and the
minimum indicators in the array of drivers according to the
research models is 2.51, which indicates a high dissipation
of the distribution. Passive road users (passengers) take the first place in
model 3 (regional city) — 36.36 %, and the lowest specific
weight (third place) was in model 1 (22.47 %). The difference
in the specific weight of passengers in models 1 (22.47 %)
and 2 (23.49 %) is only 1.02 %, which is within the statistical
margin of error. Research methods The analysis of the actual material was carried out using
the methods of parametric and non-parametric statistics, the
law of formal logic and fractal analysis. The probability of regularity and connection was assessed
by calculating the polychoric correlation and Pearson’s test
(χ2), followed by a comparison of the obtained data with the
indicator according to Snedecor’s tables. The above determined the direction, expediency and ne
cessity of this research. Its results will be a fundamental basis
for the formation of adequate and effective clinical routes
and protocols for the provision of medical assistance to the
victims of road accidents. The comparison was made according to the clinical and
epidemiological characteristics of participation in traffic in
gender groups, in groups by age and the outcome of the trau
matic process. The aim of the research: to determine the characteristics
of the victims of traffic accidents depending on the sign of
participation in traffic and living conditions of the victims. The Statistica computer program was used to calculate
the data. Object of the research Summarizing the above, we can conclude that the cha-
racteristics of the research models have a direct impact on
the formation of the array of victims of road accidents,
namely, the properties of model 1 in women have the grea-
test impact on drivers and pedestrians, and in men, they
have a much smaller impact on all road users. In addition,
the very low specific weight of female drivers of 4.55 % in
the formation of the research array in model 2 (rural area)
draws attention. As a result of the conducted polychoric analysis, a posi
tive (φ2 = 0.0606), strong (C = 0.2390) and highly probable
relationship (χ2 = 102.72) was found, and the above provi
sions are contained within the field of probability. To establish a more detailed impact of research models on
the occurrence of traffic injuries, we conducted an integrated
comparative analysis of research models based on gender and
traffic participation. Its results are shown in Table 2. The above points to a significant influence of the charac
teristics of the research models on the formation of the array
of road accident victims, depending on the sign of participa
tion in traffic and gender. Male drivers have the highest specific weight (first
rank) in model 1 — 55.18 %, the lowest specific weight
is observed in model 2 — 19.28 %. The ratio of the maxi
mum and the minimum indicators in the array of male
drivers is 2.86. Female drivers have the highest specific
weight in model 3 — 24.39 %, the lowest specific weight
is recorded in model 2 — 4.55 %. The ratio of the maxi
mum and the minimum indicators in the array of female
drivers is 5.36. As a result of the conducted polychoric analysis, a posi
tive (φ2 = 0.1061), strong (C = 0.3097) and highly probable
relationship (χ2 = 179.93) was found, and the above provi
sions are contained within the field of probability. An important characteristic of any injury is the result of
the traumatic process. In order to verify the influence of the
characteristics of the research models on the outcome of the
traumatic process and the formation of the array of victims in
the resulting groups, we conducted an integrated comparative
analysis based on the sign of participation in traffic and the
outcome of the traumatic process in the study models. Object of the research The ratio of the maximum and the minimum
indicators in the array of passengers according to the research The research was carried out in accordance with the plan
of the dissertation work on the topic “Traffic injury (clinical- 36
Vol. 20, No. 1, 2024
Emergency Medicine (Ukraine), ISSN 2224-0586 (print), ISSN 2307-1230 (online)
Table 1. Comparative structural analysis of arrays of victims based on participation
in traffic in research models
Models
Drivers
Passengers
Pedestrians
%
rank
%
rank
%
rank
1
40.65
1
22.47
3
36.88
2
2
16.19
3
23.49
2
60.32
1
3
36.36
2
36.36
1
27.28
3 Table 1. Comparative structural analysis of arrays of victims based on participation
in traffic in research models Table 1. Comparative structural analysis of arrays of victims based on participation
in traffic in research models 36 Оригінальні дослідження / Original Researches Оригінальні дослідження / Original Researches models is 1.62, which indicates a moderate dissipation of the
distribution. Male pedestrians have the highest specific weight in mo-
del 2 — 59.44 %, in the second ranking place, there are the
victims of model 1 — 31.90 %, in the last ranking place — of
model 3 (25.00 %). The ratio of the maximum and the mini
mum indicators in the array of male pedestrians is 2.38. Fe
male pedestrians occupy the first rank in model 2 — 63.63 %,
and the last rank — in model 3 (31.71 %). The ratio of the
maximum and the minimum indicators in the array of female
pedestrians is 2.01. Among pedestrians, the first ranking place belongs to
model 2 — 60.32 %, and the lowest specific weight (third
place) is observed in model 3 — 27.28 %. The ratio of the
maximum and the minimum indicators in the array of pe
destrians according to the research models is 2.21, which
indicates a high dissipation of the distribution. Summarizing the above data, it should be noted that re
search models definitely have a direct impact on the occur
rence of traffic injuries in all road users, but such an impact
is most pronounced in drivers and pedestrians. https://emergency.zaslavsky.com.ua Оригінальні дослідження / Original Researches As a result of the conducted polychoric analysis, a
positive (φ2 = 0.2219), strong (С = 0.4262) and highly pro-
bable relationship was found between the sign of participa
tion in traffic, the outcome of the traumatic process, and
the properties of research models in road accident victims
(χ2 = 376.39), and the above-mentioned provisions are within
the limits of the probability field. In order to verify in detail the influence of the characte-
ristics of the research models on the occurrence of mortality
in road accident victims, we conducted a comparative analy
sis of the mortality indicators in traffic participants. Its results
are shown in Table 4. The comparative analysis of the data in Table 4 demon
strated that among all traffic participants, the lowest morta
lity was detected for model 1 (metropolis), and the highest
for model 2 (rural area). The fact that pedestrians have the
highest mortality rate compared to other road users is also
noteworthy. The ratio of the maximum and the minimum
mortality rates in the group of drivers is 8.44, for passen
gers — 19.92, for pedestrians — 4.90. On the other hand, the structural characteristics of the
array of those who survived and those who died do not coin
cide in any research model, and this confirms the influence
of the properties of study models on the occurrence of a
negative outcome of the traumatic process depending on the
victim’s participation in traffic. At the same time, drivers survive most often in model 1
(metropolis) — 41.23 %, and least often in model 3 (regional
city) — 18.27 %. The peak-to-minimum ratio of surviving
drivers is 2.26. In the array of deceased drivers, the highest
specific weight is observed in model 1 — 28.00 %, and the
lowest specific weight is observed in model 2 — 12.15 %. The
ratio of the maximum to the minimum indicators in the array
of deceased drivers is 2.30. Therefore, it can be asserted that the properties of re
search models have an impact on the occurrence not only of
traffic injuries, but also of a negative outcome of the trauma
tic process. This influence is especially pronounced among
passengers. Such data indicate the effect of the infrastructural
characteristics of the models on the outcome of the traumatic
process. Оригінальні дослідження / Original Researches Оригінальні дослідження / Original Researches survived is 2.26. In the array of dead pedestrians, the hig
hest indicator is found in model 2 (66.36 %), and the lowest
specific weight was in model 3 — 39.40 %. The ratio of the
maximum to the minimum indicators in the array of dead
pedestrians is 1.68. Among the victims in model 1 who survived, drivers have
the highest specific weight (41.23 %), passengers are in the
last place (23.14 %). The ratio of the maximum to the mini
mum indicators in model 1 is 1.78. Among the victims in
model 2 who survived, pedestrians have the highest specific
weight (57.21 %), and drivers are in the last place (18.27 %). The ratio of the maximum to the minimum indicators in
model 2 is 3.13. Among the model 3 victims who survived,
the highest specific weight was recorded among drivers
(37.80 %), and the lowest among pedestrians (25.36 %). The
ratio of the maximum to the minimum indicators in model
3 is 1.49. The data presented above confirm the fact that the pro
perties of the models have a direct impact on the forma
tion of the research array in both result groups depending
on participation in traffic. It should also be noted that the
properties of the models have a negative impact on the oc
currence of a negative outcome of the traumatic process,
especially in passive road users. In the mass of the dead in model 1, pedestrians have the
highest specific weight (64.00 %), and passengers (8.0 %) are
in the last place. The ratio of the maximum to the minimum
indicators in the deceased of model 1 is 8.0. Among the dead
victims in model 2, pedestrians have the highest percentage
(66.36 %), drivers have the lowest (12.15 %). The ratio of
the maximum to the minimum indicators in model 2 among
the dead is 5.46. Among the deceased victims in model 3,
pedestrians have the highest specific weight (39.40 %), dri-
vers have the lowest specific weight (27.27 %). The ratio of the
maximum to the minimum indicators in model 3 among the
dead is 1.44. Therefore, in all research models, pedestrians
have the highest specific weight among the dead victims. Object of the research Its
results are shown in Table 3. Male passengers occupy the first place in model 3 —
32.50 %, and the last ranking place is recorded in model
1 — 12.92 %. The ratio of the maximum and the minimum
indicators in the array of male passengers is 2.52. Female
passengers also take the first place in model 3 — 43.90 %,
and the last (third) rank is recorded in model 2 — 31.82 %. The ratio of the maximum and the minimum indicators in
the array of female passengers is 1.38. Data in Table 3 indicate that among all traffic partici
pants, there is a difference in specific weight in the effective
groups of study models. Table 2. Integrated comparative analysis of arrays of victims based on participation in traffic and gender
in research models
Models
Drivers
Passengers
Pedestrians
Men
Women
Men
Women
Men
Women
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
1
55.18
1
13.39
2
12.92
3
40.40
2
31.90
2
46.21
2
2
19.28
3
4.55
3
21.28
2
31.82
3
59.44
1
63.63
1
3
42.50
2
24.39
1
32.50
1
43.90
1
25.00
3
31.71
3 ated comparative analysis of arrays of victims based on participation in traffic and gender
in research models Table 2. Integrated comparative analysis of arrays of victims based on participation in traffic a
in research models Models
Drivers
Passengers
Pedestrians
Survived
Dead
Survived
Dead
Survived
Dead
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
1
41.23
1
28.00
1
23.14
3
8.00
3
35.63
2
64.00
2
2
18.27
3
12.15
3
24.52
2
21.49
2
57.21
1
66.36
1
3
37.80
2
27.27
2
36.84
1
33.33
1
25.36
3
39.40
3
Table 3. Integrated comparative structural analysis based on participation in traffic according
to research models in effective groups Table 3. Integrated comparative structural analysis based on participation in traffic according
to research models in effective groups https://emergency.zaslavsky.com.ua Vol. 20, No. 1, 2024 37 Оригінальні дослідження / Original Researches From a scientific and practical point of view, a compara
tive analysis on the impact of research models on a negative
outcome of the traumatic process in different age groups is
of particular interest. From a didactic point of view, such an
analysis was conducted in groups by age separately for each
array of traffic participants. In the array of passengers who survived, the highest spe
cific weight was recorded in model 3 — 36.84 %, and the lo-
west in model 1 — 23.14 %. The ratio of the maximum to the
minimum indicators for passengers who survived is 1.59. The
highest specific weight of deceased passengers is observed in
model 3 — 33.33 %, and the lowest in model 1 — 8.00 %. The ratio of the maximum to the minimum indicators in the
array of deceased passengers is 4.17. A comparative analysis of drivers by age in research mo
dels based on the outcome of the traumatic process is given
in Table 5. In the array of pedestrians who survived, the highest spe
cific weight of victims is observed in model 2 — 57.21 %, and
the lowest in model 3 (25.36 %). The ratio of the maximum
to the minimum indicators in the array of pedestrians who When analyzing the data of Table 5, it was found that in
all age groups, there was a difference in mortality rates, but
of different degrees. In the group under 20 years, the highest 38
Vol. 20, No. 1, 2024
Emergency Medicine (Ukraine), ISSN 2224-0586 (print), ISSN 2307-1230 (online)
Table 4. Comparative analysis of mortality rates in traffic participants according to research models
Models
Drivers
Passengers
Pedestrians
%
rank
%
rank
%
rank
1
3.02
3
1.56
3
7.62
3
2
25.49
1
31.08
1
37.37
1
3
10.23
2
12.50
2
19.70
2 Table 4. Comparative analysis of mortality rates in traffic participants according to rese mparative analysis of mortality rates in traffic participants according to research models 38 Оригінальні дослідження / Original Researches Оригінальні дослідження / Original Researches specific weight of the deceased is observed in model 3 —
15.91 %, and the lowest in model 1 (4.97 %). The ratio of the
maximum to the minimum indicators in this age group is 3.2. Оригінальні дослідження / Original Researches Analysis of the cases proved that it was due to the violation
of the speed limit by the drivers. Data of the comparative analysis of passengers in
age groups according to research models are given in
Table 6. When analyzing the data of Table 6, a difference was
found in the distribution of specific weight in all age groups,
but such indicators vary of spe according to research models. In the group under 20 years, the highest specific weight of
dead passengers was recorded in model 3 (regional city) —
32.96 %, the lowest was in model 1 (metropolis) — 8.21 %. The ratio of the maximum to the minimum indicator in this
age group is 4.01. The highest specific weight of deceased
passengers in the group of 21–30 years is observed in model Table 5. Comparative analysis of the distribution of the array of dead drivers according
to research models in age groups
Models
Age, years
Under 20
21–30
31–40
41–50
51–60
61–70
Over 71
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
1
4.97
3
28.29
1
30.89
1
19.00
2
10.80
2
4.32
2
1.73
2
2
9.80
2
23.53
3
25.49
2
31.38
1
5.88
3
3.92
3
–
3
3
15.91
1
26.13
2
15.91
3
15.91
3
15.91
1
6.82
1
3.41
1 Table 5. Comparative analysis of the distribution of the array of dead drivers according
to research models in age groups Models
Age, years
Under 20
21–30
31–40
41–50
51–60
61–70
Over 71
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
1
8.21
3
24.61
2
20.31
1
13.28
2
12.11
1
14.45
1
7.03
1
2
20.27
2
28.38
1
20.27
2
16.22
1
2.70
3
8.11
3
4.05
2
3
32.96
1
22.73
3
11.36
3
11.36
3
11.36
2
9.09
2
1.14
3
Table 6. Comparative analysis of the distribution of the array of dead passengers according
to research models in age groups Table 6. Comparative analysis of the distribution of the array of dead passengers according
to research models in age groups Table 7. Оригінальні дослідження / Original Researches In the group of 21–30 years, the specific weight of deceased
drivers was the highest in model 1 (28.29 %), and the lowest
in model 2 (23.53 %); the difference is not large enough. The
ratio of the maximum to the minimum indicators in this age
group is 1.2. In the group of 31–40 years, the highest spe
cific weight of deceased victims was recorded in model 1 —
30.89 %, and the lowest in model 3 (15.91 %). The ratio of
the maximum to the minimum indicators in this age group is
1.94. The highest specific weight in the group of 41–50 years
is observed in model 2 — 31.38 %, and the lowest in model 3
(15.91 %). The ratio of the maximum to the minimum indi
cators in this age group is 1.97. In the group of 51–60 years,
the highest specific weight of the deceased was recorded in
model 3 — 15.91 %, and the lowest in model 2 (5.88 %). The
ratio of the maximum to the minimum indicators in this age
group is 2.71. The highest specific weight of victims in the
group of 61–70 years was recorded in model 3 — 6.82 %,
the lowest in model 2 (3.92 %). The ratio of the maximum
to the minimum indicators in this age group is 1.74. Among
those older than 71 years, the highest number of victims was
recorded in model 3 — 3.41 %, and the lo-west — in model 1
(1.73 %). The ratio of the maximum to the minimum indica-
tors in the age group is 1.97. On the other hand, the ratio of the maximum to the mi-
nimum indicators in model 1 is 17.86, which indicates a high
dissipation of the distribution, in model 2, this parameter is
8.01, in model 3 — 7.66. Therefore, taking into account the above data, it can be
stated that the properties of research models have a direct
impact on a negative outcome of the traumatic process in
drivers. This influence is especially pronounced in model
1, and in models 2 and 3, it is smaller and almost similar. On the other hand, the properties of research models have a
pronounced effect on mortality in the age groups of up to 20
and 51–60 years, a little less — in victims aged 31–50 years. https://emergency.zaslavsky.com.ua Discussion When analyzing the data of Table 7, it was found that
pedestrians have the same uneven distribution in groups by
age in research models, as other traffic participants. In the
group under 20 years, the highest specific weight of dead
pedestrians is observed in model 3 — 15.15 %, and the lo-
west in model 1 — 7.15 %. The ratio of the maximum to the
minimum indicator in deceased pedestrians under the age
of 20 is 2.12. The highest specific weight of dead pedestrians
in the group of 21–30 years is noted in model 3 (21.21 %),
and the lowest in model 2 — 18.95 %. The ratio of the maxi
mum to the minimum indicator in this age group is 1.12. In the 31–40 years group, the highest specific weight was
recorded in model 1 — 19.52 %, and the lowest in model 3
(15.15 %). The ratio of the maximum to the minimum in
dicator in this age group is 1.29. The highest specific weight
of dead pedestrians in the age group of 41–50 years is ob
served in model 3 — 21.21 %, and the lowest is recorded
in model 2 — 14.21 %. The ratio of the maximum to the
minimum indicator in this age group is 1.49. In the group of
51–60-year-olds, the highest specific weight of dead pedes
trians was recorded in model 2 — 20.00 %, and the lowest
specific weight was in model 3 — 3.03 %. The ratio of the
maximum to the minimum indicator in this age group is 6.60. In the group of 61–70 years, the highest specific weight of
dead pedestrians is noted in model 3 — 15.15 %, the lowest in
model 2 — 11.05 %. The ratio of the maximum to the mini
mum indicator in this age group is 1.37. The highest specific
weight in the group older than 71 years is recorded in model
1 — 9.76 %, and the lowest indicator in model 2 — 7.89 %. The ratio of the maximum to the minimum indicator in this
age group is 1.24. Оригінальні дослідження / Original Researches Comparative analysis of the distribution of the array of dead pedestrians according
to research models in age groups
Models
Age, years
Under 20
21–30
31–40
41–50
51–60
61–70
Over 71
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
%
rank
1
7.15
3
19.76
2
19.52
1
20.24
2
10.71
2
12.86
2
9.76
1
2
11.58
2
18.95
3
16.32
2
14.21
3
20.00
1
11.05
3
7.89
3
3
15.15
1
21.21
1
15.15
3
21.21
1
3.03
3
15.15
1
9.10
2 Table 7. Comparative analysis of the distribution of the array of dead pedestrians according
to research models in age groups https://emergency.zaslavsky.com.ua Vol. 20, No. 1, 2024 39 Оригінальні дослідження / Original Researches 2 — 28.38 %, the lowest specific weight is recorded in model
3 — 22.73 %. The ratio of the maximum to the minimum
indicator in this age group is 1.25. The highest specific
weight of victims in the group of 31–40 years is noted in
model 1 — 20.31 %, and the lowest in model 3 (11.36 %). The ratio of the maximum to the minimum indicator in
this age group is 1.79. In the group of 41–50 years, the
highest specific weight is in model 2 — 16.22 %, and the
lowest — in model 3 (11.36 %). The ratio of the maximum
to the minimum indicator in this age group is 1.43. The
highest specific weight of dead passengers in the age group
of 51–60 years is observed in model 1 — 12.11 %, and the
lowest — in model 2 (2.70 %). The ratio of the maximum
to the minimum indicator in this age group is 4.49. In the
age group of 61–70 years, the highest specific weight of pas
sengers who died was recorded in model 1 — 14.45 %, and
the lowest in model 2 (8.11 %). The ratio of the maximum
to the minimum indicator in this age group is 1.78. The
highest specific weight of dead passengers over the age of 71
was recorded in model 1 — 7.03 %, and the lowest in model
3 — 1.14 %. The ratio of the maximum to the minimum
indicator in this age group is 6.17. Оригінальні дослідження / Original Researches At the same time, as with other road users, there is a dis
crepancy in the distribution of deceased victims according
to models in age groups, with a simultaneous discrepancy
in the ratio coefficients. The ratio of the maximum to the
minimum indicator in model 1 is 2.83, in model 2 — 2.53,
in model 3, it is 3.0. Therefore, in all research models, there
is a moderate dissipation of the distribution of the mass
of dead pedestrians in age groups, that is, the properties
of the research models have an almost uniform effect on
pedestrians. As a result of the conducted polychoric analysis, it was
found that there is a positive (φ2 = 0.2054), strong (C =
0.4128) and highly probable relationship between the signs
of participation in traffic, age, the outcome of the traumatic
process, and the properties of research models in road acci
dent victims (χ2 = 348.37), and the above-mentioned provi
sions are within the limits of the probability field. Summarizing the above data, it should be noted that the
greatest influence of the model property (taking into account
age groups) on a negative outcome of the traumatic process
is observed in passengers, to a lesser extent in drivers and
insignificant in pedestrians. The greatest influence of the
model properties is noted in the age groups under 20 and
51–60 years among all traffic participants. This is related
to the psychophysiological characteristics of a person at a
certain age. It is worth noting that, in general, the difference
by age groups is more pronounced in passive road users (pas
sengers). At the same time, there is a mismatch of distribution
indicators in the array of deceased according to models in
age groups with a simultaneous mismatch of ratio coeffi
cients. The ratio of the maximum to the minimum indicator
in model 1 is 3.50, in model 2 — 7.01, in model 3 — 28.91. References victims is observed in the metropolis (40.65 %), and the
smallest in rural areas — 16.19 %, which indicates non-ob
servance of road safety rules, in particular, the speed limit in
the metropolis compared to the countryside. Among pas
sengers, victims in the conditions of the regional center have
the highest specific weight — 36.36 %, while the specific
weight of passengers in rural areas and the metropolis is very
close (22.47 and 23.49 %, respectively). Analysis of the cases
proved that this is due to the neglect to wear seat belts at a
fairly high speed. A very significant share of victims-pedestri
ans (60.32 %) is observed in rural areas, which is significantly
higher than the indicators of the metropolis and the regional
center (36.88 and 27.28 %, respectively). As the analysis of
the cases proved, this is due to poor road infrastructure in
rural areas. victims is observed in the metropolis (40.65 %), and the
smallest in rural areas — 16.19 %, which indicates non-ob
servance of road safety rules, in particular, the speed limit in
the metropolis compared to the countryside. Among pas
sengers, victims in the conditions of the regional center have
the highest specific weight — 36.36 %, while the specific
weight of passengers in rural areas and the metropolis is very
close (22.47 and 23.49 %, respectively). Analysis of the cases
proved that this is due to the neglect to wear seat belts at a
fairly high speed. A very significant share of victims-pedestri
ans (60.32 %) is observed in rural areas, which is significantly
higher than the indicators of the metropolis and the regional
center (36.88 and 27.28 %, respectively). As the analysis of
the cases proved, this is due to poor road infrastructure in
rural areas. 1. Ilchenko L. In Ukraine, the number of road accidents with inju
ries and deaths is increasing: three regions are in the anti-rating. Avail
able from: https://www.epravda.com.ua/news/2023/09/13/704253/. Ukrainian. 1. Ilchenko L. In Ukraine, the number of road accidents with inju
ries and deaths is increasing: three regions are in the anti-rating. Avail
able from: https://www.epravda.com.ua/news/2023/09/13/704253/. Ukrainian. 2. Centers for Disease Control and Prevention (CDC); National
Center for Injury Prevention and Control. Road Traffic Injuries and
Deaths - A Global Problem. Available from: https://www.cdc.gov/injury/
features/global-road-safety/index.html. 3. Naqvi G, Johansson G, Yip G, Rehm A, Carrothers A, Stöhr K. References Mechanisms, patterns and outcomes of paediatric polytrauma in a UK
major trauma centre. Ann R Coll Surg Engl. 2017 Jan;99(1):39-45. doi:
10.1308/rcsann.2016.0222. 4. Onieva-García MÁ, Martínez-Ruiz V, Lardelli-Claret P, et al. Gender and age differences in components of traffic-related pedestrian
death rates: exposure, risk of crash and fatality rate. Inj Epidemiol. 2016
Dec;3(1):14. doi: 10.1186/s40621-016-0079-2. The dependence of the influence of gender on all traf
fic participants in all research models was also detected,
which is most pronounced in the metropolis and in rural
areas. 5. Whitaker J, O'Donohoe N, Denning M, et al. Assessing trauma
care systems in low-income and middle-income countries: a systematic re
view and evidence synthesis mapping the Three Delays framework to inju
ry health system assessments. BMJ Glob Health. 2021 May;6(5):e004324. doi: 10.1136/bmjgh-2020-004324. This study demonstrated the influence of models on the
emergence and formation of the array of victims with a nega
tive outcome of the traumatic process, depending on the age
and participation in traffic. For drivers, this impact is most
pronounced in model 1 (metropolis), for passengers in model
3 (regional city), and for pedestrians in all models this influ
ence is approximately similar. 6. Vitalis N, Runyoro A, Selemani M. Assessing Factors for Oc
currence of Road Accidents in Tanzania Using Panel Data Analysis:
Road Safety Perspective. Journal of Transportation Technologies. 2022
Jan;12(1):123-136. doi: 10.4236/jtts.2022.121008. 6. Vitalis N, Runyoro A, Selemani M. Assessing Factors for Oc
currence of Road Accidents in Tanzania Using Panel Data Analysis:
Road Safety Perspective. Journal of Transportation Technologies. 2022
Jan;12(1):123-136. doi: 10.4236/jtts.2022.121008. 7. Melnychenko OI, Grysjuk JuS, Garanskyj OV, Chechet AM. Traf
fic accidents in Kyiv and their consequences. Upravlinnja proektamy,
systemnyj analiz i logistyka. 2011;(8):234-238. Ukrainian. It was found that the above-mentioned combination has
an impact on a negative outcome of the traumatic process
and the formation of an array of victims as a whole. 7. Melnychenko OI, Grysjuk JuS, Garanskyj OV, Chechet AM. Traf
fic accidents in Kyiv and their consequences. Upravlinnja proektamy,
systemnyj analiz i logistyka. 2011;(8):234-238. Ukrainian. 8. Nilsbakken IMW, Cuevas-Østrem M, Wisborg T, Sollid S, Jeppe
sen E. Effect of urban vs. remote settings on prehospital time and mortality
in trauma patients in Norway: a national population-based study. Scand J
Trauma Resusc Emerg Med. 2023 Oct 5;31(1):53. doi: 10.1186/s13049-
023-01121-w. 8. Nilsbakken IMW, Cuevas-Østrem M, Wisborg T, Sollid S, Jeppe
sen E. Effect of urban vs. References remote settings on prehospital time and mortality
in trauma patients in Norway: a national population-based study. Scand J
Trauma Resusc Emerg Med. 2023 Oct 5;31(1):53. doi: 10.1186/s13049-
023-01121-w. Research limitations Certain limitations of the study are related to the large
number of people who are injured due to road accidents —
up to 45,000 every year. Therefore, it is practically impossible
to conduct research in the scope of the general population
of the phenomenon. In addition, the exclusion criterion was
two-wheel and rail injury. 9. Han W, Zhao J, Chang Y. Driver behaviour and traffic acci
dent involvement among professional heavy semi-trailer truck drivers in
China. PLoS One. 2021 Dec 2;16(12):e0260217. doi: 10.1371/journal. pone.0260217. 9. Han W, Zhao J, Chang Y. Driver behaviour and traffic acci
dent involvement among professional heavy semi-trailer truck drivers in
China. PLoS One. 2021 Dec 2;16(12):e0260217. doi: 10.1371/journal. pone.0260217. Vol. 20, No. 1, 2024 Discussion The results of the study make it possible to reveal the like
ly influence of the sign of participation in traffic of victims of
road accidents both on the formation of road traffic trauma,
as a medical and sanitary consequence of an emergency, and
on the course of the traumatic process. It has been found that
this sign, which causes damage and a negative outcome of
the traumatic process, is the most dangerous for pedestrians. The relationship was also noted between the influence of
the living conditions of the victims (metropolis, rural area,
regional city) and signs of participation in traffic on the for
mation of injuries as a consequence of an emergency and
on the outcome of the traumatic process. Such a study, as
evidenced by the analysis of open and accessible sources of
scientific information, has not been found and is unique. It is this combination that determines the role and place of
the sign of participation in traffic by victims in the origin of
a traffic injury as a medical and sanitary consequence of an
emergency of a man-made nature. The comparative analysis proved that the combination
of living conditions and signs of participation in traffic of
victims in rural areas has the greatest impact. This leads to a
more frequent occurrence of trauma as a result of road ac
cidents and a more likely negative outcome of the traumatic
process in them. As the analysis of the cases proved, this is
primarily due to the infrastructural imperfection of rural
areas, including the infrastructure of the health care system. Such imperfection is characteristic of many countries of the
world [5, 16–20]. Regarding the signs of participation in traffic, it is worth
noting that the largest specific weight of drivers among the Vol. 20, No. 1, 2024 40 Emergency Medicine (Ukraine), ISSN 2224-0586 (print), ISSN 2307-1230 (online) Оригінальні дослідження / Original Researches Information about authors Information about authors
Sergiy Guryev, MD, PhD, Professor, Deputy Director for Research, State Institution “Ukrainian Scientific and Practical Center of Emergency Medical Care and Disaster Medicine of the Ministry of Health
of Ukraine”, Kyiv, Ukraine; e-mail: gurevsergej1959@gmail.com; fax: +380(44)5185708; https://orcid.org/0000-0003-0191-945X O.S. Soloviov, MD, PhD, Professor, Research Fellow, State Institution “Ukrainian Scientific and Practical Center of Emergency Medical Care and Disaster Medicine of the Ministry of Health of Ukraine”,
Kyiv, Ukraine; e-mail: a.soloviov71@gmail.com; https://orcid.org/0000-0002-6615-4868 y
Natalia Iskra, MD, PhD, Professor, Head Teacher of the Department of Disaster Medicine and Military Medical Training, Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine; e-mail:
mk_kafedra@ukr.net; https://orcid.org/0000-0001-5976-3625 Conflicts of interests. Authors declare the absence of any conflicts of interests and own financial interest that might be construed to influence the results or interpretation of the manuscript. Authors’ contribution. Guryev S.O. — concept and design of the study; Kushnir V.A. — collection and processing of materials, writing the text; Soloviov O.S. — processing of materials; Iskra N.I. —
data collection. Гур’єв С.О.1, Кушнір В.А.2, Соловйов О.С.1, Іскра Н.І.3 Гур’єв С.О.1, Кушнір В.А.2, Соловйов О.С.1, Іскра Н.І.3 ур
у
р
р
1ДЗ «Український науково-практичний центр екстреної медичної допомоги та медицини катастроф МОЗ України»,
м. Київ, Україна 1ДЗ «Український науково-практичний центр екстреної медичної допомоги та медицини катастроф МОЗ У
м. Київ, Україна 2Інститут державного управління та наукових досліджень з цивільного захисту, м. Київ, Україна
3Національний університет охорони здоров’я України імені П.Л. Шупика, м. Київ, Україна Дорожньо-транспортна травма як медико-санітарний наслідок надзвичайної ситуації
техногенного характеру в Україні. Повідомлення третє: аналіз та характеристика масиву
постраждалих залежно від ознаки участі в русі Дорожньо-транспортна травма як медико-санітарний наслідок надзвичайної ситуації
техногенного характеру в Україні. Повідомлення третє: аналіз та характеристика масиву
постраждалих залежно від ознаки участі в русі місті (27,28 %). Крім того, встановлено вплив характеристик
моделей на формування масиву дослідження залежно від ознаки
участі в русі та статі постраждалих. Привертає увагу дуже низька
питома вага жінок-водіїв (4,55 %) у сільській місцевості, в об
ласному місті цей показник становить 24,39 %. Також доведено
вплив властивостей моделей та ознаки участі в русі на виникнен
ня негативного результату травматичного процесу в постраж
далих унаслідок дорожньо-транспортних пригод. У результаті
аналізу встановлено дію моделей дослідження на виникнення
та формування масиву постраждалих із негативним результатом
травматичного процесу залежно від їхнього віку та участі в русі. Висновки. Виявлено достовірний та прямий вплив умов життєді
яльності та ознаки участі в русі на формування масиву постраж
далих унаслідок дорожньо-транспортних пригод. Conclusions care systems in low-income and middle-income countries: a systematic re
view and evidence synthesis mapping the Three Delays framework to inju
ry health system assessments. BMJ Glob Health. 2021 May;6(5):e004324. doi: 10.1136/bmjgh-2020-004324. care systems in low-income and middle-income countries: a systematic re
view and evidence synthesis mapping the Three Delays framework to inju
ry health system assessments. BMJ Glob Health. 2021 May;6(5):e004324. doi: 10.1136/bmjgh-2020-004324. 16. Seresirikachorn K, Singhanetr P, Soonthornworasiri N, Amorn
petchsathaporn A, Theeramunkong T. Characteristics of road traffic
mortality and distribution of healthcare resources in Thailand. Sci Rep. 2022 Nov 24;12(1):20255. doi: 10.1038/s41598-022-24811-4. 20. Núñez-Samudio V, Mayorga-Marín F, López Castillo H, Lan
dires I. Epidemiological Characteristics of Road Traffic Injuries Involving
Children in Three Central American Countries, 2012-2015. Int J Environ
Res Public Health. 2020 Dec 23;18(1):37. doi: 10.3390/ijerph18010037. 17. Shahsavari S, Mohammadi A, Mostafaei S, et al. Analysis of
injuries and deaths from road traffic accidents in Iran: bivariate regres
sion approach. BMC Emerg Med. 2022 Jul 18;22(1):130. doi: 10.1186/
s12873-022-00686-6. 21. Hyder AA, Hoe C, Hijar M, Peden M. The political and social
contexts of global road safety: challenges for the next decade. Lancet. 2022 Jul 9;400(10346):127-136. doi: 10.1016/S0140-6736(22)00917-5. 21. Hyder AA, Hoe C, Hijar M, Peden M. The political and social
contexts of global road safety: challenges for the next decade. Lancet. 2022 Jul 9;400(10346):127-136. doi: 10.1016/S0140-6736(22)00917-5. 18. El Mestoui Z, Jalalzadeh H, Giannakopoulos GF, Zuidema WP. Incidence and etiology of mortality in polytrauma patients in a Dutch level
I trauma center. Eur J Emerg Med. 2017 Feb;24(1):49-54. doi: 10.1097/
MEJ.0000000000000293. Received 02.01.2024
Revised 12.01.2024
Accepted 20.01.2024 19. Whitaker J, O'Donohoe N, Denning M, et al. Assessing trauma Conclusions 10. Antonio Obregón Biosca S, Luis Reyes Araiza J, Angel Pérez
Lara y Hernández M. Probability to Be Involved in a Road Accident:
Transport User Socioeconomic Approach. In: Jaoudé AA, editor. Applied
Probability Theory - New Perspectives, Recent Advances and Trends. London, UK: IntechOpen; 2023. doi: 10.5772/intechopen.106325. 1. Probable and direct interdependence between living
conditions and signs of participation in traffic in victims
of traffic accidents was found. Although active road users
(drivers and pedestrians) prevail in all models, this indica
tor is the largest in the metropolis, and the smallest in the
countryside. 11. Degais W, Awooda H, Elnimeiri M, Kaddam L. Epidemio
logical Pattern of Injuries Resulting from Road Traffic Accidents in
Khartoum, Sudan. Health. 2018 Jun; 10(6):816-822. doi: 10.4236/
health.2018.106061. 2. The gender feature has a significant impact in all re
search models and is most pronounced in the conditions of
the metropolis in male victims. 12. Sichembe W, Manyozo SD, Moodi R. The epidemiology of Road
Traffic Crashes in Rural Zambia: A Retrospective Hospital-Based Study
at Monze Mission Hospital. Medical Journal of Zambia. 2019;46(4):264-
276. doi: 10.55320/mjz.46.4.235. 3. The sign of participation in traffic has a significant
impact on the outcome of the traumatic process. This ef
fect is especially pronounced in victims who were injured in
rural areas. 13. Guryev SO, Kushnir VA, Soloviov OS. Clinical and epidemio
logical characteristics of road traffic injuries in the conditions of a me
tropolis. Travma. 2023;24(1):14-19. Ukrainian. doi: 10.22141/1608-
1706.1.24.2023.926. 4. The highest fatality rate among all road users is
observed among pedestrians in rural areas — 37.37 %,
and the lowest among drivers in metropolitan areas —
3.02 %. 14. Khorshah H, Eri M, Honarvar MR, et al. Epidemiological Study
of Road Traffic Accidents and Detection of Accident Hot Spot in Golestan
Province, Northern Iran. JCBR. 2018;2(4):15-22. 5. In general, the largest number of victims is observed
among all participants of the traffic aged 21–50 years, and
this tendency is also expressed in pedestrians who were in
jured in rural areas. Province, Northern Iran. JCBR. 2018;2(4):15-22. 15. Guryev SO, Kushnir VA, Kushnir HP. Traffic injury as a medical
and sanitary consequence of a man-made emergency in Ukraine. Report 41 Vol. 20, No. 1, 2024 https://emergency.zaslavsky.com.ua Оригінальні дослідження / Original Researches one: clinical and epidemiological characteristics. Medicina neotložnyh
sostoânij. 2023;19(5):370-377. Ukrainian. doi: 10.22141/2224-
0586.19.5.2023.1613. one: clinical and epidemiological characteristics. Medicina neotložnyh
sostoânij. 2023;19(5):370-377. Ukrainian. doi: 10.22141/2224-
0586.19.5.2023.1613. Information about authors Стать відіграє
суттєву роль в усіх моделях дослідження, це найбільш виражено в
умовах мегаполісу серед осіб чоловічої статі. Ознака участі в русі
має достовірний вплив на результат перебігу травматичного про
цесу в постраждалих унаслідок дорожньо-транспортних пригод. Особливо такий вплив виражений у тих, кого було травмовано
в умовах сільської місцевості. Найвища летальність серед усіх
учасників руху спостерігається в пішоходів, які отримали до
рожньо-транспортну травму в сільській місцевості (37,37 %), а
найменша — у водіїв в мегаполісі (3,02 %). Резюме. Актуальність. Дорожньо-транспортна травма зали
шається значною медико-соціальною проблемою для всього
людства в будь-який час. Мета дослідження: визначити характе
ристику постраждалих унаслідок дорожньо-транспортних пригод
залежно від ознаки їхньої участі в русі та умов життєдіяльності. Матеріали та методи. Стаття є продовженням попередніх публі
кацій у журналі «Медицина невідкладних станів». Цей компонент
комплексного дослідження проведено із використанням єдиного
фактичного матеріалу (1696 випадків дорожньо-транспортної
травми), що був обраний методом безповоротної рандомізації. Моделлю 1 реального типу став мегаполіс із обсягом матеріалу
1139 постраждалих із дорожньо-транспортною травмою, модель
2 (сільська місцевість) — це 315 осіб, модель 3 (обласне місто) —
242 постраждалих. Вивчення фактичного матеріалу проводилося
із використанням методів параметричної та непараметричної
статистики, закону формальної логіки та фрактального аналізу. Результати. Установлено, що властивості моделей дослідження
мають суттєвий вплив на виникнення дорожньо-транспортної
травми залежно від ознаки участі в русі. Найбільша питома вага
водіїв зафіксована в мегаполісі (40,65 %), найменша — в сільській
місцевості (16,19 %). Питома вага пасажирів була найбільшою в
обласному місті (36,36 %), сільській місцевості (23,49 %), а най
меншою — в мегаполісі (22,43 %). Найбільшу питому вагу пішо
ходів зареєстровано в сільській місцевості — 60,32 %, у мегаполісі
показник становить 36,88 %, а найменшим він є в обласному Ключові слова: дорожньо-транспортна пригода; дорожньо-тран
спортна травма; участь у русі; постраждалі; порівняльний аналіз Vol. 20, No. 1, 2024 Emergency Medicine (Ukraine), ISSN 2224-0586 (print), ISSN 2307-1230 (online) 42 Vol. 20, No. 1, 2024
Emergency Medicine (Ukraine), ISSN 2224-0586 (print), ISSN 2307-1230 (online)
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Implementasi Market Basket Analysis Pada Toko Retail Menggunakan Algoritma Apriori
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KOPERTIP: Jurnal Ilmiah Manajemen Informatika dan Komputer
Vol. 07 No. 01 Bulan Februari 2023, pp.29-34
https://jurnal.kopertipindonesia.or.id/index.php/kopertip
E-ISSN : 2549-9351
P-ISSN : 2549-211X
IMPLEMENTASI MARKET BASKET ANALYSIS PADA TOKO
RETAIL MENGGUNAKAN ALGORITMA APRIORI
Mia Fijriani1, Umi Hayati2 , Gifthera Dwilestari3 Ade Rizki Rinaldi4
Faturrohman5
Program Studi Teknik Informatika, STMIK IKMI Cirebon, Kota Cirebon Indonesia 12
Program Studi Sistem Informasi, STMIK IKMI Cirebon, Kota Cirebon Indonesia3
Email: miafijriani149@gmail.com1, umi.haya41@gmail.com2, @ggdwilestari@gmail.com3, aderizkirinaldi@ikmi.ac.id4,
fathurrahman.ikmi@gmail.com5
E-mail Penulis Korespondensi (ggdwilestari@gmail.com)
Abstract— Utilization of data can now be done in the field of
marketing with the help of technology. Technology helps humans
manage data and turn it into useful knowledge. Toko Rizieq Putra
2 is a retail store located in Kuningan Regency, the problem is that
it does not utilize sales data which is only stored as an archive. This
results in the data being useless and getting more and more.
Therefore, historical sales data needs to be reprocessed into useful
knowledge. One of the problems that occurs is the lack of
knowledge about the use of transaction data to analyze what
products consumers often buy together. Therefore, it is necessary
to do a Market Basket analysis to find out the sales pattern at the
Rizieq Putra 2 Store. The method used is Market Basket analysis
using the Apriori algorithm. The stages include problem
identification, data preparation and preprocessing, determining
the minimum value of support items and confidence values,
implementing the a priori algorithm, and analyzing the results.
The results achieved were 14 sales pattern rules with the highest
confidence value in purchasing toiletries and basic necessities
tending to buy household needs with a value of 66.01%.
Kata Kunci: Market Basket Analysis, Sales Pattern, Apriori,
Rizieq Putra 2 Store, Transaction Data
Abstrak— Pemanfaatan data kini bisa dilakukan dalam bidang
pemasaran dengan bantuan teknologi. Teknologi membantu
manusia mengelola data dan menjadikannya sebagai
pengetahuan yang bermanfaat. Toko Rizieq Putra 2 adalah
sebuah toko retail yang terletak di Kabupaten Kuningan,
permasalahannya
yaitu
kurang
memanfaatkan
data
penjualannya yang hanya disimpan sebagai arsip saja. Ini
mengakibatkan data tersebut tak berguna dan semakin lama
semakin banyak. Oleh karena itu, data historis penjualan perlu
diolah kembali menjadi pengetahuan yang berguna. Salah satu
permasalahan yang terjadi adalah kurangnya pengetahuan
tentang pemanfaatan data transaksi untuk menganalisis produk
apa saja yang sering dibeli bersama oleh konsumen. Oleh karena
itu, perlu dilakukan Market Basket Analysisuntuk mengetahui
pola penjualan di Toko Rizieq Putra 2. Metode yang digunakan
adalah analisis Market Basket dengan menggunakan algoritma
apriori. Tahapannya meliputi identifikasi masalah, persiapan dan
preprocessing data, menentukan nilai minimum support item dan
nilai confidence, implementasi algoritma apriori, dan analisis
hasil. Hasil yang dicapai adalah 14 aturan pola penjualan dengan
nilai confidence tertinggi pada pembelian alat mandi dan sembako
cenderung akan membeli kebutuhan rumahtangga dengan nilai
66,01%.
Kata Kunci— Market Basket Analysis, Pola Penjualan, Apriori,
Toko Rizieq Putra 2, Data Transaksi
I. PENDAHULUAN
Perkembangan teknologi, penjualan menjadi faktor
yang sangat penting dalam mencapai tujuan bisnis. Dalam
melakukan kegiatannya, para pelaku bisnis berusaha untuk
memenuhi kebutuhan konsumen dengan sebaik-baiknya. [1]
Dalam situasi perkembangan dunia usaha yang semakin
beragam dan kompetitif saat ini, sulit untuk memprediksi laju
pertumbuhan yang akan terjadi. Oleh karena itu, memiliki
strategi yang tepat sangatlah penting.[2] Ini dilakukan untuk
pertahanan dalam persaingan bisnis yang semakin ketat dengan
kompetitor lain[3]. Toko retail merupakan bidang usaha yang
sudah berdiri cukup lama. Industri retail adalah salah satu
industri yang sangat penting dan memegang peran yang besar
dalam perekonomian. Saat ini toko retail sangat beredar luas di
Indonesia.
Keberadaan toko retail memberikan kemudahan bagi
masyarakat dalam memenuhi kebutuhan mereka. Dari sini kita
bisa melihat bahwa toko retail memiliki peran yang sangat
penting dalam memenuhi kebutuhan masyarakat dan dalam
bidang perekonomian. Toko Rizieq Putra 2 adalah sebuah toko
ritel yang berlokasi di Kuningan Jawa Barat. Seperti toko ritel
pada umumnya, toko Rizieq Putra 2 juga menawarkan berbagai
kebutuhan masyarakat seperti bahan makanan, perawatan bayi,
popok dan lainnya. Pelanggan toko ini biasanya berasal dari
lingkungan desa setempat[4], [5].
Hasil observasi yang dilakukan pada bisnis Toko Rizieq
Putra 2 menunjukkan bahwa ada masalah pada data transaksi
penjualan. Saat ini, data-data tersebut hanya disimpan sebagai
arsip saja dan terus bertambah setiap hari, tetapi tidak ada
pengolahan yang lebih lanjut yang dilakukan, sehingga hanya
menumpuk tanpa memiliki arti. Setelah melakukan wawancara
dengan pemilik toko, penulis menyimpulkan bahwa Toko
Rizieq Putra 2 membutuhkan implementasi analisis keranjang
belanja (Market Basket Analysis). Penulis menggunakan
29
Mia Fijriani : Implementasi Market Basket ……..
metode data mining yang menggunakan algoritma apriori untuk
mengetahui pola penjualan yang terjadi selama toko ini
beroperasi. Tujuannya adalah untuk mengoptimalkan
pemanfaatan data transaksi sehingga menjadi informasi dan
pengetahuan baru. Penggunaan Market Basket Analysis adalah
bagian dari teknik data mining yang bergerak dalam aturan
asosiasi yang bertujuan untuk menemukan hubungan atau pola
yang berasal dari data transaksi penjualan, sehingga dapat
mempelajari isi keranjang belanja yang dibeli dalam satu
transaksi.
Market Basket Analysis adalah sebuah teknik yang
mempelajari perilaku pembeli atau konsumen secara mendalam,
berdasarkan data dari kelompok-kelompok tertentu.[8] Market
Basket Analysis mempelajari tren belanja pelanggan dengan
mengidentifikasi barang yang sering dibeli bersama dalam satu
transaksi. Ini dilakukan untuk memahami perilaku konsumen
dalam membeli barang yang terkait dan membantu perusahaan
dalam membuat keputusan bisnis yang lebih baik[6], [7].
Dalam implementasi apriori, nilai support digunakan
untuk menentukan frekuensi munculnya suatu data
dibandingkan dengan jumlah data secara keseluruhan[7], [8].
Keuntungan dari algoritma ini adalah sangat mudah digunakan
dan diterapkan, serta dapat menangani data yang besar.
Berdasarkan masalah yang teridentifikasi, implementasi
Market Basket Analysis dilakukan untuk menemukan pola
penjualan barang yang saling terkait. Penelitian ini penting
dilakukan karena data transaksi yang tidak dimanfaatkan
dengan baik dan kurangnya manajemen produk. Pemilik toko
ingin memahami pola penjualan dan perilaku pembeli di Toko
Rizieq Putra 2. Harapannya, melalui Market Basket Analysis,
dapat tercipta pengetahuan baru yang dapat dijadikan dasar
bagi pemilik Toko Rizieq Putra 2 dalam mengambil keputusan
bisnis. Oleh karena itu, peneliti melakukan penelitian bertajuk
"Impementasi Market Basket Analysis Pada Toko Retail
Menggunakan Algoritma Apriori" dengan alasan bahwa
urgensi penelitian sangat penting bagi keberlangsungan bisnis
yang efektif dan efisien pada Toko Rizieq Putra 2.
II. METODOLOGI PENELITIAN
Pada bagian ini akan dijelaskan mengenai metodologi
penelitian untuk melakukan implementasi Market Basket
Basket Analysis Menggunkan Algoritma Apriori[8], [9].
A. Tahap Identifikasi Masalah
Sebagai langkah awal sebelum melakukan penelitian,
dilakukan identifikasi masalah pada Toko Rizieq Putra 2.
Tujuannya adalah untuk mengetahui isu yang ada dan
menjadikannya sebagai bahan penelitian. Teknik pengumpulan
data transaksi ini menggunakan metode wawancara dan
dokumentasi[10], [11].
B. Tahap Data Preparation dan Preprocessing
Proses Data preparation dan processing dalam penelitian
ini mencakup pengumpulan dan pembersihan data transaksi
penjualan Toko Rizieq Putra 2. Tujuannya adalah untuk
memastikan bahwa data yang akan digunakan dalam penelitian
akurat dan siap untuk dianalisa menggunakan market basket
analysis. Tahap-tahap yang dilakukan meliputi pengumpulan
data dari toko, membersihkan data yang tidak relevan, dan
menghapus data duplikat[12]. Setelah selesai dibersihkan, data
akan disusun dan diformat sesuai dengan kebutuhan penelitian
dan siap untuk dianalisis dengan menggunakan algoritma
apriori[12].
C. Tahap Menentukan Nilai Minimum Support dan
Confidence
Pada tahap ini dilakukan menentukan nilai minimum
support dan confidence, dilakukan pengukuran atas setiap item
atau itemset dalam transaksi. Tujuannya adalah untuk
mengetahui frekuensi dari setiap item atau itemset.
Berdasarkan frekuensi tersebut, dapat ditentukan nilai
minimum support dan confidence. Nilai minimum support
adalah nilai minimum dari frekuensi item atau itemset yang
diperbolehkan untuk muncul dalam transaksi[12]. Ini
digunakan untuk menyaring item atau itemset yang tidak
memiliki frekuensi yang cukup tinggi dan tidak memiliki
signifikansi dalam analisis. Sedangkan, nilai confidence
mengukur tingkat keyakinan dari suatu asosiasi antara dua item
atau lebih[13]. Ini menunjukkan tingkat keterkaitan antara item
atau itemset yang dianalisis. Kedua nilai ini berguna untuk
memfilter item atau itemset yang tidak relevan dan
memfokuskan pada item atau itemset yang memiliki
keterkaitan yang kuat dan signifikan. Tahap ini dilakukan untuk
membatasi jenis barang yang memenuhi kriteria untuk
dianalisis lebih lanjut dan membentuk kandidat-kandidat item
set yang akan diolah.
Berikut adalah rumus menghitung nilai support dan Confidence:
𝐽𝑢𝑚𝑙𝑎ℎ 𝑡𝑟𝑎𝑛𝑠𝑎𝑠𝑘𝑖 𝑚𝑒𝑛𝑔𝑎𝑛𝑑𝑢𝑛𝑔 𝐴
𝑇𝑜𝑡𝑎𝑙 𝑇𝑟𝑎𝑛𝑠𝑎𝑘𝑠𝑖
𝐽𝑢𝑚𝑙𝑎ℎ 𝑡𝑟𝑎𝑛𝑠𝑎𝑠𝑘𝑖 𝑚𝑒𝑛𝑔𝑎𝑛𝑑𝑢𝑛𝑔 (𝐴 ∩ 𝐵)
𝐶𝑜𝑛𝑓𝑖𝑑𝑒𝑛𝑐𝑒 (𝐴 ∩ 𝐵) =
𝑇𝑜𝑡𝑎𝑙 𝑇𝑟𝑎𝑛𝑠𝑎𝑘𝑠𝑖 𝑀𝑒𝑛𝑔𝑎𝑛𝑑𝑢𝑛𝑔 𝐴
𝑆𝑢𝑝𝑜𝑟𝑡 (𝐴) =
Identifikasi Masalah
Data Preparation dan
Processing
Menentukan Nilai Minimum
Support dan Confidence
Implementasi Algoritma
Apriori
D. Tahap Implementasi Algoritma Apriori
Pada tahap ini, penulis menerapkan algoritma apriori dengan
menggunakan paket yang tersedia di Rstudio sebagai software
bantu dalam melakukan analisis asosiasi untuk memahami pola
penjualan dari data transaksi yang telah diolah. Algoritma
apriori merupakan bagian dari teknologi data mining untuk
mencari aturan asosiasi[14]. Dalam asosiasi, data mining
berperan dalam mencari aturan yang belum terdeteksi untuk
mendapatkan hubungan antara dua atau lebih atribut.[8]
Asosiasi dalam data mining merujuk pada gabungan,
Hasil
Gambar 1. Metodologi Penelitian
30
DOI: https://doi.org/10.32485/kopertip.v7i1.252
pengumpulan, atau pengorganisasian data Misalnya ketika
produk A dibeli, maka produk B juga dibeli, atau ketika produk
B dibeli maka produk C juga dibeli, dan seterusnya.
Keberadaan atribut yang muncul secara bersamaan dapat
diukur menggunakan nilai confidence. Algoritma untuk
mencari asosiasi dalam data mining sering disebut dengan
Association Rules. Hal ini memungkinkan penemuan item set
yang memiliki keterkaitan yang kuat antar item yang memenuhi
syarat minimum support dan confidence yang sudah ditentukan
sebelumnya. Berikut adalah hasil implementasi algoritma
apriori pada Rstudio.
E. Hasil
Bagian hasil merupakan tahapan terakhir dalam metodologi
penelitian yang memaparkan hasil dari implementasi algoritma
apriori terhadap data transaksi penjualan pada Toko Rizieq
Putra2.
III. HASIL DAN PEMBAHASAAN
Berdasarkan metodologi penelitian yang terlah diuraikan
berikut adalah hasil implementasi Market Basket Analysis
menggunakan algoritma apriori yang dijalankan menggunakan
tools Rstudio.
a.
Identifikasi Masalah
Berdasarkan wawancara dan pengumpulan data yang telah
dilakukan, ditemukan permasalahan yang timbul adalah
kurangnya pemahaman tentang pola penjualan yang ada,
sehingga kurang maksimal dalam melakukan kegiatan
operasional toko. Oleh karena itu, Toko Rizieq Putra
memerlukan Implementasi Market basket Analysis Toko Retail
Menggunakan Algoritma Apriori.
b.
Data Preparation dan Preprocesssing
Tahap ini menghasilkan data yang telah dibersihkan dan siap
digunakan dalam implementasi algoritma apriori, sehingga
hasilnya menjadi lebih akurat dan valid. Terdapat tiga tahapan
dalam mempersiapkan data, yaitu: (1) membersihkan data yang
tidak relevan dengan penelitian, (2) mengelompokkan item ke
dalam jenis-jenis barang tertentu yang telah ditentukan, dan (3)
menghapus data duplikat menggunakan Microsoft Excel.
Selain itu, data transaksi yang terdeteksi menggunakan
separator (;) diubah menjadi (,) agar dapat diimpor ke dalam
RStudio dengan menggunakan fungsi Replace All pada
Notepad. Pengelompokan item dilakukan agar implementasi
Market Basket Analysis lebih efektif dan efisien, serta untuk
memperjelas pola-pola penjualan yang muncul, berikut adalah
17 contoh sebagian data transaksi yang sudah melalui tahap
prepocessing dari total.
Tabel 1. Data Transaksi
Id
Penjualan
4374
Jenis Barang
Rokok
4374
Sembako
4374
Sembako
Id
Penjualan
4375
Jenis Barang
baby care
4376
Sembako
4376
Sembako
4376
Sembako
4376
Snack
4376
Snack
4376
Snack
4377
alat mandi
4378
baby care
4378
Minuman
4378
Sembako
4378
Sembako
4378
Snack
4379
kebutuhan rumah tangga
c. Menentukan Nilai Minimum Support dan Confidence
Sebelum menentukan nilai minimum support dan confidence,
langkah pertama yang dilakukan penulis adalah menghitung
nilai support pada setiap item data transaksi penjualan di Toko
Rizieq Putra 2. Hal ini bertujuan untuk mengetahui kandidat
item mana yang memenuhi atau tidak memenuhi nilai
minimum support yang telah ditetapkan. Penghitungan
kandidat item set dilakukan setelah pengolahan data yang
digabungkan berdasarkan transaksi pembelian produk yang
terjadi secara bersamaan di toko. Setelah nilai support
ditemukan, kandidat item set digabungkan menjadi bentuk
kombinasi itemset baru untuk mengidentifikasi frekuensi
kemunculan data. Namun, tidak semua data transaksi
digunakan dalam penelitian ini. Hanya data yang memenuhi
nilai minimum support sebesar 3% dan nilai confidence sebesar
30% yang digunakan untuk pengolahan data. Berikut adalah
kandidat item set yang lulus dan tidak lulus sesuai nilai
minimum support yang telah ditentukan:
Tabel 2. Kandidat Itemset
No
Jumlah
Transaksi
522
Nilai
Support
0,1435
Dalam %
14,35%
Lulus
2
Jenis
Barang
alat
mandi
atk
386
0,1061
10,61%
Lulus
3
baby care
168
0,0462
4,62%
Lulus
4
diapers
406
0,1116
11,17%
Lulus
5
elektronik
11
0,0030
0,3%
6
kebutuhan
rumah
tangga
kosmetik
dan
skincare
573
0,1575
15,75%
Tidak
Lulus
Lulus
378
0,1039
10,39%
Lulus
1
7
Status
31
Mia Fijriani : Implementasi Market Basket ……..
8
minuman
359
0,0987
9,87%
Lulus
9
p3k
82
0,0225
2,25%
rokok
560
0,1540
15,40%
Tidak
Lulus
Lulus
10
11
sembako
860
0,2365
23,65%
Lulus
No
12
13
snack
susu dan
makanan
bayi
1490
86
0,4097
0,0235
40,97%
2,35%
Lulus
Tidak
Lulus
1
d.
Implementasi Algoritma Apriori
Langkah berikutnya adalah menerapkan Market Basket
Analysis dengan menggunakan algoritma Apriori pada data
transaksi dari Toko Rizieq Putra 2 yang sudah diproses melalui
tahap data preparation dan preprocessing . Algoritma Apriori
akan membantu dalam menemukan asosiasi antara item-item
yang memenuhi nilai minimum support dan confidence yang
telah ditetapkan sebelumnya. RStudio digunakan sebagai
perangkat lunak pendukung untuk melaksanakan proses
implementasi. Berikut adalah hasil implementasi algoritma
apriori:
Tabel 3. Hasil Pola Penjualan
2
3
4
5
6
7
Gambar 2. Implementasi Algoritma Apriori
Dari gambar diatas terlihat bahwa ada 14 rules yang
dihasilkan dari proses implementasi algoritma apriori tersebut.
e.
Hasil
1) Uraian Hasil
Berikut adalah hasil dari basket rule yang
menampilkan asosiasi produk pada Toko rizieq Putra
2.
8
9
10
11
12
13
14
Gambar 3. Hasil Basket Rules
Untuk
menyederhanakan
informasi
diatas
penulis
sederhanakan melalui tabel dengan format persentase sebagai
berikut:
Rules
alat mandi,
sembako =>
kebutuhan
rumahtangga
kebutuhan
rumahtangga
=> sembako
alat mandi,
kebutuhan
rumahtangga
=> sembako
alat mandi
=>
kebutuhan
rumahtangga
minuman =>
snack
kebutuhan
rumahtangga
=> alat
mandi
kebutuhan
rumahtangga
=> alat
mandi
kosmetik
dan skincare
=> alat
mandi
alat mandi
=> sembako
kosmetik
dan skincare
=>
kebutuhan
rumahtangga
diapers =>
sembako
sembako =>
kebutuhan
rumahtangga
minuman =>
sembako
alat mandi
=> kosmetik
dan skincare
Support
CoverAge
5,66%
Lift
3,74%
Confidence
66,01%
4,1
Count
136
8,27%
52,53%
15,75%
2,2
301
3,74%
51,51%
7,26%
2,1
136
7,26%
50,57%
14,35%
3,2
264
4,56%
46,23%
9,87%
1,1
166
7,26%
46,07%
15,75%
3,2
264
3,74%
45,18%
8,27%
3,1
136
4,56%
43,91%
10,39%
3
166
5,66%
39,46%
14,35%
1,6
206
4,07%
39,15%
10,39%
2,4
148
4,04%
36,20%
11,16%
1,5
147
8,27%
35%
23,65%
2,2
301
3,43%
34,81%
9,87%
1,4
125
4,56%
31,89%
14,35%
3
166
Dapat dilihat dari tabel bahwa pola penjualan yang dihasilkan
ada 14 rules dengan informasi lengkap mengenai nilai support,
confidence, coverage lift dan countnya. Cara membaca data di
atas yaitu sebagai contoh perhatikan data pada baris pertama
tabel diatas. Peluang terbelinya alat mandi, sembako bersamaan
dengan kebutuhan rumah tangga sebesar 3,74% dengan tingkat
nilai kepercayaan 66,01%. Kemudian peluang terbelinya alat
mandi, sembako dalam seluruh transaksi sebesar 5,66% dan
kecenderungan terbelinya kebutuhan rumah tangga setelah alat
mandi, semabako yaitu 4,1 kali lebih besar dibanding produk
lain, serta jumlah pembelian alat mandi, sembako terhadap
kebutuhan rumahtangga sebanyak 135 transaksi.
32
DOI: https://doi.org/10.32485/kopertip.v7i1.252
2) Visualisasi Data
2.
Gambar 4. Scatter Plot
Pada scatter plot, setiap titik merepresentasikan sebuah
transaksi tertentu dan lokasinya menunjukkan bahwa dua set
item tersebut dibeli secara bersamaan. Scatter plot tersebut
diurutkan berdasarkan nilai lift, dan semakin pekat warnanya
menunjukkan semakin tinggin nilai liftnya.
[1]
[2]
[3]
Gambar 5. Diagram Penjualan Terlaris
Selain Scatter plot yang telah disebutkan sebelumnya,
penulis juga menyajikan diagram yang menampilkan 10 jenis
barang yang paling laris di Toko Rizieq Putra 2. Diagram
tersebut dapat digunakan oleh pemilik toko untuk mengelola
produk sesuai dengan jumlah penjualan. Berikut adalah
diagram yang menunjukkan hasil penjualan terlaris:
IV.
KESIMPULAN
Dari hasil penelitian yang telah dijelaskan pada bab
sebelumnya, penulis dapat menyimpulkan hal-hal berikut ini :
1. Penerapan Market Basket Analysis menggunakan
algoritma apriori pada data penjualan Toko Rizieq
Putra 2 membantu pemilik toko dalam memahami
pola penjualan. Hal ini terbukti pada hasil yang
memuat aturan-aturan berdasarkan data transaksi yang
diolah melalui bantuan tools Rstudio yang
menampilkan hasil dari kombinasi item set yang
saling berkaitan satu sama lain. Dari data yang
disajikan dapat diketahui bahwa produk yang
cenderung dibeli bersama memiliki nilai support,
[4]
[5]
[6]
[7]
confidence, lift, coverage dan menampilkan informasi
berapa banyak transaksi itemset tersebut yang keluar.
Melalui Market Basket Analysis dengan algoritma
apriori, dapat ditemukan 14 aturan pola belanja
konsumen atau pola penjualan yang mucul secara
bersamaan dengan nilai confidence/tingkat keyakinan
tertinggi terdapat dalam pembelian produk-produk
alat mandi dan sembako, yang kemungkinan besar
akan membeli produk-produk kebutuhan rumah
tangga memiliki nilai confidence sebesar 66%. Nilai
ini paling tinggi juga lift nya sehingga frekuensi dapat
dikatakan lebih tinggi daripada item set yang lainnya.
Hasil dari penelitian ini dapat digunakan oleh Pemilik
Toko Riezieq Putra 2 sebagai acuan untuk mengambil
sebuah keputusan dalam menyusun strategi penjualan
agar bisnis Toko Rizieq Putra 2 dapat berkembang dan
mampu bersaing dengan kompetitor dengan
memanfaatkan data yang sudah diolah sebelumnya
dalam penelitian.
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34
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The Value of Recreational Ecosystem Services in India
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1. Introduction Forests provide a number of ecosystem services to human beings [1–4]. Forest eco-
system services are classified by four major types such as provisioning services (wild
foods, raw materials, etc.), regulating services (climate regulation and weather, etc.), cul-
tural services (spiritual and recreational services), and supporting services (habitat ser-
vices) [3]. Forest ecosystem services have provided a vital benefit for, for instance, poverty
reduction [5–7] and environmental benefits [8–11]. Forest ecosystem services have also
contributed to household income for the forest depended communities [12–14]. Cultural
ecosystem services have played a vital role in human well-being (see Table 1). However,
the 28% income earned from forest in the developing countries. Further, forest ecosystem
services have provided recreational ecosystem services [15]. Recreational ecosystem ser-
vices have been described as the nonmaterial benefits people obtain from ecosystems
through spiritual enrichment, cognitive development, reflection, recreation, and aesthetic
experience [3]. Recreational ecosystem services have a strong association between hu-
man–nature interaction [16] However, there are a number of studies have been estimated
the economic value of ecosystem services in India for example, the economic value and
stock of six tiger reserves at US $128 million to US $271 million and US $344 million to US
$10.08 billion, respectively [17]. The value of ecosystem services has provided US $13–148
million based on various economic valuation methods for the Nagarhole National Park in
Karnataka [4]. The value of recreational ecosystem services at US $167,619 and the value
of carbon sequestration at US $63.6 million for the Corbett Tiger Reserve [18]. The eco-
nomic value of recreation services at Rs 4.4 million provided by Lalbagh botanical garden
based on the travel cost method [19]. Moreover, considering that India accounts for a ma-
jor global biological diversity, ecosystems like forests, wetlands etc., provide a larger
number of benefits to human beings. However, the existing economic growth models be-
ing followed the world over have increasingly led to the degradation of ecosystems and Citation: Balasubramanian, M. The
Value of Recreational Ecosystem
Services in India. Environ. Sci. Proc. 2021, 3, 80. https://doi.org/10.3390/
IECF2020-08030 Academic Editors: Angela Lo
Monaco, Cate Macinnis-Ng and
Om P. Rajora Academic Editors: Angela Lo
Monaco, Cate Macinnis-Ng and
Om P. Rajora
Published: 15 November 2020 Academic Editors: Angela Lo
Monaco, Cate Macinnis-Ng and
Om P. Rajora Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations. Muniyandi Balasubramanian Centre for Ecological Economics and Natural Resources, Institute for Social and Economic Change,
Bangalore 560 072, India; balasmku@gmail.com Centre for Ecological Economics and Natural Resources, Institute for Social and Economic Change,
Bangalore 560 072, India; balasmku@gmail.com Centre for Ecological Economics and Natural Resources, Institute for Social and Economic Change,
Bangalore 560 072, India; balasmku@gmail.com
† Presented at the 1st International Electronic Conference on Forests—Forests for a Better Future: Sustainability,
Innovation, Interdisciplinarity, 15–30 November 2020; Available online: https://iecf2020.sciforum.net. g
,
;
g
† Presented at the 1st International Electronic Conference on Forests—Forests for a Better Future: Sustainability,
Innovation, Interdisciplinarity, 15–30 November 2020; Available online: https://iecf2020.sciforum.net. g
g
† Presented at the 1st International Electronic Conference on Forests—Forests for a Better Future: Sustainability,
Innovation, Interdisciplinarity, 15–30 November 2020; Available online: https://iecf2020.sciforum.net. Abstract: Forest ecosystem services have played a vital role in human well-being. Particularly, rec-
reational ecosystem services are creating physical and mental well-being for human beings. There-
fore, the main objective of the paper is to estimate the economic value of recreational ecosystem
services provides by recreational sites such as Nandi Hills and Nagarhole National Park based on
the individual travel cost method in Karnataka, India. This study has used a random sampling
method for 300 tourist visitors to recreational sites. The present study has also estimated the con-
sumer surplus of the visitors. The results of the study have found that (i) economic value of two
creational sites has been estimated at US $323.05 million, (ii) the consumer surplus has been esti-
mated for Nandi Hills at US $7.45 and Nagarhole National Park at US $3.16. The main implication
of the study is to design the entry fees for the recreational site and sustainable utilization of recrea-
tional ecosystem services for the present and future generations. Keywords: forest; ecosystem services; travel cost method; India www.mdpi.com/journal/environsciproc Citation: Balasubramanian, M. The
Value of Recreational Ecosystem
Services in India. Environ. Sci. Proc.
2021, 3, 80. https://doi.org/10.3390/
IECF2020-08030 Proceeding Paper
The Value of Recreational Ecosystem Services in India † Muniyandi Balasubramanian 1. Introduction Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (http://crea-
tivecommons.org/licenses/by/4.0/). Environ. Sci. Proc. 2021, 3, 80. https://doi.org/10.3390/IECF2020-08030 Environ. Sci. Proc. 2021, 3, 80 2 of 10 2 of 10 their valuable services. Therefore, there is a need for a larger number of economic valua-
tion studies for a better understanding of the importance of ecosystem services, as well as
sustainable use of ecological resources. Moreover, most of the studies focus mainly on the
tangible benefits of very few ecosystem services. their valuable services. Therefore, there is a need for a larger number of economic valua-
tion studies for a better understanding of the importance of ecosystem services, as well as
sustainable use of ecological resources. Moreover, most of the studies focus mainly on the
tangible benefits of very few ecosystem services. Table 1. Cultural ecosystem services and related goods and services. Cultural Ecosystem Services
Examples of Related Goods and Services
Opportunities for recreation and tourism
Hiking, camping, nature walks, jogging, winter sports, wild watching, horse riding, hunting, etc. Aesthetic values
Enjoyment of rural, unique and colorful landscapes, individual habitats and species, and tranquility
supporting mental well-being. Inspiration for the art, science, and technology Writing, painting, design, documentaries, movies, engineering materials, and architecture
Information for education and research
Education trips by schools and other groups; employee training; research related to ecosystem
function, publications and patents. Spiritual and religious experience
Natural and built sacred places, philosophy and faith; support to mental well-being. Cultural identify and heritage
Landscape and habitats formed by human activities, species of spiritual importance, traditional and
indigenous knowledge
Source: [3,20,21]. Table 1. Cultural ecosystem services and related goods and services. The recent global studies on the value of recreational ecosystem services, for exam-
ple, the value of Amazon forest ecosystem services at US $68.47 to US $822.76 million that
includes the value of nuts, rubber, timber, livestock, energy, CO2 sequestration, etc. [22]. The value of ecosystem services provided by the Andassa watershed of the Upper Blue
Nile basin of Ethiopia has been estimated at US $22.58 × 106 in 2000 [23]. The economic
value of 11 ecosystem services in China has been estimated at US $5.63 trillion for 2010. 1. Introduction Among the 11 ecosystem services, regulating ecosystem services has contributed the high-
est value at 71% in the respect of China [24]. In the India context, a number of studies have
estimated the value of recreational ecosystem services, based on the travel cost method
and contingent valuation method. The economic value of recreational ecosystem services
has been estimated at US $0.41 million, based on willingness to pay method for the Na-
garhole National Park in Karnataka [4]. Further, the economic value of recreation ecosys-
tem services provided by Little Rann Kachchh has been estimated at US $4.6 million,
based on individual travel cost and contingent valuation method [25]. On the other side
the value of recreational services, based on secondary data, has been estimated at US $6.5
million for the PeriyarTiger Reserve in Kerala [17]. The Corbett Tiger Reserve accounts for
the economic value of recreational services at US $167,619, based on the individual travel
cost method [26]. The recreational value of coastal and marine ecosystem services, based
on the zonal travel cost method, has been estimated at US $531 billion for 2012-13 [27]. Furthermore, some studies have estimated the value of urban park recreational services. For example, the value of recreation services provided by Lal Bagh botanical garden based
on the individual travel cost method has been estimated at Rs 4.4 million [19]. There are
studies that have dealt with the valuation of recreational sites in Karnataka. The recrea-
tional value has estimated the protected areas of Western Ghats [28] based on the travel
cost method the average consumer surplus per visit Rs. 290. A similar study carried out
in the valley of a national park shows that the net recreational benefit was Rs. 588,332, and
the average consumer surplus Rs. 194.68 [29]. The total recreation value of Dandeli wild-
life sanctuary using the travel cost method for 2004-05has been estimated atRs. 37,142.86
per Sq. km, and the total value of Rs. 17,643,600 [30]. Most of the Indian studies has estimated consumer surplus for recreational sites, for
instance, consumer surplus for the domestic visitors at Rs 227 and foreign visitors at Rs
1384 for Nagarhole National Park in Karnataka [4]. Jala and Nandagiri (2015) calculated
consumer surplus at Rs 238 for Pilikula Lake in Karnataka. Further, the value of consumer
surplus has been estiamted at the US $2.5 for Corbett Tiger Reserve in Uttarakhand [18]. 2.1. Study Area Karnataka has been a number of economic valuation studies conducted with respect
to protected areas, but there is no economic value of recreational services study Nandi
Hills. Nandi Hills are an old hill fortress or evergreen forest patch at the top ofthe Chikka-
ballapur district of Karnataka. It is 10 km away from Chikkaballapur town and approxi-
mately 60 km away from the city of Bangalore. Bangalore is home to a number of private
companies with people from differentparts of the country, and working there exhausts
them and so, on weekends, people like to visit different places in and around Bangalore,
andNandi Hills is one suchnatural outdoor recreation place. Moreover, it is close to Ban-
galore, and hence many people visit it during weekends, as a quick getaway from their
routine life. The Department of Horticulture is maintaining the hill top and climate with
several plant species introduced across an experimental garden, a large-scale exotic bo-
tanical garden (140 acres), music stage (three-and-a-half-acre), food court, and temple. The
hills are very rich in fauna, making this location popular for birdwatchers and bird pho-
tographers. The climate during winter is the best, with the hill top covered with dense
attracting and people to visit, and also this place is home to many migrant bird species
(ex. Warblers, flycatchers), such as Nilgiri woodpigeon and some species of Western
Ghats (Uropeltid snakes, malbar whistling thrush). The hill slopes are home to endemic
species of peninsular India (yellow-throated bulbul). Nandi Hills is one of the best places
for hiking as well as trekking for beginners. Tourists come from various parts of Banga-
lore, the surrounding districts, and other states for viewing the unique nature of the hill. Second, Nagarhole National Park is located in the Kodagu and Mysore districts in Karna-
taka. There are more number of visitors has been visited to the park during the week days
and weekend days. 2.2. Data Collection In order to estimate the economic value of recreational ecosystem services being pro-
vided by the Nandi Hills and Nagarhole National Park, we undertook a field survey in
December 2019 for obtaining empirical data on the number of visits, travel expenditures,
and other socio-economic variables. The field data collection was undertaken through in-
person interviews (Nandi Hills, n = 150 and Nagarhole National Park, n = 150) of individ-
uals who were randomly selected near the entrance or inside the Nandi Hills and Nagar-
hole National Park.However, this study did not cover any foreign tourists due to their
unavailability in the recreation site during the study period. 2.3. Data Analysis 1. Introduction The value consumer surplus for the four tiger reserves in India. Among the four tiger
reserves, Kanha Tiger Reserve (KTR) has the highest consumer surplus value at approxi-
mately Rs 2558, while Kaziranga Tiger Reserve has the second highest consumer surplus Environ. Sci. Proc. 2021, 3, 80 3 of 10 3 of 10 at about Rs 187. Third and fourth place were occupied by Periyar Tiger Reserve and Cor-
bett Tiger Reserve with consumer surplus estimated at about Rs 147.38 to Rs 161.32 and
Rs 150, respectively [31]. The economic value of consumer surplus for three protected ar-
eas such as BiligiriRangaswamy Temple Wildlife Sanctuary (Rs 38.24), Bannerghatta Na-
tional Park (Rs 191.73), and Nagarhole National Park (Rs 557.33) per visit to the recrea-
tional site [32] 2.3.3. Consumer Surplus Consumer surplus has been described as “the difference between the total travel costs
incurred by a visitor to a tourist site and the highest amount the visitor is willing to pay
to make a visit to the site” [37]. The consumer surplus has been obtained [38]. 𝐶𝑜𝑛𝑠𝑢𝑚𝑒𝑟 𝑆𝑢𝑟𝑝𝑙𝑢𝑠= −1
𝛽் 𝐶𝑜𝑛𝑠𝑢𝑚𝑒𝑟 𝑆𝑢𝑟𝑝𝑙𝑢𝑠= −1
𝛽் 𝐶𝑜𝑛𝑠𝑢𝑚𝑒𝑟 𝑆𝑢𝑟𝑝𝑙𝑢𝑠= −1
𝛽் The total annual recreational value of the site can be estimated by multiplying the
individual consumer surplus with the total number of visits during the year. 𝐶𝑆=
ିଵ
ఉ் × total number of visitors per year to the recreational site. 2.3.2. Econometric Model The travel cost method makes the evaluation of individual preferences for expendi-
ture on non-market goods possible. The travel cost method uses the cost of travelling to a
non-priced entertaining location in order to presume the recreational benefits provided
by the site [36]. The present study interviewed 300 visitors to the Nandi Hills and Nagar-
hole National Park. A basic econometric model used in this study shows the number of
visitors is the independent variable to Nandi Hills and NagarholeNatioal Park as func-
tional factors such as travel cost, age, residential location, household income, age, residen-
tial local, household income, household size, educational status, and quality of the park. Hence, the trip-generating functions for the entire datasets are described below. + 𝛽ସ ℎ𝑜𝑢𝑠𝑒ℎ𝑜𝑙𝑑 𝑖𝑛𝑐𝑜𝑚𝑒+ 𝛽ହ ℎ𝑜𝑢𝑠𝑒ℎ𝑜𝑙𝑑 𝑠𝑖𝑧𝑒 + 𝛽𝑒𝑑𝑢𝑐𝑎𝑡𝑖𝑜𝑛𝑎𝑙 𝑠𝑡𝑎𝑡𝑢𝑠+ 𝛽 𝑞𝑢𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑎𝑟𝑘+ 𝑒 where ri is the dependent variable that stands for the number of visits by the ith individual
to Nandi Hills and Nagarhole National Park per period of time; travel cost denotes the
round trip total cost of an individual’s residence to and from the site, and includes the
opportunity cost of travel time and stay at the park. 2.3.1. Travel Cost Method The purpose of this study is to estimate the recreational benefit of Nandi Hills and
Nagarhole National Park, using travel cost method (TCM). TCM is used to calculate the
value of some goods or services that cannot be obtained through market prices such as
forest parks, ecosystems, beaches, etc. The economic value is measured purely based on
people’s preferences. Thus, the theory of economic valuation is based on individual pref-
erences and choices. People express their preferences through choices and trade-offs that
they make, given certain constraints, such as those related to income or time availability
(Ecosystem Valuation, 2013). Travel cost method was first introduced by Hotelling in 1947 Environ. Sci. Proc. 2021, 3, 80 4 of 10 for valuation of protected areas [33], and TCM is one of the best valuation methods for
estimating the economic value of recreational ecosystem services [34]. Travel cost method
is defined by a ‘trip-generating function’, and this study has used the following formula
by [35]: for valuation of protected areas [33], and TCM is one of the best valuation methods for
estimating the economic value of recreational ecosystem services [34]. Travel cost method
is defined by a ‘trip-generating function’, and this study has used the following formula
by [35]: (1) 𝑉= 𝑓ሺ𝐶, 𝑋ሻ
(1) 𝑉 is the number of visits to the site, 𝐶 is the visitor cost, and 𝑋 is the other socio-
economic indicators that are considerably described in 𝑉. Travel cost method has defined
the independent variables (𝑉) as the number of visits made by each visitor to a national
park or wildlife sanctuary or any other recreational site over a specific period. The number
of visits to the Nandi Hills has been estimated based on the time and cost incurred on
travelling to the hill. The time and costs of travel vary from visitor to visitor depending
on the point of origin. The value of a site also depends on how many people are willing
to pay to visit that place. It is called revealed preference method, because the actual be-
havior and choices are used to account for the environmental values. 3. Results and Discussion This section highlights the results and discussion. The result of the study is found
that young age respondents more often visit the recreational site. In addition, the two
study areas such as the National Park and Nandi Hills have received many young visitors. Further, this study has estimated that the university level educated respondents have a
higher number of visits compared to other levels of education. Furthermore, 65 percent of Environ. Sci. Proc. 2021, 3, 80 5 of 10 respondents are earning Rs 25,000 to Rs 50,000, and married respondents created a higher
number of visits to the recreational site (Table 2). respondents are earning Rs 25,000 to Rs 50,000, and married respondents created a higher
number of visits to the recreational site (Table 2). Table 2. Socio-economic status of tourist respondents. Nagarhole National Park
Nandi Hills
Age
18–40
78
66.66
41–60
20
29.33
Above 60
1.3
4
Education
Illiterate
1.3
6
Primary
2
18
Secondary
24.7
70
University level
72
5.33
Household Income
Rs 10,000–Rs 25,000
13.3
14.7
Rs 25,000–Rs 50,000
78.7
47.3
Rs 50,000–Rs 75,000
8
38
Rs 75,000 and above
0
0
Marital Status
Single
8.1
44.67
Married
86.7
54
Widow
0
1.3
Household Size
2 to 5
80
75.3
6 to 10
17.3
20
Above 10
2.7
5.7
Source: Author’s estimates. Figure 1 highlights that respondent’s willingness to pay for visiting to the parks. The
tourist visitors are ready to pay from Rs 10 to the above Rs 500 for visiting the recreational
sites. Nagarohle National Park has received the highest willingness to pay compared to
the Nandi Hills in Karnataka. Figure 1 shows that 80 percent of tourist visitors are ready
to willing pay more than Rs 500 and above for visiting the Nagarhole National Park. More-
over, 25 percent of the visitors are ready to willing to pay between Rs 100 to Rs 150, and
48 percent of the visitors are willing to pay Rs 10 to Rs 50 for the Nandi Hills. This figure
clearly shows that a few tourist respondents are ready to pay the range between Rs 250 to
Rs 350 visiting to the recreational sites. Overall, this study has found the visitors’ average
willingness to pay in the range between Rs 150 to Rs 200 to both the Nagarhole National
Park and Nandi Hills in Karnataka. 3. Results and Discussion Figure 2 highlights the frequency of visits to the rec-
reational site, the minimum one visit, and maximum more than visits. Seventy-five per-
cent and 65 percent of the respondents have visited the Nagarhole National Park and
Nandi Hills once, respectively. Twenty-five percent of the tourist visitors are visiting at
least two times to the recreational sites. Moreover, less than 10 percent of the tourist re-
spondents are visiting three to four times to the tourist areas. Further, less than 5 percent of
the respondents are visiting more than five times to the recreational sites. 6 of 10 Environ. Sci. Proc. 2021, 3, 80 Figure 1. Respondent’s willingness to pay. Source: Author’s estimates. Figure 2. Frequency of visits to the site. Source: Author’s estimate. 0
10
20
30
40
50
60
70
80
90
Rs 10 to
Rs 50
Rs 50 to
Rs 100
Rs 100
to Rs
150
Rs 150
to Rs
200
Rs 200
to Rs
250
Rs 250
to Rs
300
Rs 350
to Rs
400
Rs 450
to 500
Rs 500
above
% Visitors
NNP
Nandi Hills
0
10
20
30
40
50
60
70
80
1
2
3
4
5
Above 5
% visitors
Number of visits
NNP
Nandi Hills Figure 1 Respondent’s willingness to pay Source Author’s estimates
0
10
20
30
40
50
60
70
80
90
Rs 10 to
Rs 50
Rs 50 to
Rs 100
Rs 100
to Rs
150
Rs 150
to Rs
200
Rs 200
to Rs
250
Rs 250
to Rs
300
Rs 350
to Rs
400
Rs 450
to 500
Rs 500
above
% Visitors
NNP
Nandi Hills Figure 1. Respondent s willingness to pay. Source: Author s estimates. Figure 2. Frequency of visits to the site. Source: Author’s estimate. 0
10
20
30
40
50
60
70
80
1
2
3
4
5
Above 5
% visitors
Number of visits
NNP
Nandi Hills Figure 2. Frequency of visits to the site. Source: Author’s estimate. Frequency of visits to the site. Source: Author’s estim Table 3 highlights the regression results of the individual travel cost method for both the
recreational sites in Karnataka. Travel cost is significant and negatively correlated. Table 3
indicates that one percent increase in the travel cost has impacts on 1.7 percent decrease
in the number of trips for Nagarhole National Park and 3 percent decrease in the Nandi
Hills trips, respectively. 3. Results and Discussion Age and number of trips are statistically significant and nega-
tively correlated, old age people have less visits to the recreational site compared to the
young age respondents. Residential locations and visiting to the recreational site are sta-
tistically significant, and a one percent increase in the distance impacts on 1.9 percent and
1.7 percent Nagarhole National Park and Nandi Hills. In addition, household income is
statistically significant with the number of trips, for example, one percent increases in re-
spondent’s income impact on 2 percent increase number of visits to the Nagarhole Na-
tional Park and Nandi Hills in Karnataka. In addition, household size, marital status, qual-
ity of the park, and educational status are not statistically significant, but positively cor-
related with the recreational site. Table 4 shows the consumer surplus and economic value
of the two recreational sites for Nagarhole National Park and Nandi Hills. The consumer
surplus has been estimated for Nagarhole National Park at Rs 247 and Nandi Hills at Rs
557, respectively. In addition, the economic value of NNP has been estimated at 55.8 mil-
lion and 2.47 billion for the Nandi Hills. Environ. Sci. Proc. 2021, 3, 80 7 of 10 Table 3. Regression results of recreational values of Nagarhole National Park (NNP) and Nandi Hills. Variables
Coefficient
t-Statistics) NNP
Coefficient
(t-Statistics) Nandi Hills
Intercept
0.980
(2.761)
1.823
(4.037)
Travel Cost
−1.014 × 10−5
(−1.716) **
−0.247
(−3.074) ***
Age
−0.009
(−2.136) **
−0.175
(−2.212) **
Marital Status
0.113
(1.110)
0.431
(2.301)
Household size
0.060
(1.264)
0.049
(2.386)
Educational status
−0.017
(−1.285)
0.983
(2.487)
Residential location
0.139
(1.969) **
0.140
(1.750) *
Household Income
3.880 × 10−6
(2.108) **
0.149
(1.846) *
Quality of the park
−0.47
(−1.258)
−0.32
(−1.130)
R2
14.0
12.9
F-Statistics
2.837
2.273
Note: ***, **, * denote significance at 1%, 5%, 10% levels, respectively. Table 3. Regression results of recreational values of Nagarhole National Park (NNP) and Nandi Hills. Table 4. Economic value of recreational ecosystem services in Nandi Hills and Nagarhole National
Park. Components
Nandi Hills Value in (Rs) Nagarhole National Park
Individual Average Consumer Surplus
Rs 247
Rs 557.33
Total Economic Benefits
Rs. 2.47 billion
Rs 55.8 million
Source: Author’s estimate based on primary survey. Table 4. Economic value of recreational ecosystem services in Nandi Hills and Nagarhole National
Park. 4. 3. Results and Discussion The recreational
value of coastal and marine ecosystem services, based on the zonal travel cost method,
has been estimated at US $531 billion for 2012–2013 [27]. Whereas, the economic value of
Dachigam National Park in Jammu and Kashmir, based on the travel cost method, has
been estimated at US $4.5 million. Furthermore, some studies have estimated the value of
urban park recreational services. For example, the value of recreation services provided
by Lal Bagh botanical garden based on the individual travel cost method has been esti-
mated at Rs 4.4 million [19]. There are studies that have dealt with the valuation of recre-
ational sites in Karnataka. The recreational value in respect of the protected site of Western
Ghats [28] based on the relationship between travel cost and visitation rate and willing-
ness to pay, has been estimated at Rs. 26.7 per visitor, and the average consumer surplus
per visit Rs. 290. A similar study carried out in the valley of a national park shows the net
recreational benefit at Rs. 588,332, and the average consumer surplus at Rs. 194.68 [29]. The total recreation value of Dandeli wildlife sanctuary using the travel cost method for
2004-2005 has been estimated at Rs. 37,142.86 per Sq. km, and a total value of Rs. 17,643,600
[30]. Similarly, a study based on the willingness to pay for the preservation of watershed
in Karnataka indicates a value of Rs.125.45 per hectare, and a total value of Rs. 480 million
(for 2004–2005). Further, [32] estimated the value of recreational ecosystem services based
on individual travel cost method for BilgiriRangaswamy Temple Wildlife Sanctuary (Rs
3.8 million), Nagarhole National Park (Rs 55.8 million), and Bannerghatta National Park
(Rs 19 million) for Karnataka. 3. Results and Discussion Economic value of recreational ecosystem services in Nandi Hills and Nagarhole National The result of the study has found that (i) economic value of two creational sites has
been estimated at US $323.05 million, (ii) the consumer surplus has been estimated for
Nandi Hills at US $7.45 and Nagarhole National Park at US $3.16. Similar results have
found that the various recreational sites, for example, the value of tourism 525 million
euros in the six German national parks based on travel cost method [39]. An economic
value of recreational ecosystem services has been estimated at $AUD 3.3 billion per year
provided by New South Wales in south-eastern Australia [33]. Further, the value of rec-
reational ecosystem services has been estimated at US $31.8 million for McKenzie Lake,
Fraser Island based on travel cost method [40]. Economic value of Gold Cost beaches has
been estimated at US $500 million per year based on travel cost method for Australia [41]. The value of recreational ecosystem services provided by Coorong, Australia has been
estimated at US $30.5 per year [42]. The value of recreational benefits has been estimated
at 359 to 574 euro per visit to the Marine Nature Reserve (MNR) in the United Kingdom
[43]. Economic value of the Poseidon temple in Sounio, Greece has been estimated at 1.5–
24.5 million per year based on travel cost method [44]. In India, there are a number of studies that have investigated the value of recreational
ecosystem services for national parks and wildlife sanctuaries. For instance, the value of
recreational ecosystem services at Rs 773.45 million for the Kaziranga National Park based
on travel cost method [45]. The economic value of Nagarhole National Park, especially
recreational ecosystem services, has been estimated at US $0.41 million, based on willing-
ness to pay method [4]. The economic value of recreation ecosystem services provided by
Little Rann Kachchh has been estimated at US $4.6 million, based on individual travel cost Environ. Sci. Proc. 2021, 3, 80 8 of 10 8 of 10 and contingent valuation method [25]. On the other side, the value of recreational services,
based on secondary data, has been estimated at US $6.5 million for PeriyarTiger Reserve
in Kerala [17]. The Corbett Tiger Reserve accounts for the economic value of recreational
services at US $167,619, based on the individual travel cost method [46]. g
3.
Pascual, U.; Muradian, R.; Brander, L.; Gómez-Baggethun, E.; Martín-López, B.; Verma, M.; Armsworth, P.; Christie, M.; Cor-
nelissen, H.; Eppink, F.; et al. The economics of valuing ecosystem services and biodiversity. In Economics of Ecosystems and
Biodiversity: Ecological and Economic Foundations; Routledge: Oxfordshire, UK, 2010; pp. 183–256. 4. Conclusions Recreational ecosystem services play vital roles towards human well-being. Most of
the developing and developed people are more interested in tourism and recreation. Rec-
reational ecosystem services have an important role in the mental and physical well-being
of people. However, recreational ecosystem services have created a larger number of eco-
nomic and employment opportunities for local people. The present study has estimated
the value of recreational ecosystem services for two recreational sites such as Nagarhole
National Park and Nandi Hills in Karnataka based on the Individual Travel Cost Method
(ITCM). In addition, this study has also estimated the consumer surplus for the two rec-
reational sites. This study has estimated that (i) the economic value of two creational sites
at US $323.05 million, and (ii) the consumer surplus for Nandi Hills at US $7.45 and Na-
garhole National Park at US $3.16. The main policy implication of the study is to design
(1) land use and land cover policy, (2) designing entry fees for the various protected areas
for sustainable tourism, and (3) achieving sustainable development goals (SDGs) at the
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Patient Safety Culture and Nursing Shortage among Nurses in Al-Medina Hospitals
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Journal of Health, Medicine and Nursing
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Patient Safety Culture and Nursing Shortage among Nurses in
Al-Medina Hospitals Dr. Saeed S. Banawas
Assistant Professor in Microbiology
Department of Medical Laboratories
College of Applied Medical Sciences Majmaah University, Saudi Arabia Dr. Jehan Yehia AbdElhaliem ElRazkey
Assistant Professor in Microbiology
Department of Medical Laboratories
College of Applied Medical Sciences Majmaah University, Saudi Arabia Kayd Ayed Al_Erwi
Department of nursing science
College of Applied Medical Sciences Majmaah University, Saudi Arabia Bander Saleh Alrasheedi
Department of nursing science
College of Applied Medical Sciences Majmaah University, Saudi Arabia Abstract This article explores the current relationship of patient safety culture to the nursing shortage in general hospitals
in the city of Al-Medina, Saudi Arabia. A mixed methods article was conducted on 335 respondents drawn from
10 government hospitals in the region. We utilized an AHRQ Questionnaire for data collection, and we analyzed
the data with statistical approaches such as the mean, percentages, and standard deviation. This analysis
established significant relationship between patient safety cultures and nursing shortages. Furthermore, we found
a weak and positive relationship between some of patient SC’s components and nursing shortages in the
hospitals studied; these components included communications (r=0.144, p<0.05), the frequency of event reports
(r=0.151, p<0.05), and hospital work area (r=0.329, p<0.05). Most of the nurses who participated in the current
article had low incident report rates, with only three to five event reports in one year. However, among the
dimensions of patient safety culture, it was found in this article that the nurses had a high level of
communication (3.91±0.54/High). The overall grading of patient safety culture was moderate (3.27±0.93) in Al-
Medina City. The findings of this investigation will help the Ministry of Health (MOH) decide how to improve
patient safety. Moreover, to increase the condition of nursing care provided to the patients and to boost patient
safety culture, the healthcare sector must consider the factors and other causes. It is also crucial that nurse
leaders learn to maintain and secure patient safety and that nurses learn to frequently report incidents, including
any actual errors. Keywords: Patient Safety, Culture, Nursing Shortage
DOI: 10.7176/JHMN/100-07
Publication date:May 31st 2022 Publication date:May 31st 2022 Journal of Health, Medicine and Nursing Journal of Health, Medicine and Nursing Journal of Health, Medicine and Nursing
ISSN 2422-8419
An International Peer-reviewed Journal
Vol.100, 2022 www.iiste.org Patient Safety Culture and Nursing Shortage among Nurses in
Al-Medina Hospitals
Dr. Saeed S. Banawas
Assistant Professor in Microbiology
Department of Medical Laboratories
College of Applied Medical Sciences Majmaah University, Saudi Arabia
Dr. Jehan Yehia AbdElhaliem ElRazkey
Assistant Professor in Microbiology
Department of Medical Laboratories
College of Applied Medical Sciences Majmaah University, Saudi Arabia
Kayd Ayed Al_Erwi
Department of nursing science
College of Applied Medical Sciences Majmaah University, Saudi Arabia
Mansor Abdulrahman Abu Hussain
Department of nursing science
College of Applied Medical Sciences Majmaah University, Saudi Arabia
Bander Saleh Alrasheedi
Department of nursing science
College of Applied Medical Sciences Majmaah University, Saudi Arabia 1. Introduction Moreover, ongoing organizational training of various types, including
analysis, supervision, and giving feedback, is also a common strategy discussed by risk management staff. Dedobbeleer (1998) proposed another critical component of SC: the attitude of workers concerning safety. Safety problems can develop if attitudes and perceptions about safety are not recognized and taken into account. Recent studies have shown that health care human resources are a dominant PS challenge in several
countries such as Taiwan, Ethiopia, and the United States (Mekonnen, Mclachlan, Brien, Mekonnen & Abay,
2017; Chen & Li, 2010; Famolaro, Yount, Burns, Flashner& Liu, 2011). Allocating nurses to meet demand
while not jeopardizing PS presents major challenges (Jye, Hing, Peter, Bartholomew, & Senok, 2019). In several
countries, staffing is not a major concern, particularly when standard operating procedures, policies, and systems
on the lack of nurses and nurse workloads are given utmost attention (Sorra, & Nivea, 2004; Bartoníčková et al.,
2019). Fatigue can be a consequence of overworked nurses due to nurse shortages, and this can cause errors and
compromise PS (Keller, 2009). p
(
,
)
PS’s relationship with nurse workloads and shortages has been explored comprehensively in other countries. However, there is little paper available regarding this concern in Saudi Arabia. Therefore, it is worth
investigating whether Al-Medina hospitals can advance a culture of care of PS, and to what extent the culture is
embodied in the hospital system. Furthermore, examining the consequences of the scarcity of nurses on PS care
by soliciting the nurses’ experiences is the most likely method to capture previous incidents that compromised
PS. This way, the identified effects can be addressed for proper action and reflected through the article’s
recommendations. The researcher brings with him his determination, dedication, and experience to make certain
that this undertaking will benefit the nursing profession together with the nursing staff and patients. g
g p
g
g
p
The researcher’s trepidation for PS as a healthcare provider is one driving force that triggered this article. As observed by the researcher, clinical errors have continued despite the resources invested in PS by health care
institutions. For example, there have been cases of medication errors, patient falls, and failure to document
interventions. The lack of nurses (as observed by the researcher personally) is an evident factor contributing to
these incidents in Saudi hospitals. 1. Introduction Various investigators have explored the impact of the nurse shortage on service delivery in the Kingdom of
Saudi Arabia. For example, the McKinsey Global Institute (2015) noted that the existing nurses’ workload is
high, which leads to burnout, fatigue, and work-related stress. Indeed, turnover among nurses is also high in the
United States. Another impact of nurse shortage is compromised patient safety (PS) programs. For example, if a
nurse is expected to care for over 100 patients, patient-centered care will become impossible and patients’ safety
will be jeopardized. Nurse shortages can also cause misdiagnoses because unqualified nurses are often employed
to fill the gap. Moreover, there is a shortage in the number of supervisors, which further risks the safety of the
patients. However, this investigation will focus on the nurse shortage and its possible effects on patient safety in
the KSA. The term “safety culture” was first introduced after the nuclear explosion at Chernobyl in 1986. The
International Atomic Energy Agency (IAEA) presented the term in its initial evaluation after the accident. 105 Journal of Health, Medicine and Nursing www.iiste.org w.iiste.org ISSN 2422-8419
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Vol.100, 2022 Researchers further developed the meaning of the word, and a review of the literature reveals several
connotations. The IAEA characterizes one of the two basic concepts of safety culture as “a set of characteristics
and attitudes in organizations and individuals classified as priorities, where the safety aspects of nuclear power
plants pay attention to their interests” (IAEA, 1991). A second characterization was adopted by the British Health and Safety Commission (HSC). The
organization supported and laid out the characteristics of positive safety cultures, defining the idea as “group
values and individual outcomes, attitudes, perceptions, behavioral models, driving skills involvement that
determines the skills and style and management of health and safety of the organization” (Safety and Health
Committee, 1993). Institutions that possess a good safety culture rely on communication based on mutual trust,
which is in turn based on trust in organizational effectiveness. Safety cultures promote preventative actions and a
general understanding of the importance of safety (HSC, 1993). Pidgeon and O’Leary (1994) argued that a “good” security culture possesses and promotes four factors,
including “senior management's commitment to security” and policies that are practical, flexible, and that
address vague and unclear threats. 3. Hypothesis There is a significant relationship that exists between the patient safety cultures to the nursing shortage in the
general hospitals of Al-Medina City* Based on the aforementioned objectives, lone null hypothesis was made: H:
There is no significant relationship exists between the patient safety cultures to the nursing shortage in hospitals
of Al-Medina city. 2. Aim, Objectives and Paper Question ,
j
p
Q
The aims of this article to determine the patient SC along with the effects of nursing shortage on PS as perceived
by the nurses in select Al-Medina hospitals in 2019. The article specifically addresses the following objectives: 1. j
p
The aims of this article to determine the patient SC along with the effects of nursing shortage on PS as perceived
by the nurses in select Al-Medina hospitals in 2019. The article specifically addresses the following objectives: 1. Determine the overall grading of the patient SC by the nurses in general hospitals of Al-Medina. 2. Determine the relationship between patient safety culture and the nursing shortage. Determine the relationship between patient safety culture and the nursing shortage. Determine the relationship between patient safety culture and the nursing shortage. "Is there relationship between patient safety culture and the nursing shortage in Al-Medina Hospitals" Determine the relationship between patient safety culture and the nursing shortage. "Is there relationship between patient safety culture and the nursing shortage in Al-Medina Hospitals" "Is there relationship between patient safety culture and the nursing shortage in Al-Medina Hospitals" 1. Introduction The number of staff nurses often does not match the proportion of patients
under their care. Also, despite the established standard operating procedures and measures, some health care
institutions have failed to strictly implement policies. Such lax implementation creates a confusing double
standard for PS. These observations and incidents demonstrate the gap that this article aims to fill 5.1 Patient Safety (PS) 5.1 Patient Safety (PS) 5.1 Patient Safety (PS) PS is a worldwide issue that requires multiple skills and knowledge areas, including human factors and system
design. Numerous theoretical approaches and methods may be used to examine, prevent, and mitigate medical
errors. Due to the complexity of job systems and healthcare processes, robust partnerships are necessary to
increase PS in the health sciences. These collaborations will improve PS, introducing the program reforms
required to strengthen the “work systems and processes” necessary for PS in the health care context (Carayon &
Wood, 2010). Wood, 2010). 4. Significance of the Article g
This article aims to provide scientific proof that nurse shortages negatively affect patients’ safety, helping policy
makers in the quality department and hospital management develop means of ensuring that there are enough
nurses in hospitals. It can also help students to identify the effect of nurse shortages and how to address the 106 Journal of Health, Medicine and Nursing .iiste.org ISSN 2422-8419
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Vol.100, 2022 problem. Clients. Patients will be assured of safe and quality care, which will guarantee patient satisfaction. They will
develop confidence and trust in the healthcare sector. Nurses. Nurses will be able to deliver high-quality, safe nursing care consistently, as the number of patients
that they care for will be within the standards of the patient-to-nurse ratio. They will be able to manage their time
effectively and efficiently to provide care, compassion, competence, and commitment. y
y
p
p
p
Other Healthcare Professionals. Collaboration with the nurses will become more effective and efficient as
the proper healthcare professional-to-patient proportion is achieved. Teamwork will improve, and the team will
be able to manage the patients safely. g
p
y
Hospital Administrators. Administrators can make use of the recommendations of this article to come up
with policies to cover the patient-to-nurse proportion. They will be able to adopt measures to address workforce
requirements to ensure PS. q
Policymakers. This article will help policymakers in the quality department develop means of ensuring that
there are enough nurses in hospitals. New policies, standards, regulations, and laws may be drafted,
recommended, and implemented to guarantee positive patient outcomes and PS. p
g
p
p
Researcher. Through this work, the researcher will gain new knowledge about the variables being studied
and contribute to the nursing profession through the output of the article. Furthermore, the researcher will gain
expertise in conducting paper studies. Future Researchers. This article will help students to identify the effect of nurse shortages and how to
address the problem. The findings of the article can serve as future paper topics. To fortify the article’s findings,
a replication of the article may be done by future researchers to cover a wider area and a greater number of
participants. A validation may also be performed using a different paper design. 6. Nurse Staffing and PS g
The diligence of nursing staff at the bedside is vital to their capacity to safeguard PS. It is inevitable that
excessively high workloads jeopardize nurses’ ability to deliver safe care. Several publications have revealed a
correlation between the nurse-to-patient proportion and PS, showing that a lack of staff increases PS-related risks
of illness and death. In 2004 in CA, such data led many states to develop legally mandated “minimum nurse-to-
patient proportions.” In CA, a registered nurse will only be assigned to handle five patients (AHRQ, 2019). Nurse-to-patient proportions are just one element of the connection between nurse workload and PS. The
cumulative workload in nursing is also likely to be related to patient outcomes. A article in 2011 revealed that
an increase in client turnover was also linked to an increased risk of death, despite adequate nursing staff. Nurse
staffing is an extremely complicated procedure that varies on a shift-by-shift basis and requires intimate
collaboration amongst the administration, nurse administrators, and nursing heads. Staffing must account for
perception, retention, provision accessibility, and the proficiency mix, among other influences. The application
of nursing skills and training can also be related to patient outcomes. One article revealed lower inpatient
death rates for different surgical clients with nurses with superior educational attainment. This finding led to
recommendations that nurses gain at least a baccalaureate degree. Independently of educational level, the caliber
of OJT nurse training can significantly influence clinical outcomes (AHRQ, 2019). The correlation between nurse-to-patient proportions and client outcomes is likely due to higher workloads
and excessive stress and burnout risk among nurses. Failure to provide nursing care—an oversight in which the
necessary nursing care essentials are not provided or completed—is somewhat widespread in hospital inpatient
units. According to a British investigation, nursing incidents are closely linked with very high numbers of
patients assigned to each nurse. Burnout among both nurses and doctors has been consistently linked with risks
to PS, and other studies reveal that an increased number of clients per nurse correlates with a high risk of nurse
burnout (AHRQ, 2019). (
Q
)
The high-intensity nature of nursing ensures that nurses are at greater risk of making errors when delivering
routine care to their clients. 5.2 Safety Culture (SC) The subsequent items are the dimensions of patient SC: “(a) Communication openness is the degree to
which the staff speak freely when they see anything that concerns a patient and feel free to ask more informed
questions; (b) Feedback and communication about errors is the extent to which staff are informed of errors that
occur, provide feedback on changes, and discuss ways to prevent errors;(c) Frequency of events reported is the 107 Journal of Health, Medicine and Nursing
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extent to which errors of the following types are reported: (a) errors spotted and remedied before impacting the
client; (b) errors with no potential to damage the client; and (c) errors that may harm the client but do not. (d)
Handoffs and transitions (e) PS management support is the degree to which the management of the hospital
provides a working environment that allows safety of patients and displays that PS is of utmost importance. (f)
Non-punitive response to errors is the degree to which employees believe that their errors and incident reports
are not against them and that errors are not kept in their personnel file. (g) Organizational Learning —
Continuous improvement is the degree to which the errors have contributed to positive changes and the success
of the changes is measured. (h) Public expectations of PS are to the degree the processes and systems are good at
preventing accidents and PS concerns are missing. (i) the degree that there are adequate staff to manage the
workload and working hours, it is reasonable to provide patients with the best possible care. (j) The degree to
which supervisors / managers accept staff suggestions for improving PS, applaud staff for pursuing PS protocols
and do not ignore PS concerns is the standards and actions of supervisors / managers supporting PS. (k) The
extent to which the hospital departments collaborate and communicate with each other in order to provide
patients with the best possible care. 5.2 Safety Culture (SC) (l) The degree to which employees support each other, treat others with
respect and play as a team (Famolaro et al., 2018).” Journal of Health, Medicine and Nursing w.iiste.org ISSN 2422-8419
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Vol.100, 2022 extent to which errors of the following types are reported: (a) errors spotted and remedied before impacting the
client; (b) errors with no potential to damage the client; and (c) errors that may harm the client but do not. (d)
Handoffs and transitions (e) PS management support is the degree to which the management of the hospital
provides a working environment that allows safety of patients and displays that PS is of utmost importance. (f)
Non-punitive response to errors is the degree to which employees believe that their errors and incident reports
are not against them and that errors are not kept in their personnel file. (g) Organizational Learning —
Continuous improvement is the degree to which the errors have contributed to positive changes and the success
of the changes is measured. (h) Public expectations of PS are to the degree the processes and systems are good at
preventing accidents and PS concerns are missing. (i) the degree that there are adequate staff to manage the
workload and working hours, it is reasonable to provide patients with the best possible care. (j) The degree to
which supervisors / managers accept staff suggestions for improving PS, applaud staff for pursuing PS protocols
and do not ignore PS concerns is the standards and actions of supervisors / managers supporting PS. (k) The
extent to which the hospital departments collaborate and communicate with each other in order to provide
patients with the best possible care. (l) The degree to which employees support each other, treat others with
respect and play as a team (Famolaro et al., 2018).” 5.2 Safety Culture (SC) f y
(
)
Safety culture is a multifaceted subject. The healthcare system and independent health care facilities have
described safety culture, polled staff and medical personnel, created performance enhancement strategies for
safety results, and built models and resources to direct and assist in the process. Nevertheless, both hospitals and
the public still have unanswered questions, such as whether hospitals can provide a safe environment for their
patients and people in the community. Questions may also be raised if the changes introduced to guarantee safety
cause changes in the community. Several steps can be taken by government officials in order to guarantee a
culture of safety. McCarthy and Blumenthal (2006) affirmed that “policymakers could help stimulate a culture of
safety by linking regulatory goals to SC expectations, sponsoring collaborations, rewarding safety improvements,
better using publicly reported data, encouraging consumer involvement, and supporting paper and education,”
(Sammer, Lykens, Singh, Mains & Lackan, 2010). (
y
g
)
Hansen et al. (2003) suggested that policymakers update the nurse-to-patient proportion guidelines and
identify provider functions and accountabilities, particularly care “extenders” such as nurses. Leaders must
examine the links between workplace culture, a rapidly changing labor force, and economic and quality
achievements (Gelinas & Loh, 2004). Researchers believe that nursing, other health professions, and auxiliary
academics should integrate SC values in educational curricula. 5.3 Patient SC Composites This article’s “Hospital Survey on Patient SC” included two queries that solicited participants to assign their
“work area or unit” a general rating on “PS” and to provide the frequency of incidents they recorded in the past
twelve months. Furthermore, participants were asked to provide a partial contextualization of their personal data,
including their work area or unit, their respective positions, their direct patient interaction, and their tenure in
their respective work area or unit (Famolaro et al., 2018). 6. Nurse Staffing and PS The theory of human factors technology suggests that when a person tries to perform
a difficult task, such as giving medicine to a sick patient, the working conditions should be as favorable as
possible to perform the job. However, predictable factors such as disruptions and equipment defects can interfere
with the ability of nurses to execute their duties; a few publications have already established that disruptions are
standard in nursing jobs. These disruptions are linked to an increase in errors, particularly in medication
administration. Although some disruptions are clearly imperative for clinical care, the connection between
disruptions and errors is one example of how shortcomings in nurses’ day-to-day workplace are closely related
to PC (AHRQ, 2019). Longer hours and overtime were also linked with an increased risk of error. In a well-known case, an
extremely tired nurse committed an error because this nurse had already worked a double shift. This error
concluded in a criminal trial of the nurse. Nurses who commit errors are in jeopardy of being punished for those
errors. A trend toward thorough documentation is also linked to an increase in self-reported failure. This tends to
lead nurses and other medical and paramedical professionals to leave their profession. Nurses are often subjected
to intrusive or inappropriate behaviors by the rest of the medical team, particularly the doctors. This type of
experience contributes to emotional stress that leads to burnout, as it ruins nurses’ mental health and pushes them
to quit. Given that the job becomes high-risk with an increased workload, pressures from different sources like
negative behaviors from colleagues or supervisors and the mental burden from committing errors create retention
issues. A low client proportion is dangerous, particularly when nurses are exposed to other forms of negative
stress. In the end, the nurse will feel burnout and resign. In the so-called Swiss cheese model of medical errors, 108 Journal of Health, Medicine and Nursing ISSN 2422-8419
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Vol.100, 2022 nurses who commit active errors are predicted to cover up their errors rather than declare them so as to avoid
potential damage to themselves (AHRQ, 2019). 9. Analysis Frequency Distribution and Percentage Frequency distribution is a statistical proportion displaying how many times a variable occurs for each of its
probable values. This was used to present the data on hospitals and personal features. Percentage was also used
to go along with the frequency distribution. g
This technique was used to rank the identified effects of staffing shortages on PS as identified by the nurses. St
d d D
i ti This technique was used to rank the identified effects of staffing shortages on PS as identified by the nurses. Standard Deviation The standard deviation (SD) describes the spread of scores around the mean. This was used to determine the
extent to which the hospitals’ measures differed from the mean. If the hospitals included in the article resulted in
the same measures, then the mean would signify that the measures were flawless and the value of standard
deviation was zero. If the hospitals’ measures were extremely near to the mean, then the value of SD would be
close to zero. Additionally, if the measures were dissimilar from the mean, then the SD would be high. When the
distribution of hospital measures is normal and forms a bell-shaped curve (“where most of the measures fall in
the middle of the distribution, with fewer measures at the lower and superior ends of the distribution”), the mean,
plus or minus the standard deviation, will comprise around 68% of all hospital measures. 8. Methods This article utilized a descriptive design supported by Quantitively data. Descriptive paper is employed
whenever there is limited information about a certain event (Burns & Grove, 2005; Walker, 2005). The
researcher of the article only observes, explains, and records the features of the event; in this type of design,
variables are not manipulated to cause effects in the subject being studied. In addition, such studies simply
explain the phenomenon under investigation, describe how often it occurs, and classify the data. In other terms,
descriptive researchers pose Level I paper questions (Polit, Beck, & Hungler, 2001; Seers & Crichton, 2001;
LoBiondo-Wood & Haber, 2002). This article descriptively determines the hospital and personnel features
related to patient SC (in all its dimensions), together with perceptions of the effects of nursing shortages on PS
among Al-Medina nurses in 2019. Recommendations will be proposed based on the results of the article. g
p
p
The participants of the present article were the nurses from the 10 Government hospitals in
Probability sampling methods we used through simple random technique. To proportionately distribute the sampling, total population of 3,840 requires a sample of size of 350 nurses
I use (Sample Size Calculator, confidence interval and confidence level) to get sample size of total population. To proportionately distribute the sampling, please refer to the table below for the sample size for each of the
hospital. The intended size of the sample was based on the following: random selection, 50 percent response rate,
and +/−5 percent confidence interval. 7. Nursing Shortage in Saudi Arabia The shortage of nurses has proved a challenge in Saudi Arabia. The report of the Ministry of Health (2009)
stated that the nurse shortage crisis led to a heavy reliance on expatriates. Previous studies have shown that Saudi
Arabia recruits foreign nurses of different origins (AlYami and Watson, 2014), demonstrating that Saudi Arabia
is very short-handed in nursing staff. The problem with hiring expatriates is that a language barrier is present,
affecting the nurses’ quality of care. Foreign nurses face a handicap since they cannot establish good
communication because they cannot speak the native language of Saudi Arabia (Al-Mahmoud & Mullen, 2013). Several factors may have caused the crisis of the nurse shortage. One factor that contributes to the nursing
shortage is the work environment. In Saudi Arabia, working night shifts is considered an undesirable condition
in the profession, leading to a high turnover rate (Aboshaiqah, 2016). In addition, the nurse shortage results in a
heavier workload and longer hours for the nurses who remain in their job. AlYami and Watson (2014) also noted
that nurses are hindered by unpleasant working conditions. Similarly, studies have shown that many nurses were
dissatisfied with their workplace, and the supervisors could not meet their needs (Almalki, 2011). These factors
all lead to the resignation of nurses, thus contributing to the nurse shortage in the country. 11. Findings This article found that the aspect of communication was the highest compared to the other elements of patient
safety, with a mean score of 3.91±0.54. Most nurses reported that they had always been informed whenever
modifications occurred based on their event reports. These results indicate that the nurses were well informed of
the changes that were happening inside the institutions. However, most reported that they rarely asked questions
of those in higher positions. This is due to the fact that there is a huge gap between the hierarchical status of the
nurses and the supervisors. This outcome is in agreement with the results of a article conducted in Africa, which
found that doctors, as one of the healthcare professionals who are slightly higher in position compared to staff
nurses, tend to disobey safety policies with impunity because they have the authority to control their behavior
(Aveling et. al 2015). This difference in hierarchy is a possible reason why it is difficult for nurses to question
the actions of employees with more authority, as they are afraid that it will compromise their status in the
institution. In terms of the hospital work area, it was found out in the present article that the nurses perceived it as
moderate. Most nurses never let the safety of the patients be jeopardized in order to get more work done
(3.94±0.98). However, most nurses disagreed that their procedures were good and were designed to prevent
future errors (2.60±1.14). The majority of nurses also felt that whenever they made mistakes, it was held against
them. This outcome is the same as that of Chegini et al. (2020) and Alqattan (2018), who found that nurses’
mistakes would be remembered and used against them in the near future. However, this contrasts with the article
conducted by Alrowely and Baker (2019), who found that most of their participants did not feel the same
predicament because most of the participants were actively engaged in the improvement of patient safety in their
field. The nurses in the present article might have observed and felt that they were in a workplace with an intense
and strict atmosphere, making it difficult to forget the mistakes they committed for the fear that it may affect
their job. The participants in the present article also agreed that patient safety problems occur in their unit. 11. Findings These
areas also seem to be problematic in Bhutan, where several concerns have been identified, such as errors in
diagnosis and patient identification and in medical aspects as well (Pelzang & Hutchinson, 2018). These
problems in the hospital work area make it clear that it needs improvement and requires urgent attention. This
finding suggests that a friendly working environment for the nurses is essential in order enrich the culture of
patient safety in the hospital. Supervision was also seen to be moderate, as shown by the item that says the nurses were encouraged by
the supervisors to work faster when in times of pressure. This means that they were given opportunities to take
shortcuts in order to get the work done. Nurses also noted that their supervisors overlooked problems that
seemed to happen repeatedly, which may affect the patient safety. It is in such cases that the leadership role of
supervisors becomes crucial. Competent and reflective leaders contribute to the enhancement of safety culture. They are required to comprehend the flaws that exist in each process (Clarke et al., 2007) that could potentially
lead to failures; if this is overlooked, the patients will be put at risk. In essence, a leader should always prioritize
creating and maintaining a patient safety culture (Leonard & Frankel, 2012) as they have critical roles to
strengthen the quality of care being delivered by the nurses to the patients. For example, in a article conducted in
Iran, some nurse leaders did not exhibit commitment to intensifying and maintaining safety culture. Thus, the
leaders must take into account the constant supervision and guidance in providing safety culture, as it is one of
the key roles of nurse managers (Farokhzadian et al., 2018). The nurses also perceived the frequency of events as moderate. However, mistakes and errors that could
possibly harm the patient often went unreported. The same is true of events that were prevented before they
affected the patient. Nurses usually did not report mistakes and errors because they were more worried about the
consequences of reporting the error incidents; this could lead to punishment, salary deduction, or termination
(Farokhzadian et al., 2018). The present article also reported that there has been a low rate of event reporting,
with only three to five events reported over the past 12 months. This weakness in reporting errors also poses a
challenge in other countries. 10. Limitations The article utilized random sampling interpretations and descriptive article of a sample population. Random
sampling means that nurses from the selected 10 hospitals had equal chances to participate in the article. Because
of this design, the results of the article are representative of the entire population of nurses in the general
hospitals of Al-Medina City. The article also utilized a descriptive paper design to describe the relationship
between the nursing shortage and the patient safety culture without affecting the subjects being studied. 109 Journal of Health, Medicine and Nursing
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Furthermore, the article only focused on the nurses from the general hospitals in the Al-Medina region. Therefore, the results might not hold for other settings and contexts. Lastly, the sample population of the article
was slightly too small to draw conclusions that could represent all nurses in Al-Medina City. Conflicts may arise
as well, as the responses of the participants in each item of the survey questionnaire may be subject to bias and
personal issues. Journal of Health, Medicine and Nursing ISSN 2422-8419
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Vol.100, 2022 Furthermore, the article only focused on the nurses from the general hospitals in the Al-Medina region. Therefore, the results might not hold for other settings and contexts. Lastly, the sample population of the article
was slightly too small to draw conclusions that could represent all nurses in Al-Medina City. Conflicts may arise
as well, as the responses of the participants in each item of the survey questionnaire may be subject to bias and
personal issues. 11. Findings For example, in Australia, a article reported that there were some failures to report
errors and mistakes committed by doctors and nurses (Kingston et al., 2004). In Kuwait, staff members reported
only one to two events in a period over one year (Ghobashi et al., 2014). Low reporting rates may also reflect the
fact that nurses do not perceive reports as a way to make changes. As a result, nurses may be reluctant to report. Another reason that may contribute to low reporting is the lack of feedback (Whitaker and Ibrahim, 2016). Nurses might not take reporting as a serious matter since it does not make any difference to them. These findings
suggest that nurses must learn to report errors frequently in order to boost patient SC. 110 Journal of Health, Medicine and Nursing www.iiste.org www.iiste.org ISSN 2422-8419
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Vol.100, 2022 Nurses must have a workplace that promotes safe nursing care for the patients. The aspect of hospital
environment in this article was also perceived to be moderate, since the nurses expressed that the hospitals cared
about and prioritized their patients. However, most agreed that essential patient information was lost during shift
changes. This is not a good sign, since vital data about patients that could affect their treatment and medication
was being disregarded. Moreover, most nurses reported that the units in the hospital did not coordinate well with
each other. This indicates that problems frequently occur whenever the units exchange information with one
another. Teamwork culture among units when changing shifts must be strengthened and professionalism may be
required to carry out this task (Lee at al., 2016). The findings of the present article indicate that problems
occurred whenever nurses’ hand off responsibility to other units right after their shift. Therefore, professionalism
should be invoked to enhance patient SC. If effective, this transfer of information maintains the continuity of
appropriate care and treatment, thereby reinforcing patient safety culture. pp
p
y
g p
y
Nurses also expressed that there was an inadequate number of staff to manage the workload, as provided by
the mean score of 2.59±1.29. This indicates that the studied hospitals are currently facing a shortage of nurses. The workload must be divided among the nurses so that the number of working hours that they complete in a
week is at an optimum level. 11. Findings The results showed that 51.6% of the participants in this article worked for 40 to 59
hours per week. This finding alone indicates that the nurses’ work exceeds the expected working hours of a
typical nurse. The only possible reason for this is a heavy workload assigned to each nurse due to the shortage of
staff. Heavy workloads can lead to burnout among nurses, which impacts the type of care being given to the
patients. p
This article also negated the null hypothesis, which stated that there was no relationship between nursing
shortages and patient SC. The present article revealed that there is, in fact, a correlation between nurses
shortages and some areas of patient SC. First, the article revealed a weak and positive relationship between
nursing shortage and hospital work area (r=0.329, p<0.05). This is clear evidence that whenever the staff is
short-handed in the hospital work area, nurses are unable to create a support system for one another, as they are
more preoccupied with heavy workloads such as attending to the patients. This leads to working longer hours
than expected. A typical nursing workload is 40 hours per week. However, in this case, most nurses work from
40 to 59 hours per week. This means that nurses in this setting sometimes work overtime, which exceeds the full-
time standard work for registered nurses. Previous studies have shown that the longer nurses spend at work, the
more errors they commit, endangering the lives of patients (Wu et al., 2013, Rogers et al, 2004). In contrast, a
article found that nurses who were working for 35 hours per week exhibited a more positive attitude towards
patient safety compared to those who were working for long hours (Elsous et al., 2017). Other studies previously
reported that exhausted workers tend to commit mistakes in their work, putting the lives of the patients at risk
(Leung et al., 2013, Hamdan et al., 2013, Kamran et al., 2018, Abu-El-Noor et al., 2017). Nurses should avoid
such incidents because they greatly affect patient safety culture. This result suggests that the total number of
nurses working relates to the number of hours that each nurse works, and that it therefore affects the efficacy of
the nurses in delivering care to their patients. This is likely why the hospital work area is correlated weakly with
nursing shortage. 11. Findings g
g
Communication was also observed to be weakly correlated with nursing shortage (r=0.144, p<0.05). Good
communication affects the condition and status of the nurses working in the hospital. If good communication is
established, nurses will be more informed and open to one another’s opinions, thus preventing errors. There was also a weak and positive relationship between the frequency of reports to nursing shortage
(r=0.151, p<0.05). If more reports are made, then more actual errors are being reported, making the health care
institution a more reliable one because patients could be reassured that the nurses do not frequently commit
mistakes and that they would receive an optimum quality of care (Wolf and Hughes, 2008). This shows that
whenever these negative incidents are reported, employers tend to remove nurses who are less efficient. The
sudden removal of inefficient nurses could lead to a nursing shortage. It is only logical that the supervisors
would remove less efficient workers, as it may affect the quality of nursing care to the patients. After all, one
wrong move by a nurse could prove fatal for a patient. Preventing such incidents is the reason why supervisors
need incident reports. This seems to suggest that the supervisors need to rectify the errors of nurses by providing
educational trainings in order to avoid career mistakes. References qah, A. (2016). Strategies to address the nursing shortage in Saudi Arabia. International nursing review,
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Health Services Paper (2019) 19:550. Doi: 10.1186/s12913-019-4374-9 Abu-El-Noor, N.I., Abu-El-Noor, M.K., Abuowda, Y.Z., Alfaqawi, M. and Bottcher, B. (2019). Patient safety
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Health Services Paper (2019) 19:550. Doi: 10.1186/s12913-019-4374-9 p
(
)
Abu-El-Noor N.I., Hamdan, M.A., Abu-El-Noor, M.K., Radwan, A-K.S. and Alshaer, A.A. (2017). Safety
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,
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,
,
,
,
y
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,
,
(
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The effects of nurse staffing and nurse education on patient deaths in hospitals with different nurse work
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10.1186/s12992-015-0096-x Balamurugan, E. and Flower, Josephine L. (2014). A article on patient safety culture among nurses in a tertiary
care hospital of pudcherry. 12. Conclusions Based on the results of the current article, the following conclusions were drawn:
The nurses from the general hospitals selected in the Al-Medina Region perceived patient safe
as acceptable.
The nurses in the article had a high communication rate, and they were well informed about errors.
Nurses from the selected hospitals had a low incident report rate because of the fear of consequenc The nurses in the article had a high communication rate, and they were well informed about errors. Nurses from the selected hospitals had a low incident report rate because of the fear of consequences. Nurses from the selected hospitals had a low incident report rate because of the fear of consequences. The nursing shortage weakly correlates with communications, frequency of reported events, and
hospital work area p
p
q
The nursing shortage weakly correlates with communications, frequency of reported events
hospital work area 111 Journal of Health, Medicine and Nursing ISSN 2422-8419
An International Peer-reviewed Journal
Vol.100, 2022
There was significant relationship between Nursing shortage and Patient Safety in
There was significant relationship between Nursing shortage and Patient Safety in
al-Medina Hospitals.
al-Medina Hospitals. There was impact of nursing shortage on patient safety.
There was impact of nursing shortage on patient safety.
There was impact of nursing shortage on patient safety. 13. Recommendation On the basis of the results of the article, the following list of recommendations was enumerated to improve the
patient safety culture in hospitals: On the basis of the results of the article, the following list of recommendations was enumerated to improve the
patient safety culture in hospitals: y
p
Healthcare institutions should foster a friendly environment where nurses can freely express their
opinions. Healthcare institutions should foster a friendly environment where nurses can freely express their
opinions.
Good communication should be established among the healthcare units in order to make the handoff as
effective as possible.
Good communication should be established among the healthcare units in order to make the handoff as
effective as possible.
The hospitals should add more staff to lessen the workload of each nurse, so that nurses can efficiently
carry out their tasks, focus on their patients, and provide a support system to one another.
The hospitals should add more staff to lessen the workload of each nurse, so that nurses can efficiently
carry out their tasks, focus on their patients, and provide a support system to one another.
Training and other programs should be employed for the staff nurses regarding the crucial role of
submitting and reporting actual errors and incidents. Parallel interventions must also be designed for
nurse leaders to properly supervise the institution in order to avoid the repetition of mistakes.
Training and other programs should be employed for the staff nurses regarding the crucial role of
submitting and reporting actual errors and incidents. Parallel interventions must also be designed for
nurse leaders to properly supervise the institution in order to avoid the repetition of mistakes.
The Future Paper Recommendation to be larger population Including whole Hospitals in the region and
anther sitting Include also Healthcare providers.
The Future Paper Recommendation to be larger population Including whole Hospitals in the region and
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Comparative Tandem Mass Tag-Based Quantitative Proteomic Analysis of Tachaea chinensis Isopod During Parasitism
|
Frontiers in cellular and infection microbiology
| 2,019
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cc-by
| 7,728
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Comparative Tandem Mass
Tag-Based Quantitative Proteomic
Analysis of Tachaea chinensis Isopod
During Parasitism
Yingdong Li, Xin Li, Zhibin Han, Weibin Xu, Xiaodong Li and Qijun Chen*
College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China Parasitic isopods perforate and attach to the host integument via the mandibles and
then feed on hemolymph and exudate from the wounds. Such isopods attack a variety
of commercially important fish and crustacean hosts. Similar to other hematophagous
parasites, isopods may also employ biomolecules that affect host blood conglutination
and defense systems. In the present study, a tandem mass tag-based quantitative
proteomic approach was used to identify differentially expressed proteins in Tachaea
chinensis parasites of shrimp, by comparing parasitic (fed) and pre-parasitic (unfed)
individuals. We identified 888 proteins from a total of 1,510 peptides, with a significant
difference in 129 between the fed and unfed groups. Among these, 37 were upregulated
and 92 were downregulated in unfed T. chinensis. This indicates that T. chinensis
may require more energy before parasitism during its search for a host. In addition,
as is the case for other blood-sucking parasites, it might secrete antihemostatic,
anti-inflammatory, and immunomodulatory molecules to facilitate blood meal acquisition. To our knowledge, this study is the first to use a TMT-based proteomic approach to
analyze the proteome of isopod parasites, and the results will facilitate our understanding
of the molecular mechanisms of isopod parasitism on crustaceans. *Correspondence:
Qijun Chen *Correspondence:
Qijun Chen
qijunchen759@syau.edu.cn Keywords: comparative proteomic, isopod parasite, Tachaea chinensis, blood-sucking, during parasitism Specialty section:
This article was submitted to
Parasite and Host,
a section of the journal
Frontiers in Cellular and Infection
Microbiology Specialty section:
This article was submitted to
Parasite and Host,
a section of the journal
Frontiers in Cellular and Infection
Microbiology Keywords: comparative proteomic, isopod parasite, Tachaea chinensis, blood-sucking, during parasitism INTRODUCTION Hematophagous parasites such as ticks, mosquitoes, fleas, and leeches can bypass the clotting
system of their vertebrate hosts by secreting anticoagulant compounds (Shu et al., 2010; Santiago
et al., 2017). Isopod parasites mainly feed on blood (hemolymph) from hosts after perforating the
integument with their mandibles, which can cause stress, tissue damage, secondary infection, and
mortality (Wilson, 2008; Poore and Bruce, 2012; Williams and Boyko, 2012; Smit et al., 2014). There is evidence that isopod parasites may also inject anticoagulants or other compounds directly
into the host so as to evade defensive responses (Nair and Nair, 1983; Manship et al., 2012). At
present, only a few proteins for this purpose have been identified, including trypsin inhibitors and
anticoagulants in Paragnathia formica. Identification of isopod anticoagulant agents may be useful
in fully understanding parasite modulation of the host physiological system. This information may
be valuable for the identification of novel target antigens and antithrombotic drug development. Received: 23 July 2019
Accepted: 30 September 2019
Published: 11 October 2019 ORIGINAL RESEARCH
published: 11 October 2019
doi: 10.3389/fcimb.2019.00350 Edited by:
Itabajara Silva Vaz Jr.,
Federal University of Rio Grande do
Sul, Brazil Itabajara Silva Vaz Jr.,
Federal University of Rio Grande do
Sul, Brazil
Reviewed by:
Marina Amaral Xavier,
Federal University of Rio Grande do
Sul, Brazil
Ben J. Mans,
Agricultural Research Council,
South Africa
*Correspondence:
Qijun Chen
qijunchen759@syau.edu.cn Reviewed by:
Marina Amaral Xavier,
Federal University of Rio Grande do
Sul, Brazil
Ben J. Mans,
Agricultural Research Council,
South Africa Protein Digestion and TMT Labeling Protein Digestion and TMT Labeling
Briefly, six protein concentrates (300 µg each) were mixed in
ultrafiltration filtrate tubes (30 kDa cut-off, Sartorius, Gottingen,
Germany) with 200 µL urea buffer (8 M urea, 150 mM Tris-
HCl, pH 8.0), and the sample was centrifuged at 14,000 × g at
20◦C for 30 min. The sample was washed twice by adding 200
µL UA and centrifuged at 14,000 × g at 20◦C for 30 min. The
flow-through from the collection tube was discarded. Next, 100
µL of indole-3-acetic acid (IAA) solution (50 mM IAA in UA
buffer) was added to the filter tube and vortexed at 600 rpm
in a Thermomixer comfort incubator (Eppendorf, Germany)
for 1 min. Subsequently, the sample was incubated at room
temperature for 30 min in the dark and centrifuged at 14,000 × g
for 30 min at 20◦C. Next, 100 µL UA was added to the filter unit,
which was centrifuged at 14,000 × g for 20 min; this was carried
out three times. The protein suspension in the filtrate tube was
subjected to enzyme digestion with 52 µL of trypsin (Promega,
Madison, WI, USA) buffer [6 µg trypsin (0.5 µg/µL) in 40 µL
of dissolution buffer] for 16–18 h at 37◦C. Finally, the filter unit
was transferred to a new tube and centrifuged at 14,000 × g for
30 min. Peptides were collected in the filtrate and the peptide
concentration was measured based on the optical density at 280
nm (OD280). We performed tandem mass tag (TMT)-based quantitative
proteomic profiling of T. chinensis to compare protein expression
in fed and unfed parasites and gain insight into their integrated
molecular mechanisms and host responses. This will provide an
empirical basis for disease prevention and control efforts and
support further research on the molecular biology of isopods. Ethics Statement Our study did not involve endangered or protected species. In
China, the capture of isopod parasites and their host shrimp from
rice fields does not require specific permits. All efforts were made
to minimize animal suffering and discomfort. The experimental
protocol was approved by the Animal Ethics Committee of
Shenyang Agriculture University. TMT labeling was performed using the TMT6plexTM Isobaric
Label
Reagent
Set
(Thermo
Scientific)
according
to
the
manufacturer’s instructions. The proteins in the unfed group
were labeled with reagents 119, 127, and 128, and those in
the infected group were labeled with reagents 129, 130, and
131. The labeling solution reaction was then incubated at room
temperature for 1.5 h prior to further analysis. Citation: Li Y, Li X, Han Z, Xu W, Li X and
Chen Q (2019) Comparative Tandem
Mass Tag-Based Quantitative
Proteomic Analysis of Tachaea
chinensis Isopod During Parasitism. Front. Cell. Infect. Microbiol. 9:350. doi: 10.3389/fcimb.2019.00350 October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Li et al. Isopod Proteomic Analysis During Parasitsm One microliter of the underlayer liquid of each sample was used
for BCA quantitative analysis, and 1 µg of the protein sample
from each group was subjected to SDS-PAGE (12.5% resolving
gels) analysis (Figure S1). Tachaea chinensis, one of the most common ectoparasites
of farm-raised shrimps, is widely distributed in China and
neighboring countries (Lu et al., 2007; Hua et al., 2018; Li et al.,
2018). It is normally approximately 0.7 cm in size and can easily
be observed with the naked eye following parasitization. After
attacking its host, this isopod invariably remains attached until
either the shrimp dies or another shrimp comes within close
proximity. Over the past 2 years, T. chinensis has been shown
to parasitize over 80% of aquacultured Palaemonetes sinensis (Li
et al., 2018) and 90% of Exopalaemon carinicauda (Li et al.,
2019). Additional research on isopod parasitization mechanisms
is necessary to develop effective methods of control for use in
shrimp aquaculture. Animals Tachaea chinensis isopod parasites (0.82 ± 0.17 cm) and their
host shrimp P. sinensis (3.48 ± 0.35 g) used in this study were
collected from a rice field in Panjin City, Liaoning Province,
China, in November 2018, and transported to the aquaculture
laboratory at Shenyang Agricultural University. They were
acclimated in two 300 L2, fiberglass recirculation tanks with a
circular flow system. Water temperature was maintained at 24
± 0.5◦C, and the photoperiod was set at a 12:12 h light:dark
cycle. After 2 weeks of acclimatization, 20 healthy T. chinensis
were transferred to individual plastic tanks (15.8 cm diameter
and 32.1 cm height), each containing 5 L water, with the same
environmental conditions as during the acclimatization period. Data Analysis y
Raw files were analyzed using the Proteome Discoverer
2.1 software (Thermo Fisher Scientific). A search for the
fragmentation spectra was performed using the MASCOT
search engine embedded in Proteome Discoverer against
the
NCBI_Peracarida_91190_20190313.fasta
database. The
following search parameters were used: monoisotopic mass,
trypsin as the cleavage enzyme, two missed cleavages, TMT
labeling,
and
carbamidomethylating
of
cysteine
as
fixed
modifications. Peptide charges of 2+, 3+, and 4+ and the
oxidation of methionine were specified as variable modifications. The mass tolerance was set to 20 ppm for precursor ions and to
0.1 Da for fragment ions. The results were filtered based on a
false discovery rate of no more than 1%. The relative quantitative
analysis of the proteins in the samples based on the ratios of
TMT reporter ions from all unique peptides representing each
protein was performed using Proteome Discoverer (version
2.1). The relative peak intensities of the TMT reporter ions
released in each of the MS/MS spectra were used. Then, the final
ratios obtained from the relative protein quantifications were
normalized based on the median average protein quantification
ratio. The fold change was set to >1.2 for protein upregulation
and <0.85 for protein downregulation (P < 0.05). Protein
functional annotation was conducted using the Universal
Protein (UniProt) database. Gene Ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) databases were
also used. FIGURE 1 | Total numbers of tandem mass spectrometry spectra (MS/MS
spectrum), peptide-spectrum matches (PSMs), peptides, unique peptides,
and proteins in Tachaea chinensis. of B liquid from 18 to 50%; 45–48 min, linear gradient of B
liquid from 50 to 95%; and 48–60 min, B liquid maintained at
95%. The peptides were separated and subjected to targeted
PRM/MS using a Q-Exactive mass spectrometer (Thermo
Scientific) for 60 min. The parameters were set as follows:
detection mode, positive mode; parent ion scanning range,
350–1,500 m/z; capillary voltage, 1.8 kV; isolation width, 1.6
Th; first-order MS resolution, 70,000 at m/z 200; AGC target,
3 e 6; first-level maximum IT, 250 ms. Peptide secondary MS
were obtained as follows: for each full scan, target peptides of
precursor m/z were sequentially selected based on the inclusion
list for second-order MS (MS2) scan. The parameters used
were as follows: resolution, 35,000 at m/z 200; AGC target, 3
e 6; Level 2 maximum IT, 120 ms; MS2 activation type, HCD;
peptide fragmentation, nitrogen; isolation window, 2.0 Th;
normalized collision energy, 28 eV. Data Analysis Four proteins were randomly
selected from the global proteomics analysis (re-labeled peptide
GISNEGQNASIK∗as the reference standard mixture), including
phosphoglucomutase
(TFTTQETITNAATSAK),
glucose-6-
phosphate isomerase (LGAENFVFFHPR), xylose isomerase
(YFGNLMDAGR,
LSICGEESFGTGSDHIR),
and
malate
dehydrogenase (IFGVTTLDIVR, IQDAGTEVVK). Skyline 3.5
was used to generate an initial PRM transition pair list for the
four candidate DEPs. The mass spectrometry proteomics data have been deposited
in the ProteomeXchange Consortium via the PRIDE partner
repository (identifier PXD015247). All analyzed data are available
from the corresponding author upon reasonable request. SCX Fractionation and LC-MS/MS Analysis SCX Fractionation and LC-MS/MS Analysis
SCX fractionation and LC-MS/MS analysis were conducted
according to a published protocol (Xiao et al., 2019). The TMT-
labeled samples were analyzed using an Easy-nLC nanoflow
HPLC system connected to Orbitrap-Elite (Thermo Fisher
Scientific, San Jose, CA, USA). One microgram of each sample
was loaded onto each of two Thermo Scientific EASY columns
using an autosampler at a flow rate of 200 nL/min. The sequential
separation of peptides on Thermo Scientific EASY trap column
(100 µm × 2 cm, 5 µm, 100 Å, C18) and analytical column
(75 µm × 25 cm, 5 µm, 100 Å, C18) was achieved using a
segmented 1 h gradient from 5 to 28% Solvent B (0.1% formic
acid in 100% ACN) for 40 min, followed by 28–90% Solvent
B for 2 min, and 90% Solvent B for 18 min. The column was
re-equilibrated to its initial highly aqueous solvent composition
before each analysis. The mass spectrometer was operated in
positive ion mode, and MS spectra were acquired over a range
of 350–2,000 m/z. The resolving powers of the MS scan and
MS/MS scan at 100 m/z for the Orbitrap Elite were set as 60,000
and 15,000, respectively. The top 16 most intense signals in the
acquired MS spectra were selected for further MS/MS analysis. Following this, one P. sinensis each was placed in 10 of the
prepared tanks, forming the fed group, with the other tanks
kept as unfed pre-parasitism controls. After 7 d, fed and unfed
T. chinensis were removed to individual 2 mL RNAse-free tubes
and immediately frozen in liquid nitrogen for storage until
protein extraction. Protein Extraction and SDS-PAGE Analysis
Three fed and three unfed isopods were ground into powder
and vortexed in 600 µL SDT buffer (pH 8.0, 4% SDS, 150 mM
Tris-HCl, 1 mM DTT), respectively. The mixtures were heated
at 100◦C for 10 min, then sonicated at 35 W for 4 s, with 7 s
intervals, for 10 min. These were centrifuged at 14,000 × g for
30 min, and supernatants were collected into 0.22 µm filter tubes. October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 Isopod Proteomic Analysis During Parasitsm Li et al. FIGURE 1 | Total numbers of tandem mass spectrometry spectra (MS/MS
spectrum), peptide-spectrum matches (PSMs), peptides, unique peptides,
and proteins in Tachaea chinensis. SCX Fractionation and LC-MS/MS Analysis The isolation window was 2 m/z, and ions were fragmented
through high energy collisional dissociation with normalized
collision energies of 35 eV. The maximum ion injection times
were set at 10 ms for the survey scan and 100 ms for the MS/MS
scans, and the automatic gain control target values for full scan
modes was set to 1 e 6 and for MS/MS was 5 e 4. The dynamic
exclusion duration was 30 s. Verification of Protein Quantifications
Using PRM Analysis g
Parallel reaction monitoring (PRM) was used to verify the
TMT-based quantitative proteomics results. Briefly, 2 µg
of peptide from each sample was taken for LC-PRM/MS
analysis. After sample loading, chromatographic separation
was performed using a Thermo Scientific EASY-nLC nano-
HPLC system, with two buffers. Solution A was 0.1% formic
acid aqueous solution and solution B was a mixed solution
of 0.1% formic acid, 95% acetonitrile, and water. The column
was first equilibrated with 95% solution A. The sample was
injected into a Trap column (100 µm × 20 mm, 5 µm C18, Dr. Maisch GmbH) and subjected to gradient separation through
a chromatography column (75 µm × 150 mm, 3 µm C18,
Dr. Maisch GmbH) at a flow rate of 250 nL/min. The liquid
phase separation gradient was as follows: 0–25 min, linear
gradient of B liquid from 5 to 18%; 25–45 min, linear gradient Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Differentially Expressed Protein Analysis Using a 1.2-fold increase or decrease in protein expression
as a benchmark for a physiologically significant change, 129
differentially expressed proteins (DEPs, p < 0.05) were identified
between the control and parasitism groups (Figure 3). Within
these DEPs, 37 were upregulated and 92 were downregulated
(Table S1, Figure S2) before parasitism. cyclic AMP-responsive element-binding protein 1 (Table 1). Among the 92 significantly upregulated differentially expressed
proteins, 20 were related to digestive metabolism, including
7, 11, and 2 in carbohydrate, protein, and lipid metabolism,
respectively (Table 1). g
p
According to GO enrichment analysis, 1,256, 206, and 274
proteins were enriched for the categories biological process, cell
component, and molecular function, respectively, and 178, 71,
and 83 of these were statistically significant (Figure S2). The top
10 significant pathways are shown in Figure 4. Fifty-seven KEGG
proteins were enriched, and five were statistically significant. Their functions included ECM-receptor interaction, pentose
phosphate pathways, protein processing in the endoplasmic
reticulum, glutathione metabolism, and phototransduction-fly,
with 3, 3, 7, 4, and 3 proteins mapped to them, respectively
(Figure 5). According to the protein–protein interaction network
analysis, the three major clusters of interaction were protein
processing in endoplasmic reticulum, ribosomes, and glutathione
metabolism (Figure 6). Candidate Proteins Related to Blood
Sucking Three hemocyanin proteins showed the greatest difference
in expression between fed and unfed isopods (Table S1). Differentially expressed proteins related to blood sucking,
including
inorganic
pyrophosphatase-like
isoform
X1,
alpha-2-macroglobulin,
bestrophin
homolog,
calreticulin,
barrier-to-autointegration
factor
putative
salivary
alkaline
phosphatase,
inorganic
pyrophosphatase-like
isoform
X1,
neurocalcin homolog isoform X3, and insulin receptor substrate
2-B were significantly upregulated after parasitism (Table 2). Four heat shock proteins and two stress-activated proteins were
also upregulated. RESULTS SDS-PAGE Analysis and Protein Profiling
BCA results indicated protein concentrations of 15.60, 20.94, and
21.55 µg/µL in fed isopods and 19.06, 25.07, and 22.03 µg/µL
in unfed isopods. TMT analysis indicated 3,492 queries in the
133,946 spectra. Among them, a total of 888 unique proteins
were identified across 1,510 peptides (Figure 1). There were 139
proteins between 0 and 20 kDa, and 282, 241, 92, 45, and 89
proteins of 20–40, 40–60, 60–80, and 80–100 kDa, respectively. Eighty-nine proteins had a mass of over 100 kDa (Figure 2). October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 Isopod Proteomic Analysis During Parasitsm Li et al. FIGURE 3 | Volcano plot of all proteins identified in the proteome database,
for the fed and unfed parasite groups. Yellow points: differentially expressed
proteins that were significantly downregulated after parasitism (fold change >
1.2; p < 0.05). Green points: differentially expressed proteins that were
significantly upregulated after parasitism (fold change < 0.8; p < 0.05). FIGURE 2 | Distribution of identified proteins with different molecular weights
(the weight and pI values are for the target proteins from the
NCBI_Peracarida_91190_20190313.fasta database). FIGURE 2 | Distribution of identified proteins with different molecular weights
(the weight and pI values are for the target proteins from the
NCBI_Peracarida_91190_20190313.fasta database). FIGURE 2 | Distribution of identified proteins with different molecular weights
(the weight and pI values are for the target proteins from the
NCBI_Peracarida_91190_20190313.fasta database). FIGURE 3 | Volcano plot of all proteins identified in the proteome database,
for the fed and unfed parasite groups. Yellow points: differentially expressed
proteins that were significantly downregulated after parasitism (fold change >
1.2; p < 0.05). Green points: differentially expressed proteins that were
significantly upregulated after parasitism (fold change < 0.8; p < 0.05). PRM Results FIGURE 5 | KEGG annotation of the top five pathways from the proteome database of the fed and unfed parasite groups. FIGURE 4 | GO functional classification of the identified proteins of the top 10 pathways identified in the proteome database, for the fed and unfed parasite groups, in
the biological process, cell component, and molecular function categories. FIGURE 5 | KEGG annotation of the top five pathways from the proteome database of the fed and unfed parasite groups. FIGURE 5 | KEGG annotation of the top five pathways from the proteome database of the fed and unfed parasite groups. FIGURE 5 | KEGG annotation of the top five pathways from the proteome database of the fed and unfed parasite groups. PRM Results g
Some
differentially
expressed
proteins
related
to
energy
metabolism
were
significantly
upregulated
in
unfed
T. chinensis,
including
calcium-transporting
ATPase
sarcoplasmic/endoplasmic reticulum type-like proteins, NADH
dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial-
like
Na+/K+-ATPase,
partial
sodium
potassium
ATPase
alpha subunit, partial smooth endoplasmic reticulum calcium
ATPase, NADH dehydrogenase subunit 1 (mitochondrion), and To validate the proteomic data, three proteins that were
upregulated after parasitism (phosphoglucomutase, glucose-6-
phosphate isomerase, and xylose isomerase) and one that was
downregulated after parasitism (malate dehydrogenase) were
selected for PRM analysis. In both the fed and unfed groups,
the validated proteins showed expression trends similar to the
proteomic expression trends, suggesting that the proteomic data
were reliable (Figure 7). October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4 Li et al. Isopod Proteomic Analysis During Parasitsm Li et al. Isopod Proteomic Analysis During Parasitsm
FIGURE 4 | GO functional classification of the identified proteins of the top 10 pathways identified in the proteome database, for the fed and unfed parasite groups, in
the biological process, cell component, and molecular function categories. FIGURE 5 | KEGG annotation of the top five pathways from the proteome database of the fed and unfed parasite groups. DISCUSSION
Hematophagous
parasites
use
antihemostatic,
anti-
inflammatory, and immunomodulatory compounds to facilitate
blood meal acquisition during feeding (Shu et al., 2010; Hajdusek
et al., 2013; Eliane et al., 2017; Santiago et al., 2017; Jalovecka
l
) Th
h
l
d
bl h
of pathogens in the host (Boonsriroj et al., 2015; Eliane et al.,
2017). Identification and characterization of such compounds
can elucidate molecular mechanisms of interaction among
parasites, pathogens, and hosts, revealing new vaccine targets
(Mcgowan et al., 1982; Slovák et al., 2013; Oleaga et al., 2015;
Yu et al., 2015; Eliane et al., 2017; Tirloni et al., 2017). Although
h
h
d
h
i
d
i FIGURE 4 | GO functional classification of the identified proteins of the top 10 pathways identified in the proteome database, for the fed and unfed parasite groups, in
the biological process, cell component, and molecular function categories. FIGURE 4 | GO functional classification of the identified proteins of the top 10 pathways identified in the proteome database, for the fed and unfed parasite groups, in
the biological process, cell component, and molecular function categories. DISCUSSION of pathogens in the host (Boonsriroj et al., 2015; Eliane et al.,
2017). Identification and characterization of such compounds
can elucidate molecular mechanisms of interaction among
parasites, pathogens, and hosts, revealing new vaccine targets
(Mcgowan et al., 1982; Slovák et al., 2013; Oleaga et al., 2015;
Yu et al., 2015; Eliane et al., 2017; Tirloni et al., 2017). Although
many researchers have assumed that isopod parasites are Hematophagous
parasites
use
antihemostatic,
anti-
inflammatory, and immunomodulatory compounds to facilitate
blood meal acquisition during feeding (Shu et al., 2010; Hajdusek
et al., 2013; Eliane et al., 2017; Santiago et al., 2017; Jalovecka
et al., 2018). These promote the survival and establishment October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Li et al. Isopod Proteomic Analysis During Parasitsm FIGURE 6 | Protein-protein interaction network analysis of the three major network groups. FIGURE 6 | Protein-protein interaction network analysis of the three major network groups. hematophagous parasites and feed mainly on fish blood or
crustacean hemolymph, no prior studies have demonstrated
this directly. Moreover, in contrast to ticks and mosquitoes,
there is a lack of comprehensive genomic resources for isopod
parasites to investigate infection and response mechanisms. We
performed, to our knowledge, the first comparative proteomic
analysis between fed and unfed T. chinensis to gain a better
understanding of its feeding and antigen-related proteins. However, because very few protein sequences for isopods are
available in the NCBI protein database and all proteins identified
were based on homologous proteins, we were able to identify only
888 proteins. Moreover, to avoid potential contamination of the
host protein samples and to screen out proteins associated with
parasitic processing, we also searched the NCBI protein database
for proteins of Peracarida and blood-sucking insects (ticks and
mosquitos); we found that proteins from Armadillidium vulgare
(442, 51.76%), Hyalella azteca (227, 26.58%), and Hirondellea
gigas (143, 16.74%) were similar to those present in our
samples (Table S1). major contributors to hemolymph density (Simonetta et al.,
2011), in their parasites, the major digestive enzymes are thought
to be proteases. Eleven proteolytic enzymes were identified
among the 129 DEPs, and all were upregulated after parasitism
(Table 1). Seven carbohydrate metabolism-related proteins were
also upregulated, suggesting that hemolymph glucose may
also be an important nutrient for T. chinensis. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org DISCUSSION Trehalose,
which regulates energy metabolism and glucose generation via
trehalose catabolism, was also upregulated after parasitism,
suggesting that access to host trehalose is an important feature
of isopod parasites. Many researchers have assumed that isopod parasites can
feed on fish blood or crustacean hemolymph, comparable to
an “aquatic mosquito” (Wilson, 2008; Manship et al., 2012;
Smit et al., 2014; Nagler et al., 2017). We previously found
that hemocyanin in P. sinensis is significantly downregulated
following parasitism by T. chinensis (Li et al., 2019). The
present results indicate that hemocyanin in T. chinensis was
significantly upregulated after parasitism, suggesting a role
in obtaining hemolymph from P. sinensis. In hematophagous
arthropods such as ticks, mosquitos, fleas, and flies, hematophagy
evolved independently over millions of years, leading to various
morphological adaptations and diverse strategies to overcome the
barriers imposed by hosts (Ribeiro, 1995; Garcia et al., 2015). However, one example of convergent evolution and adaptation
for hematophagy is the development of highly functional
salivary glands, which produce a large diversity of anticlotting,
antiplatelet, vasodilatory, and pathogen-transporting substances
(Kim et al., 2016; Tirloni et al., 2016). Although it is known
that isopod parasite morphology has been strongly modified for
parasitism over 168 million years (Nagler et al., 2017), the type
and composition of blood-sucking-related substances remain to
be understood. Comparative proteomic analysis of unfed and fed T. chinensis indicated 129 proteins expressed significantly, with 37
upregulated and 92 downregulated before parasitism. Among the
37 upregulated proteins, seven were identified as contributing to
ATP synthesis (Table 1). These results suggest that high energy
metabolic proteins are expressed in pre-parasitism isopods while
searching for hosts. Meanwhile, previous research has shown
that genes related to ATP generation in host shrimp P. sinensis
are upregulated under parasitism, which might be induced by
a rapid depletion of ATP content in the parasite itself after
parasitism, during its development and reproduction (Li et al.,
2018). A similar profile has been demonstrated in fleas, where
energy costs for feeding are lower than those in unfed individuals
(Sarfati, 2005). Moreover, in crustaceans, because proteins are October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6 Isopod Proteomic Analysis During Parasitsm Li et al. TABLE 1 | Differentially expressed proteins involved in energy and digestive metabolism, comparing the fed and unfed Tachaea chinensis. Accession
Description
Coverage
Peptides
PSMs
AAs
MW (kDa)
Calc. DISCUSSION pI
FC Unfed/Fed
P-value
ENERGY METABOLISM
1371969814
Calcium-transporting ATPase sarcoplasmic/endoplasmic
reticulum type-like
13.89
13
36
1,001
109.50
5.52
1.20
0.04
1067084430
NADH dehydrogenase flavoprotein 1, mitochondrial-like
8.55
3
3
468
50.90
8.21
1.20
0.01
1547583175
Na+/K+-ATPase, partial
29.70
5
9
202
22.40
6.77
1.28
0.02
1246309936
Sodium potassium ATPase alpha subunit, partial
25.99
5
9
227
25.20
5.39
1.28
0.01
26324131
Smooth endoplasmic reticulum calcium ATPase
18.96
17
46
1,002
109.70
5.36
1.32
0.01
1220106012
NADH dehydrogenase subunit 1 (mitochondrion)
3.56
1
1
309
34.70
8.88
1.41
0.00
1561970059
Cyclic AMP-responsive element-binding protein 1
1.98
1
1
353
37.50
7.56
1.44
0.00
DIGESTIVE METABOLISM
Carbohydrate Metabolism
1371969117
Phosphoglucomutase
3.02
1
1
563
61.40
5.26
0.68
0.00
1562001515
Trehalase
1.46
1
1
548
62.80
5.26
0.73
0.03
262305275
Glycogen synthase, partial
6.31
2
3
317
36.20
7.64
0.85
0.01
1067085998
UDP-glucuronosyltransferase 2B14-like
3.85
1
5
156
17.80
9.57
0.76
0.00
1371968467
Fructose 1,6-bisphosphatase
8.33
2
3
276
30.00
5.35
0.78
0.01
1371966397
Kynurenine–oxoglutarate transaminase 3-like, partial
2.69
1
2
484
53.90
7.71
0.83
0.00
1562032021
Ubiquitin carboxyl-terminal hydrolase 7
1.18
1
1
1,267
145.50
5.77
0.83
0.04
Protein Metabolism
1067064770
Aminopeptidase N-like
0.59
1
2
1,851
208.50
4.91
0.63
0.00
1371969475
26S protease regulatory subunit 6B
5.56
3
3
414
46.70
5.58
0.65
0.00
1562025605
Protein disulfide-isomerase A6
2.27
1
1
441
48.30
5.57
0.68
0.01
1371970147
Nucleoside diphosphate kinase
7.73
1
2
181
20.20
8.40
0.74
0.01
1562030658
Protein disulfide-isomerase
5.17
3
4
522
58.70
4.77
0.75
0.00
1371969013
Dolichyl-diphosphooligosaccharide–protein
glycosyltransferase subunit STT3A-like
1.66
1
1
722
82.10
7.87
0.78
0.01
1562029772
Ubiquitin-protein ligase E3C
0.87
1
2
1,032
117.50
6.76
0.78
0.01
1371965865
Serine/threonine-protein phosphatase 4 regulatory
subunit 1-like
0.52
1
1
1,340
149.30
4.65
0.80
0.00
1562011562
Peptidyl-prolyl cis-trans isomerase FKBP7
13.81
4
7
210
23.50
4.79
0.80
0.00
1562027280
Proteasome subunit beta type-5
4.61
1
2
282
31.50
6.65
0.85
0.02
1561992339
Putative serine/threonine-protein phosphatase PP2A
regulatory subunit
10.44
4
5
479
53.00
5.21
0.85
0.02
Lipid Metabolism
1562029772
Ubiquitin-protein ligase E3C
0.87
1
2
1,032
117.50
6.76
0.78
0.01
1562030180
Fatty acid synthase
0.57
1
2
2,263
248.70
5.49
0.81
0.02 TABLE 1 | Differentially expressed proteins involved in energy and digestive metabolism, comparing the fed and unfed Tachaea chinensis. Lipid Metabolism The data on the relative expression of proteins, for use in the TMT analysis, were obtained
from Tables 1, 2. The data on the relative expression of proteins for use in the PRM analysis were calculated relative to the expression of the relabeled peptide
GISNEGQNASIK*. FIGURE 7 | Verification of the expression profiles of randomly selected significantly changed proteins comparing the fed and unfed T. chinensis groups, as determined
by parallel reaction monitoring (PRM) and tandem mass tag (TMT) analysis. The data on the relative expression of proteins, for use in the TMT analysis, were obtained
from Tables 1, 2. The data on the relative expression of proteins for use in the PRM analysis were calculated relative to the expression of the relabeled peptide
GISNEGQNASIK*. FIGURE 7 | Verification of the expression profiles of randomly selected significantly changed proteins comparing the fed and unfed T. chinensis groups, as determined
by parallel reaction monitoring (PRM) and tandem mass tag (TMT) analysis. The data on the relative expression of proteins, for use in the TMT analysis, were obtained
from Tables 1, 2. The data on the relative expression of proteins for use in the PRM analysis were calculated relative to the expression of the relabeled peptide
GISNEGQNASIK*. et al., 2011, 2016). However, the hosts of these parasites
are vertebrates, in which the composition of the coagulation
system differs from that of invertebrates. In shrimps, the
hemolymph clotting system comprises transglutaminase and
clotting proteins and plays an important role in the innate
immune response and prevention of blood loss during injury
and wound healing (Maningas et al., 2013). Isopod parasites
may inject anticoagulants or other compounds directly into the
blood to obtain a blood meal (Nair and Nair, 1983; Manship
et al., 2012), but the constituents of these compounds are still
unclear. Here, we identified two protein disulfide-isomerase
(PDI) proteins that participate in parasite–host cell interactions. PDI-specific antibodies may constitute part of the mucosal
antibody repertoire, which is possibly involved in defense against
parasites. The potential roles of those proteins for isopod parasite
control will be investigated in our further study. In conclusion, our results represent the first comparative
proteomic study to detect key proteins from the whole body
of fed and unfed T. chinensis isopod parasites. Owing to
the lack of genomic data, we were able to identify only 888
proteins, and all were found in both stages. Lipid Metabolism 1562030180
Fatty acid synthase 1562030180
Fatty acid synthase was also significantly upregulated after parasitism in the present
study. Furthermore, calcineurin subunit B type 2 isoform X1,
putative salivary alkaline phosphatase, insulin receptor substrate
2-B, PI-actitoxin-Aeq3a, and neurocalcin homolog isoform
X3, which are similar to substances in the salivary glands of
hematophagous parasites, were also upregulated in T. chinensis
during feeding (Kim et al., 2015). However, the interaction of
those proteins between parasites and hosts and the mechanisms
of paralyzation and inhibition of inflammation remain unclear
and require further study. During blood sucking, the salivary gland of hematophagous
parasites
produces
a
cocktail
of
anti-hemostatic,
anti-
inflammatory, and immunomodulatory molecules that facilitate
blood-meal acquisition. Saravanan et al. (2003) found that
α2-macroglobulin, which they consider to have an anticoagulant
function, is highly expressed in tick salivary glands. In the
present study, α2-macroglobulin was significantly upregulated in
isopods that had recently fed, which indicates that T. chinensis
may also secrete anticoagulatory molecules during feeding. Moreover, Saurabh et al. (2011) found that fish macroglobulin
was
downregulated
during
Argulus
siamensis
parasitism. Similarly, we previously found that shrimp macroglobulin
was downregulated during T. chinensis parasitism. Moreover,
calreticulin, which functions in hematophagy in ticks through
host immunosuppression or antihemostasis (Parizi et al., 2009), was also significantly upregulated after parasitism in the present
study. Furthermore, calcineurin subunit B type 2 isoform X1,
putative salivary alkaline phosphatase, insulin receptor substrate
2-B, PI-actitoxin-Aeq3a, and neurocalcin homolog isoform
X3, which are similar to substances in the salivary glands of
hematophagous parasites, were also upregulated in T. chinensis
during feeding (Kim et al., 2015). However, the interaction of
those proteins between parasites and hosts and the mechanisms
of paralyzation and inhibition of inflammation remain unclear
and require further study. Anticlotting-related proteins are considered potential targets
for the development of new drugs and vaccines to control
and prevent ectoparasites such as ticks (Oleaga et al., 2017),
mosquitos (Popova-Butler and Donald, 2009; Tetreau et al.,
2012), Schistosoma (Delcroix et al., 2006), and sea louse (Carpio October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7 Isopod Proteomic Analysis During Parasitsm Li et al. TABLE 2 | Differentially expressed proteins involved in blood sucking, comparing the fed and unfed Tachaea chinensis. Accession
Description
Coverage
Peptides
PSMs
AAs
MW (kDa)
Calc. Lipid Metabolism pI
FC Fed/Unfed
P-value
BLOOD SUCKING
427782987
Putative salivary alkaline phosphatase
1.68
1
1
1
1.0
535.00
0.45
6.60
1561973869
Barrier-to-autointegration factor
11.11
1
1
90
10.1
5.90
0.67
0.02
1067116995
Inorganic pyrophosphatase-like isoform X1
3.41
1
1
410
46.2
5.08
0.72
0.00
1067085871
Calcineurin subunit B type 2 isoform X1
4.69
1
1
213
23.5
4.97
0.75
0.02
1067070998
Neurocalcin homolog isoform X3
10.53
2
2
190
21.9
5.25
0.74
0.01
164632859
Alpha-2-macroglobulin, partial
3.85
2
3
571
61.7
4.94
0.75
0.00
1562026595
Calreticulin, partial
13.03
3
5
330
38.4
4.93
0.81
0.00
1202288454
Insulin receptor substrate 2-B, partial
2.35
1
1
426
46.2
6.38
0.82
0.01
1067101392
Mitogen-activated protein kinase 14B-like
4.08
1
1
392
44.0
7.30
0.82
0.03
1562031882
PI-actitoxin-Aeq3a
6.51
1
1
169
19.5
4.81
0.83
0.02
1562036618
Bleomycin hydrolase
4.76
1
1
252
29.0
6.64
0.85
0.04
1371956987
Peroxiredoxin, partial
9.65
1
1
114
12.4
5.48
1.20
0.03
1371959777
Galactose ABC transporter substrate-binding protein
3.26
1
1
337
37.1
5.03
1.50
0.00
HEAT SHOCK PROTEIN
190014500
70 kDa heat shock protein
7.64
4
10
641
70.1
5.53
0.72
0.00
1561987640
Endoplasmin
3.99
3
5
803
92.1
5.02
0.77
0.00
1561990168
60 kDa heat shock protein, mitochondrial
14.19
8
13
571
60.5
5.41
0.81
0.01
1371970216
Heat shock protein HSP 90-alpha-like
7.49
5
7
708
82.0
5.05
0.83
0.00
1371965305
Stress-activated protein kinase JNK-like
3.14
1
1
509
57.2
7.40
0.83
0.04
1562029713
Putative heat shock protein HSP 90-alpha A4
10.86
5
8
488
55.7
5.08
0.83
0.00
FIGURE 7 | Verification of the expression profiles of randomly selected significantly changed proteins comparing the fed and unfed T. chinensis groups, as determined
by parallel reaction monitoring (PRM) and tandem mass tag (TMT) analysis. The data on the relative expression of proteins, for use in the TMT analysis, were obtained
from Tables 1, 2. The data on the relative expression of proteins for use in the PRM analysis were calculated relative to the expression of the relabeled peptide
GISNEGQNASIK*. et al., 2011, 2016). However, the hosts of these parasites
PDI-specific antibodies may constitute part of the mucosal Coverage
Peptides
PSMs
AAs
MW (kDa)
Calc. pI
FC Fed/Unfed
P-value FIGURE 7 | Verification of the expression profiles of randomly selected significantly changed proteins comparing the fed and unfed T. chinensis groups, as determined
by parallel reaction monitoring (PRM) and tandem mass tag (TMT) analysis. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org REFERENCES secreted every 24 h during blood feeding. PLoS Negl. Trop. Dis. 10:e0004323. doi: 10.1371/journal.pntd.0004323 secreted every 24 h during blood feeding. PLoS Negl. Trop. Dis. 10:e0004323. doi: 10.1371/journal.pntd.0004323 Boonsriroj, H., Manalo, D. L., Kimitsuki, K., Shimatsu, T., Shiwa, N., Shinozaki,
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3675-5 Carpio, Y., Basabe, L., Acosta, J., Rodríguez, A., Mendoza, A., Lisperger, A.,
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Immunol. 82, 153–161. doi: 10.1016/j.fsi.2018.08.013 Delcroix, M., Sajid, M., Caffrey, C. R., Lim, K.-C., Dvorak, J., Hsieh, I.,
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digestion by a platyhelminth parasite. J. Biol. Chem. 281, 39316–39329. doi: 10.1074/jbc.M607128200 Lu, X., Zou, W., Peng, H., Shu, Q., Chen, W., Xie, J., et al. (2007). New records of
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of the shrimp clotting system. Fish Shellfish Immunol. 34, 968–972. doi: 10.1016/j.fsi.2012.09.018 Eliane, E., Maruyama, S. R., Kawahara, R., Fujita, A., Martins, L. A., Righi, A. A.,
et al. (2017). Analysis of the salivary gland transcriptome of unfed and partially
fed Amblyomma sculptum ticks and descriptive proteome of the saliva. Front. Cell. Infect. Microbiol. 7:476. doi: 10.3389/fcimb.2017.00476 Manship, B. M., Walker, A. J., Jones, L. A., and Davies, A. J. DATA AVAILABILITY STATEMENT We thank Panjin Guanghe Fisheries Co., Ltd. for providing
the shrimps. We thank Panjin Guanghe Fisheries Co., Ltd. for providing
the shrimps. The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher. FUNDING require considerable energy during its search for a host. We
also found that similar to other hematophagous parasites,
T. chinensis
secretes
antihemostatic,
anti-inflammatory,
and immunomodulatory molecules to facilitate blood meal
acquisition. Our study provides valuable empirical data that
will support future molecular research on isopod parasitization
of crustaceans. This work was supported by the earmarked fund for Modern
Agro-industry Technology Research System (No. CARS-48), the
National Natural Science Foundation of China (No. 31502190),
and the Talent Introduction Program of Shenyang Agricultural
University (Nos. 880416005 and 880417024). AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fcimb. 2019.00350/full#supplementary-material YL and QC designed the experiments. XinL and ZH generated
biological
samples. XinL,
ZH,
and
WX
performed
the
experiments. YL, XinL, and QC analyzed data. YL and WX
performed statistic data analysis. YL and XiaL contributed
reagents, materials and analysis tools. YL, XinL, ZH, and
WX wrote the paper. All authors read and approved the
final manuscript. Figure S1 | Separation of proteins by SDS-PAGE from six unfed (A1, A2, and A3)
and fed (B1, B2, and B3) Tachaea chinensis. Figure S2 | Hierarchical clustering analysis of differentially expressed proteins in
unfed (A1, A2, and A3) and fed (B1, B2, and B3) Tachaea chinensis. Table S1 | Lists of proteins indented from Tachaea chinensis. Lipid Metabolism Differentially
expressed
proteins
related
to
energy
metabolism
were
upregulated in unfed individuals, particularly those involved
in ATP generation. This may indicate that T. chinensis may October 2019 | Volume 9 | Article 350 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 8 Isopod Proteomic Analysis During Parasitsm Li et al. Li et al. REFERENCES (2012). Blood
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absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Simonetta, L., Martinis, M., and Ferrero, E. A. (2011). Ecological relevance
of hemolymph total protein concentration in seven unrelated crustacean
species from different habitats measured predictively by a density-salinity
refractometer. J. Mar. Biol. 2011, 1–7. doi: 10.1155/2011/153654 Copyright © 2019 Li, Li, Han, Xu, Li and Chen. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
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A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity
|
Molecular medicine
| 2,015
|
cc-by
| 10,069
|
A Coding Variant of ANO10, Affecting Volume Regulation of
Macrophages, Is Associated with Borrelia Seropositivity Christian Hammer,1 Podchanart Wanitchakool,2 Lalida Sirianant,2 Sergi Papiol,1 Mathieu Monnheimer,1
Diana Faria,2 Jiraporn Ousingsawat,2 Natalie Schramek,3 Corinna Schmitt,4 Gabriele Margos,5
Angelika Michel,6 Peter Kraiczy,7 Michael Pawlita,6 Rainer Schreiber,2 Thomas F Schulz,4 Volker Fingerle,5
Hayrettin Tumani,3 Hannelore Ehrenreich,1,8* and Karl Kunzelmann2* 1Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany; 2Institut für Physiologie, Universität
Regensburg, Regensburg, Germany; 3Department of Neurology, University of Ulm, Ulm, Germany; 4Institute of Virology, Hannover
Medical School, Hannover, Germany; 5National Reference Center for Borrelia, Bavarian Health and Food Safety Authority,
Oberschleissheim, Germany; 6Division of Genome Modifications and Carcinogenesis, Infections and Cancer Program, German
Cancer Research Center, Heidelberg, Germany; 7Institute of Medical Microbiology and Infection Control, University Hospital of
Frankfurt am Main, Frankfurt/Main, Germany; and 8DFG Research Center for Nanoscale Microscopy and Molecular Physiology of
the Brain (CNMPB), Göttingen, Germany In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nu-
cleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and
3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a
missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl– channels and phospholipid scram-
blases. ANO10 augments volume-regulated Cl– currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages
and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages
and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca2+ signals, which may delay spiro-
chete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in
innate immune defense against Borrelia infection. Online address: http://www.molmed.org
doi: 10.2119/molmed.2014.00219 properties in the human host (5). Borre-
lia species have a highly complex ge-
nomic structure and genetic variation
may account for a large proportion of
the variability of pathogenicity (6). However, pathogens are not only de-
pending on their own fitness for a suc-
cessful establishment of infection, but
also on the genetic makeup of their
hosts. The recent years have produced a
wealth of studies elucidating the impor-
tant role of human genomic variation in
host defense mechanisms, both for viral
and bacterial infections (7). A Coding Variant of ANO10, Affecting Volume Regulation of
Macrophages, Is Associated with Borrelia Seropositivity Given the
immense phenotypic variation of Borre-
lia disease symptoms, it is likely that
part of the variation is due to differ-
ences in human immune response, orig-
inating in genomic variation. We there-
fore set out to (i) identify host genomic
variants mediating differential suscepti-
bility to Borrelia infection/seropositivity INTRODUCTION tral nervous system manifestations can
imitate a broad range of neuropsychi-
atric syndromes (3), in rare cases even
be indistinguishable from acute schizo-
phrenia (4). Borreliosis is caused by a
variety of species of Borrelia burgdorferi
sensu lato complex, some of which show
distinct differences in their pathogenic Lyme borreliosis, caused by bacteria
mainly transmitted by ticks of the
genus Ixodes, is the most common tick-
borne disease in Europe and the United
States (1). It involves many organs, pre-
dominantly skin, musculoskeletal sys-
tem, heart and nervous system (2). Cen- *HE and KK are joint senior authors. Address correspondence to Hannelore Ehrenreich, Clinical Neuroscience, Max Planck In-
stitute of Experimental Medicine, 37075 Göttingen, Germany. Phone: +49-551-3899-615;
Fax: +49-551-3899-670; E-mail: ehrenreich@em.mpg.de; or Karl Kunzelmann, Department
of Physiology, University of Regensburg, 93053 Regensburg, Germany. Phone: +49-941-943-
4302; Fax: +49-941-943-4315; E-mail: Karl.Kunzelmann@ur.de. Submitted November 3, 2014; Accepted for publication February 23, 2015; Published On-
line (www.molmed.org) February 23, 2015. 2 6
| H A M M E R E T A L . | M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 R E S E A R C H A R T I C L E tics GmbH, Eschborn, Germany). Assays
were processed automatically on BEP III
(Siemens Healthcare Diagnostics GmbH)
and interpreted (manufacturer’s instruc-
tions) as positive, negative or borderline. Positive and borderline samples were re-
analyzed using the Euroline Borrelia-
RN-AT immunoblot (Euroimmun,
Lübeck, Germany). Only the confirmed
were defined seropositive for statistical
analysis and contrasted against all oth-
ers. Titer levels, when mentioned in the
manuscript, refer to the enzyme-linked
immunosorbent assay (ELISA) results. To test for specificity of association sig-
nals, the following immunoenzymatic
assays were conducted: Novagnost
Chlamydia pneumoniae IgG; Novagnost
Chlamydia trachomatis IgG; Novagnost
Mycoplasma pneumoniae IgG; and
Enzygnost Anti-Helicobacter pylori/IgG
(all Siemens Healthcare Diagnostics
GmbH). tics GmbH, Eschborn, Germany). Assays
were processed automatically on BEP III
(Siemens Healthcare Diagnostics GmbH)
and interpreted (manufacturer’s instruc-
tions) as positive, negative or borderline. Positive and borderline samples were re-
analyzed using the Euroline Borrelia-
RN-AT immunoblot (Euroimmun, blood for genetic and serological analy-
ses. Patients were classified in three
subgroups based on clinical and sero-
logical findings (i) neuroborreliosis, (ii)
systemic borreliosis or (iii) laboratory-
based borreliosis without typical clinical
signs and symptoms. Phenotypical Analyses Of all schizophrenic (GRAS) patients,
extensive phenotypical characterization
was conducted as referenced previously
(8,12). Age of onset, age at first psychotic
episode, positive and negative syndrome
scale (PANSS) scores, chlorpromazine
equivalents (CPZ), neurological symp-
toms (Cambridge Neurological Inventory
[CNI]) including fine motor skills (Mac-
Quarrie dotting/tapping), current cogni-
tive functioning (composite score com-
prising reasoning, executive function,
verbal learning and memory), global as-
sessment of functioning (GAF), Parkin-
sonism, hard neurological signs, motor
coordination, sensory integration and
gait were employed as disease character-
istics. Moreover, patient self-rating was
performed using the Brief Symptom In-
ventory (BSI) (13). The Ulm borreliosis
patients had a comprehensive clinical
neurological, serological, and in 81 of 100
patients, also cerebrospinal fluid (CSF)
examination. CSF diagnostics included
leukocyte and differential cell count;
nephelometric determination of total
protein; CSF:serum ratios for albumin,
IgG, IgA, and IgM; enzyme-linked im-
munosorbent assay (ELISA) for Borrelia-
specific antibodies; and oligoclonal IgG
analysis in CSF and serum by immuno-
electrophoresis. Participants All subject data were collected in ac-
cordance with ethical guidelines and the
Helsinki Declaration (10). Regarding the
discovery sample (total of N = 2,495),
subject selection was unbiased, that is,
sera collection was concluded before
specific serological analysis was
planned: Schizophrenic patients (N =
1,076) were recruited between 2005 and
2011 at 23 German sites for the Göttin-
gen Research Association for Schizo-
phrenia (GRAS) data collection. Patients
fulfilling Diagnostic and Statistical Man-
ual of Mental Disorders, 4th edition
(DSM-IV) (11) criteria for schizophrenia
(81.4%) or schizoaffective disorder
(18.6%) were included regardless of dis-
ease stage (8,12). Healthy GRAS con-
trols were anonymized blood donors
(N = 1,271; Transfusion Medicine,
Göttingen, Department of Transfusion
Medicine, University Medicine of
Göttingen). Health was ensured by pre-
donation screening (questionnaires, in-
terviews, hemoglobin, blood pressure,
pulse, temperature). Patients with affec-
tive disorders (N = 146) also were in-
cluded (ongoing GRAS extension). Ex-
ploration sample (N = 100): In Ulm, a
total of 257 patients with documented
history of Borrelia infection were con-
tacted in written form, resulting in 100
individuals interested in participating. The study included (a) a comprehensive
history on tick bite and borreliosis-
specific symptoms, (b) a neurological
examination with special emphasis on
cerebellar signs and (c) drawing of Genetic Analyses A semicustom Axiom myDesign geno-
typing array (Affymetrix, Santa Clara,
CA, USA) was used. Array specifications
and quality controls have been described
in detail before (9). Principal compo-
nents were generated using GCTA
(v1.24) (14) and genetic outliers were ex-
cluded based on inspection of the first
two principal components. Genomic in-
flation was calculated using PLINK
(v1.07) (15) to ensure minimization of
population stratification, excluding
SNPs in the complex major histocompat-
ibility complex (MHC) region (chromo-
some 6, 29–33 MB). PLINK also was
used for association testing using the
following exclusion criteria: Hardy-
Weinberg P <5E-07, minor allele fre-
quency <0.01, missingness per marker
>0.05 and missingness per individual
>0.02. SNPs on sex chromosomes were
excluded from analysis. Variants in high
linkage of genome-wide significant
SNPs were identified using SNAP Proxy
Search (http://www.broadinstitute.org/
mpg/snap/) (16), using the 1000
Genomes Pilot 1 CEU population panel
and a r2 threshold of 0.8. Patients with INTRODUCTION by means of a genome-wide association
study (GWAS) and to (ii) uncover a
possible contribution of Borrelia
seropositivity to core phenotypes of
neuropsychiatric disorders. For advanc-
ing these objectives, we employed the
Göttingen Research Association for
Schizophrenia (GRAS) sample (8,9)
comprised of 1,271 healthy blood
donors and 1,224 patients suffering
from neuropsychiatric disease. Fluorescent Borrelia Red fluorescent B. garinii PRJS1009-
Cherry were used to infect macro phages. In some experiments, cells were exposed
to TNFα (100 ng/mL) for 2–6 h. THP-1
monocytes were differentiated into
macro phages by incubation with
100 nmol/L phorbol 12-myristate
13-acetate (PMA) (Sigma-Aldrich, Mu-
nich, Germany) for 48 h. Cell Culture, Animals, cDNAs,
Site-Directed Mutagenesis and
Transfection Oocytes were harvested from Xenopus
laevis according to German regulations
governing animal experiments. Oocytes
were defolliculated for 1 h at 18°C with
1.5 mg/mL collagenase type V (Sigma-
Aldrich, St. Louis, MO, USA). After
washing oocytes were injected with
cRNA encoding ANO10, R263H-ANO10
and AQP1. Preparation of cRNA and
voltage clamping of the oocytes have
been described earlier (17). Human ANO10 cDNA (NM_018075.2)
was purchased from OriGene (SC113757,
Rockville, MD, USA) and cloned in
pcDNA3.1 with a C-terminal His-Tag
(Life Technologies [Thermo Fisher Scien-
tific Inc., Waltham, MA, USA]). R263H-
ANO10, L510R-ANO10, L384fs-ANO10,
LRRC8A and AQP1 were mutated and
cloned, respectively, using standard
polymerase chain reaction (PCR) tech-
niques. All cDNAs were verified by se-
quencing. Culturing of HEK293 cells,
THP-1 cells and lymphocytes and isola-
tion of mouse macrophages has been de-
scribed earlier (17). Site-directed mutage-
nesis, transfection methods and other
used constructs have been described
previously (18). Phagocytosis Assay THP-1 cells were treated with PMA
(100 nmol/L) for 2 d. Cells were infected
with cherry-labeled B. garinii (MOI 1:10)
at 37°C. After infection, cells were
washed with PBS to remove remaining
Borrelia. Cells were visualized and fluo-
rescence was detected by using an Ax-
iovert 200 microscope and AxioVision
software (Zeiss), and mean fluorescence
intensity was quantified. Flow Cytometry, Single Cell Volume
Measurements and Migration Flow Cytometry, Single Cell Volume
Measurements and Migration nitol from an isotonic (300 mosmol/L)
modified Ringer solution to achieve a
hypotonic bath solution (Hypo, 33%,
200 mosmol/L). Patch-clamp experi-
ments were performed in the fast
whole-cell configuration as described
previously (17). confirmed diagnosis of borreliosis (N =
100) recruited in Ulm were genotyped
using the KASP genotyping system (LGC
Genomics, Berlin, Germany) after DNA
isolation from blood using the Jetquick
Blood and Cell Culture Kit (Genomed,
Loehe, Germany). Cells were washed and redissolved in
10 mL isotonic or hypotonic Ringer solu-
tion as described for patch clamp experi-
ments. Cells were analyzed at 37°C/pH
7.4 using a CASY flow cytometer (Roche
Diagnostics, Mannheim, Germany). Cells
were analyzed at a density of
106 cells/mL. For single cell volume
measurements cells were loaded with
1 μg of calcein-AM (Molecular Probes
[Thermo Fisher Scientific]) and 0.01%
pluronic in a standard bath solution
(Ringer) for 60 min at 20–22°C. Fluores-
cence intensity was measured at an exci-
tation wavelength of 485 nm and an
emission wavelength of 520–550 nm. Cell
swelling and RVD were observed for 10
to 15 min after applying hypotonic bath
solution. Cell migration was assessed in
Boyden chambers as described previ-
ously (17). Cells were washed and redissolved in
10 mL isotonic or hypotonic Ringer solu-
tion as described for patch clamp experi-
ments. Cells were analyzed at 37°C/pH
7.4 using a CASY flow cytometer (Roche
Diagnostics, Mannheim, Germany). Cells
were analyzed at a density of Measurement of TNFα Release Measurement of TNFα Release
THP-1 cells were grown in 96-well
plates and, when mentioned, treated
with PMA (100 nmol/L) for 2 d. Before
sample collection, cells were infected
with cherry-labeled B. garinii (MOI 1:10)
for 4 h at 37°C. Following a centrifuga-
tion step, the supernatant was collected
and immediately stored at –20°C. TNFα
was measured using Platinum ELISA kit
(eBioscience Affymetrix, Vienna, Aus-
tria) according to the manufacturer’s in-
structions. Patch Clamping Cells grown on cover slips were
mounted in a perfused bath on the stage
of an inverted microscope (IM35, Zeiss,
Munich, Germany) and kept at 37°C
The bath was perfused continuously
with Ringer solution (145 mmol/L
NaCl, 0.4 mmol/L KH2PO4, 1.6 mmol/L
K2HPO4, 6 mmol/L D-glucose, 1 mmol/L
MgCl2, 1.3 mmol/L Ca- gluconate, pH 7.4)
at about 10 ml/min. Cell swelling was
induced by removing 100 mmol/L man- Serological Analyses The presence of antibodies against
Borrelia was first determined using
Enzygnost Lyme link VlsE/IgG, a quan-
titative immunoenzymatic method
based on a mix of native Borrelia anti-
gens from B. afzelii strain PKo and re-
combinant VlsE obtained from three
genospecies pathogenic to humans
(B. Burgdorferi sensu stricto, B. garinii,
B. afzelii) (Siemens Healthcare Diagnos- M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 | H A M M E R E T A L . | 2 7 A N O C T A M I N 1 0 I N B O R R E L I A I N F E C T I O N Measurement of [Ca2+]i The plasma membrane-bound calcium
sensor has been modified by the addition
of a N-terminal signal peptide (20 aa)
from neuromodulin (Pl-G-CaMP2). Addi-
tion of this peptide results in posttransla-
tional palmitoylation of the protein,
which facilitates anchoring of the protein
to the plasma membrane. HEK293 cells
were transfected on coated glass cover
slips with pcDNA31 Pl-G-CaMP2 and
were mounted in a perfusion chamber
48 h after transfection. Cells were per-
fused with Ringer solution at a rate of
8 mL/min at 37°C. Cell fluorescence
measurements were measured continu-
ously with an inverted microscope
Axiovert S100 (Zeiss) using a 40× objec-
tive (Fluar 40×/1.3 oil, Zeiss) and a high
speed polychromator system
(VisiChrome, Visitron, Puchheim, Ger-
many). Pl-G-CaMP2 was excited at
485 nm and 405 nm. Emission was re-
corded between 520 nm and 550 nm
using a CCD-camera (CoolSnap HQ, Vis-
itron). Control of experiments, imaging
acquisition and data analysis were done
with the software package Meta-Fluor
(Universal Imaging, New York, USA). Al-
ternatively, cells were loaded with Fura2
and intracellular Ca2+ concentrations
were determined as described earlier (17). Western Blotting, Biotinylation and
Immunocytochemistry Protein was isolated from THP-1 cells
grown in the absence or presence of
PMA (100 nmol/L) and transfected with
siRNA-ANO10 (ID# s30237, s30238, Am-
bion, Life Technologies [Thermo Fisher
Scientific]). Cells were lysed using lysis
buffer containing 150 mmol/L NaCl,
50 mmol/L Tris, 1 mmol/L EDTA,
100 mmol/L DTT, 0,5% NP-40 and 1%
protease inhibitor cocktail (Roche,
Mannheim, Germany). Protein separa-
tion, transfer, blotting and detection have
been described previously (17). A poly-
clonal rabbit anti-Ano10 antibody (Aviva
Systems Biology, San Diego, CA, USA)
was used at a dilution of 1:500. Rabbit
anti β-actin antibody (Sigma-Aldrich)
was used at a dilution of 1:1000. For bi-
otinylation of plasma membrane proteins
EZ-Link Sulfo-NHS-SS-Biotin (#89881,
Pierce, Thermo Fisher Scientific) was pre-
pared at a concentration of 1 mg/mL in
ice-cold phosphate-buffered saline (PBS). Biotinylated cells were lysed and 100-μL
streptavidin beads (Thermo Fisher Scien-
tific) were added to the supernatant after
centrifugation. After incubation
overnight at 4°C, beads were washed
five times with cold lysis buffer and bi-
otinylated proteins were eluted by boil-
ing the sample for 5 min at 95°C in SDS
sample buffer. For immunocytochemistry
of ANO10, the anti-ANO10 antibody was
used at a dilution of 1:500. All supplementary materials are available
online at www.molmed.org. RESULTS In a principal component analysis, 19
subjects showed non-European ancestry
and were consequently excluded from
genetic analyses (Supplementary Figure
S3). We finally analyzed a total of 2,376
individuals with available complete
genotype and serological data, fulfilling
all inclusion criteria. Of these, 162 (6.8%)
were seropositive and 2,214 (93.2%) sero-
negative. With the use of an allelic model,
580,108 autosomal SNPs were tested and
genomic inflation was low (λ = 1.016,
Supplementary Figure S4). Two SNPs
(rs17850869, rs41289586) exceeded the
threshold for genome-wide significance,
when correcting for the number of tested Statistics in mean age (Supplementary Table S2),
which has to be considered because the
likelihood of a past Borrelia infection and
subsequent antibody formation increase
with age (Supplementary Figure S1). We
did not find a difference in mean titer
levels of seropositive subjects between
patient groups and controls (Supplemen-
tary Table S3). Overall, seropositive and
seronegative schizophrenia patients do
not show differences with respect to
major disease phenotypes of schizophre-
nia including neurological signs as deter-
mined by the CNI, which should also
cover symptoms of borreliosis (Supple-
mentary Table S4). Interestingly, how-
ever, AB carriers score significantly
worse throughout all scales of the BSI
(corrected for age and sex as a proxy for
gender) (13), an instrument based on pa-
tients’ self-evaluation (Supplementary
Figure S2). Group differences in categorical and
continuous variables were assessed using
chi-square (χ2 or Mann-Whitney U tests. A generalized linear model was em-
ployed upon covariate inclusion. At nor-
mal distribution of continuous variables,
t tests were performed (paired and un-
paired tests, respectively, for experiments
in oocytes, HEK293 cells, lymphocytes
and macrophages). A basic allelic test,
implemented in PLINK, was used to test
for association between single nucleotide
polymorphisms (SNPs) and Borrelia sero-
logical status. P values < 0.05 were con-
sidered significant and multiple testing
corrected (Bonferroni) where indicated,
but are displayed uncorrected. Data in
figures are expressed as mean ± SEM, in
tables as mean ± SD. Annexin V Binding Assay Annexin V Binding Assay
THP-1 cells treated with PMA
(100 nmol/L, 48 h) were grown in a 96- THP-1 cells treated with PMA
(100 nmol/L, 48 h) were grown in a 96- 2 8
| H A M M E R E T A L . | M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 R E S E A R C H A R T I C L E Table 1. Cross-sectional prevalence of anti-Borrelia antibodies. Anti-Borrelia AB status, N (%)
Study group
Seropositive
Seronegative
Total
P (Pearson χ2)a
GRAS patients (schizophrenia)
85 (7.9)
991 (92.1)
1,076 (100)
0.05
Affective disorder patientsb
16 (10.8)
132 (89.2)
148 (100)
0.07
GRAS controls (healthy subjects)
68 (5.4)
1,203 (94.6)
1,271 (100)
Total
169 (6.8)
2,326 (93.2)
2,495 (100)
aCorrected for age and sex. P values represent results of χ2 tests, comparing the respective
patient sample with healthy controls. bIncluded are patients with monopolar or bipolar depression. Table 1. Cross-sectional prevalence of anti-Borrelia antibodies. oss-sectional prevalence of anti-Borrelia antibodies well plate. Cells were washed twice with
cold PBS and incubated with annexin V-
FITC for 15 min at room temperature
(FITC Annexin V Detection Kit, BD Bio-
sciences, Heidelberg, Germany). Fluores-
cence intensity was detected using a
plate reader (Novostar, BMG Labtech,
Ortenberg, Germany). Cells were treated
with TNFα (10 ng/mL) or with cherry-
labeled B. garinii (MOI 1:10) for 4 h, fol-
lowed by washing with PBS and fluores-
cence detection, considered as time point
zero. For other time points, the cells were
washed to remove the remaining Borrelia
and kept with fresh media for days fol-
lowing infection. Borrelia Seropositivity in Health and
Neuropsychiatric Disease We detected anti-Borrelia antibodies
(AB) in 169 out of 2,495 individuals in
total (6.8%) (Table 1). AB prevalence
tended to be higher in schizophrenia pa-
tients (7.9%, P = 0.05) and affective disor-
der patients (11.0%, P = 0.07), when com-
pared with psychiatrically healthy
controls (5.4%). P values are corrected for
sex and age since male subjects are more
likely to be seropositive than females
(8.2% versus 4.3%, P = 1.96E-04, odds
ratio (OR) = 1.98, Supplementary Table S1). Furthermore, groups differ significantly M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 | H A M M E R E T A L . | 2 9 2 9 A N O C T A M I N 1 0 I N B O R R E L I A I N F E C T I O N Figure 1. Manhattan plot of genome-wide association analysis. The red horizontal line des-
ignates the threshold for genome-wide significance, corrected for number of tested SNPs. some 16 (NP_001188482.1, p.Leu393) and
associated with a P value of 4.17E-09
(OR = 5.36). It is in complete linkage
with only one other SNP, rs74944699, an
intronic variant in PMFBP3. Of note, the
gene upstream of ZNF821 is ATXN1L
(ataxin 1-like), a paralog of ATXN1
(ataxin 1), which is associated with spin-
ocerebellar ataxia type 1 (SCA1) (21). The other SNP, rs41289586 (P = 7.18E-
08, OR = 3.38), is a missense variant of
anoctamin 10, encoded by the gene
ANO10 (NP_060545.3, p.R263H) on chro-
mosome 3. It shows linkage (r2 > 0.8)
with two intronic SNPs, rs62250916 in
ANO10 and rs11926254 in SNRK. With
the use of software tools for a prediction
of the effect of amino acid substitutions
on protein function, the ANO10-R263H
variant was predicted to be “probably
damaging” (score 1.000) by PolyPhen-2
(http://genetics.bwh.harvard.edu/pph2/)
(22), “deleterious” (score –4.66) by
PROVEAN and “damaging” (score 0.000)
by SIFT (both http://provean.jcvi.org/)
(23,24). Notably, also mutations in
ANO10 were reported to be causative for Figure 1. Manhattan plot of genome-wide association analysis. The red horizontal line des-
ignates the threshold for genome-wide significance, corrected for number of tested SNPs. SNPs (P = 8.62E-08, Figure 1). A list of 11
SNPs with P <1.0E-05 is provided as
Supplementary Table S5, including
minor allele frequencies, association sta-
tistics, positions and SNP classifications. Borrelia Seropositivity in Health and
Neuropsychiatric Disease One of the two genome-wide signifi-
cant SNPs, rs17850869, is a synonymous
coding variant of zinc finger protein 821,
encoded by the ZNF821 gene on chromo- ANO10 were reported to be causative for
spinocerebellar ataxia (25,26). Table 2. Genotypes and MAFs of GWAS hits.a Genome-Wide Significant Hits Table 2. Genotypes and MAFs of GWAS hits.a
Subjects or populations
Genotypes
according to
Pb (allelic test)
reference SNP ID
CC
CT
TT
MAF
(OR, [95% CI])
rs17850869
Seropositive
148
14
0
0.043
Seronegative
2,177
37
0
0.008
4.17E–09 (5.36, [2.87–10.02])
1000g (CEU)
80
5
0
0.029
0.449 (1.49, [0.53–4.21])
1000g (EUR)
362
17
0
0.022
0.061 (1.97, [0.96–4.04])
EVS (EA)
4,148
50
2
0.006
1.50E–13 (6.98, [3.84–12.70])
rs41289586
Seropositive
140
21
1
0.071
Seronegative
2,118
96
1
0.022
7.18E–08 (3.38, [2.11–5.39])
1000g (CEU)
80
5
0
0.029
0.058 (2.52, [0.94–6.76])
1000g (EUR)
362
17
0
0.022
1.1E–04 (3.33, [1.75–6.32])
EVS (EA)
4,074
222
4
0.027
2.47E–06 (2.78, [1.78–4.33])
CI, confidence interval; 1000g, 1000 Genomes Project; CEU, Utah Residents (CEPH) with
Northern and Western European ancestry panel; EUR, European superpopulation panel;
EVS (EA), Exome variant server European American population panel. aGenotype distribution of GWAS hits in seropositive versus seronegative subjects, as well as
in additional control populations. bP values represent results of allelic tests, comparing the respective control sample with
the seropositive subjects. Both genome-wide significant SNPs
show a low minor allele frequency in se-
ronegative subjects, which is significantly
higher in AB carriers (rs17850869: 0.008
versus 0.043; rs41289586: 0.022 versus
0.071, Supplementary Table S5). Genotype
distributions are presented in Table 2,
where we also display results using addi-
tional open-access resources from the 1000
Genomes Project (19) and the Exome Vari-
ant Server [NHLBI GO Exome Sequenc-
ing Project (ESP); (20)]. Overall, these data
are highly similar to the distribution in
our seronegative population; hence an un-
derrepresentation of the minor alleles is
unlikely to be the source of association. As an exception, the minor allele fre-
quency (MAF) of rs17850869 is higher in
the European 1000 Genomes Project study
participants (MAF = 0.022). This may,
however, be a bias of the small number of
individuals included there (Table 2). 3 0
| H A M M E R E T A L . | M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 R E S E A R C H A R T I C L E We investigated, but did not find, an
association of either SNP with antibodies
against several other bacterial infections
(Helicobacter pylori, Mycoplasma pneumo-
niae, Chlamydia pneumoniae, Chlamydia tra-
chomatis). Genome-Wide Significant Hits They also were not associated
with a sum score including all five sero-
logical tests against bacterial infections in
a linear regression model (Supplemen-
tary Table S6). Neither SNPs is found on
commonly used genotyping arrays and
were thus not included previously in
GWAS investigating other phenotypes. In our study cohort, they were not asso-
ciated with the diagnosis of schizophre-
nia (rs41289586: Pallelic = 0.11, rs17850869:
Pallelic = 0.28). we found that Hypo induced a delayed
transient rise in intracellular Ca2+, which
was augmented by ANO10 but reduced
by R263H-ANO10. However, ANO10
does not seem to affect the filling of the
ER Ca2+ store, since the SERCA pump in-
hibitor cyclopiazonic acid (CPA) induced
a similar Ca2+ increase in the absence or
presence of ANO10 (Figure 4G). Hypo-
induced store release occurred through
dantrolene-sensitive ryanodine receptors
(40). In the presence of dantrolene, IHypo
was not augmented by ANO10 (Supple-
mentary Figure S5I, J). Taken together,
R263H-ANO10 may compromise volume
regulation by participating in an ion
channel complex or by controlling intra-
cellular Ca2+ signaling (Supplementary
Figure S8A). R263H-ANO10 (Figure 2D). It is worth
noting that activation of phospholipase
A2 by melittin, a known activator of
IHypo, also activated ANO10. Moreover,
coexpression of LRRC8A, which itself in-
duced IHypo, did not further augment
IHypo produced by ANO10 (Figures 2E, F). Taken together, ANO10 but not R263H-
ANO10 generates swelling-activated
whole cell currents in oocytes. We also expressed ANO10 in HEK293
cells and found enhanced whole cell cur-
rents activated by Hypo, which were in-
hibited by typical anoctamin blockers
such as NPPB, NS3728 and TinhA01 (Fig-
ures 3A, B). Currents could not be acti-
vated in the complete absence of Ca2+,
but were augmented, along with an in-
crease in volume regulation (regulatory
volume decrease, RVD), when only ex-
tracellular Ca2+ was reduced to We also expressed ANO10 in HEK293
cells and found enhanced whole cell cur-
rents activated by Hypo, which were in-
hibited by typical anoctamin blockers
such as NPPB, NS3728 and TinhA01 (Fig-
ures 3A, B). Currents could not be acti-
vated in the complete absence of Ca2+,
but were augmented, along with an in-
crease in volume regulation (regulatory
volume decrease, RVD), when only ex-
tracellular Ca2+ was reduced to
0.1 μmol/L (Figure 3C, Supplementary
Figures S5A, B). Genome-Wide Significant Hits IHypo was inhibited by
arachidonic acid, confirming earlier re-
ports (39) and was controlled by phos-
pholipase A2 (Supplementary Figures
S5C–F). Notably, IHypo was significantly
reduced by the expression of two
ANO10-mutants that have been reported
to cause cerebellar ataxia (25,26) (Supple-
mentary Figures S5G, H). Expression of
ANO10 augmented RVD during expo-
sure to Hypo when measured by flow
cytometry or single cell imaging of cal-
cein-loaded cells (Figures 3D–G). These
data establish a role of ANO10 for vol-
ume regulation in mammalian cells. Compromised Cellular Volume
Regulation by ANO10-R263H ANO10 belongs to a family of 10 pro-
teins which operate as Cl– channels and
phospholipid scramblases (27–31). Struc-
tural insights into TMEM16/anoctamin
proteins were provided recently (32). R263 is located close to the dimer inter-
face and is well conserved within the
anoctamin family and between species
(Supplementary Figures S8B, C). Anocta-
mins have been reported earlier to be rel-
evant for cellular volume regulation
(18,33,34), which is essential for cell
migration and immune defense (35). Anoctamins may be part of a channel or
regulatory complex that produces
volume-regulated anion currents (IHypo)
activated by hypotonic bath solution
(Hypo). An essential component of such
a complex has been identified as LRRC8
(36,37). We examined the role of ANO10
for volume regulation by coexpression
with aquaporin 1 in Xenopus oocytes,
which swell and eventually burst when
exposed to Hypo (38). Expression of
ANO10, but not R263H-ANO10, pro-
duced large outwardly rectifying whole
cell currents (IHypo) when oocytes were
exposed to Hypo (Figures 2A, B). Coex-
pression of R263H-ANO10 together with
ANO10 suppressed activation of IHypo
(Figure 2C). Moreover, bursting of
oocytes due to Hypo-induced swelling
was reduced by ANO10 but not by Compromised Macrophage Function
in the Absence of ANO10 Macrophages are within the first line
of defense during infection with Borrelia
(41). We found that ANO10 is expressed
along with ANO6 in human THP-1
macrophages as well as in freshly iso-
lated mouse peritoneal macrophages (Fig-
ures 5A, B; Supplementary Figures S7A, B). In THP-1 macrophages, ANO10 was lo-
cated mostly intracellularly (Figure 5C). RVD was examined in single cells by
loading macrophages with calcein. Re-
covery from Hypo-induced cell swelling
(RVD) was reduced after siRNA-
knockdown of ANO10 (Figures 5D, E). Similar results were obtained in mouse
macrophages in which Ano10 expression
was inhibited by siRNA or was knocked
down in Ano10lox/lox/E2A-cre mice (Sup-
plementary Figures S7C–E). The results
indicate that ANO10 is important for vol-
ume regulation also in human and
mouse macrophages. In contrast to (wild-type) wt ANO10,
R263H-ANO10 failed to produce large
IHypo and compromised RVD in HEK293
cells (Figures 4A–C). Virtually identical
results were obtained when ANO10 and
R263H-ANO10 were expressed in lym-
phocytes (Supplementary Figure S6). Im-
munocytochemistry and membrane bi-
otinylation showed weak membrane
expression of ANO10 and R263H-
ANO10 and suggested primarily a loca-
tion of ANO10 in the endoplasmic reticu-
lum (ER) (Figures 4H–J). Using the
plasma membrane-targeted Ca2+-sensi-
tive protein GCAMP2 (Figures 4D, E), or
conventional Fura2 imaging (Figure 4F), Similar to the experiments in oocytes,
in macrophages, expression of ANO10-
R263H inhibited IHypo (Figure 5F). IHypo
and volume regulation is a prerequisite
for cell migration and thus crucial for
eradication of spirochetes (35,42). We
therefore examined migration of
macrophages, which was induced by
monocyte chemoattractant protein 1 M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 | H A M M E R E T A L . | 3 1 A N O C T A M I N 1 0 I N B O R R E L I A I N F E C T I O N 2. ANO10 but not R263H-ANO10 generates volume-activated whole cell currents in Xenopus oocytes. (A) Current–voltage relationships
e cell currents activated by cell swelling (IHypo, 50 % reduced extracellular osmolarity) in Xenopus oocytes. R263H-ANO10 does not pro-
ypo. (B) Current overlay (voltage clamp [Vc] = ±100 mV) demonstrates typical time dependent inactivation of IHypo. (C) IHypo in oocytes
ing (left to right, respectively): AQP1 and ANO10; AQP1 and ANO10-R263H; or AQP1, ANO10, and ANO10-R263H. Compromised Macrophage Function
in the Absence of ANO10 Coexpression of
-R263H suppressed currents produced by wt ANO10. (D) Oocyte bursting after exposure to hypotonic bath solution. Fraction of burst
s was reduced by expression of ANO10. Oocytes survived in the absence of AQP1. (E) Summary of whole cell currents activated by
nd the PLA2-activator melittin (100 nmol/L). (F) Summary of time-dependent activation of whole cell currents in cells expressing
LRRC8A or coexpressing both. All oocytes expressed AQP1. Mean ± SEM (number of oocytes); *significant activation by Hypo (paired
significant difference (by unpaired t test) when compared with ANO10 alone (E) or with ANO10 plus AQP1 (ANO10+AQP1) (F). Figure 2. ANO10 but not R263H-ANO10 generates volume-activated whole cell currents in Xenopus oocytes. (A) Current–voltage relationships
of whole cell currents activated by cell swelling (IHypo, 50 % reduced extracellular osmolarity) in Xenopus oocytes. R263H-ANO10 does not pro-
duce IHypo. (B) Current overlay (voltage clamp [Vc] = ±100 mV) demonstrates typical time dependent inactivation of IHypo. (C) IHypo in oocytes
expressing (left to right, respectively): AQP1 and ANO10; AQP1 and ANO10-R263H; or AQP1, ANO10, and ANO10-R263H. Coexpression of
ANO10-R263H suppressed currents produced by wt ANO10. (D) Oocyte bursting after exposure to hypotonic bath solution. Fraction of burst
oocytes was reduced by expression of ANO10. Oocytes survived in the absence of AQP1. (E) Summary of whole cell currents activated by
Hypo and the PLA2-activator melittin (100 nmol/L). (F) Summary of time-dependent activation of whole cell currents in cells expressing
ANO10, LRRC8A or coexpressing both. All oocytes expressed AQP1. Mean ± SEM (number of oocytes); *significant activation by Hypo (paired
t test); #significant difference (by unpaired t test) when compared with ANO10 alone (E) or with ANO10 plus AQP1 (ANO10+AQP1) (F). cantly after siRNA-knockdown of
ANO10 (Figures 5H, I). Exposure to
B. garinii induced a strong release of the
major cytokine TNFα by THP-1 cells,
which was not affected by knockdown of
ANO10 (Figures 5J, K). No immediate
cell death was observed upon exposure
and phagocytosis of B. garinii, but apo-
ptosis of THP-1 macrophages was re-
duced 6 d after exposure to B. garinii,
which may allow B. garinii to circumvent
innate defense (data not shown). Taken (MCP-1). Migration was largely reduced
by siRNA-knockdown of ANO10 and
was inhibited by typical anoctamin
blockers (Figure 5G). Cell viability was
not affected by these procedures (data
not shown). Compromised Macrophage Function
in the Absence of ANO10 Because cell migration and
phagocytic activity of macrophages will
determine the efficacy of spirochete erad-
ication (35,42), we examined phagocyto-
sis of red-fluorescent cherry-labeled B. garinii by THP-1 macrophages. Phagocy-
tosis of B. garinii was reduced signifi- together, the present results suggest that
ANO10 is important for volume regula-
tion of macrophages and for their role in
innate immunity. Eradication of spiro-
chetes may be compromised in carriers
of the ANO10 variant R263H. Phenotypes in Borreliosis Patients In a subsequent exploratory human
study, we wondered whether patients
with laboratory-confirmed borreliosis,
carrying the ANO10-R263H variant 3 2
| H A M M E R E T A L . | M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 R E S E A R C H A R T I C L E Figure 3. ANO10 affects volume-activated whole cell currents in HEK293 cells. (A) Whole cell currents (voltage clamp [Vc] = ± 100 mV)
activated by cell swelling (IHypo, 33% reduced extracellular osmolarity) in ANO10-expressing cells. (B) Swelling induced currents (IHypo)
in ANO10-expressing cells relative to mock transfected cells and inhibition by NPPB (50 μmol/L), NS3728 (5 μmol/L) and TinhAO1
(20 μmol/L). (C) Current–voltage (I–V) curves indicating loss of IHypo with complete elimination of Ca2+ and preincubation with BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 50 μmol/L, 30 min). (D) Regulation of cell volume in the presence of Hypo
(regulatory volume decrease, RVD) in mock-transfected cells or cells overexpressing ANO10 (flow cytometry). (E) RVD in mock-trans-
fected cells or cells overexpressing ANO10 and inhibition by NPPB, NS3728 and TinhAO1. (F) Reshrinkage of cells exposed to hypotonic
bath solution (RVD), measured in single cells loaded with calcein. (G) Comparison of RVD (measured by calcein fluorescence) obtained
in mock-transfected and ANO10-overexpressing cells. Mean ± SEM (number of cells); *significant inhibition (paired t test); #significant dif-
ference to mock (unpaired t test). Figure 3. ANO10 affects volume-activated whole cell currents in HEK293 cells. (A) Whole cell currents (voltage clamp [Vc] = ± 100 mV)
activated by cell swelling (IHypo, 33% reduced extracellular osmolarity) in ANO10-expressing cells. (B) Swelling induced currents (IHypo)
in ANO10-expressing cells relative to mock transfected cells and inhibition by NPPB (50 μmol/L), NS3728 (5 μmol/L) and TinhAO1
(20 μmol/L). (C) Current–voltage (I–V) curves indicating loss of IHypo with complete elimination of Ca2+ and preincubation with BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 50 μmol/L, 30 min). (D) Regulation of cell volume in the presence of Hypo
(regulatory volume decrease, RVD) in mock-transfected cells or cells overexpressing ANO10 (flow cytometry). (E) RVD in mock-trans-
fected cells or cells overexpressing ANO10 and inhibition by NPPB, NS3728 and TinhAO1. (F) Reshrinkage of cells exposed to hypotonic
bath solution (RVD), measured in single cells loaded with calcein. (G) Comparison of RVD (measured by calcein fluorescence) obtained
in mock-transfected and ANO10-overexpressing cells. Phenotypes in Borreliosis Patients Mean ± SEM (number of cells); *significant inhibition (paired t test); #significant dif-
ference to mock (unpaired t test). would differ in any respect from noncar-
riers. Specifically, due to the potential as-
sociation of both identified SNPs with
cerebellar ataxia, we searched for a po-
tential overrepresentation of cerebellar
ataxia-like symptoms that also have been
reported previously in cases of neurobor-
reliosis (43). To address this question, we
recruited prospectively 100 patients with
laboratory-confirmed diagnosis of borre-
liosis. Patients had a mean age of 56.3
years (standard deviation: 16.0 years,
range: 15 to 86 years), 58% were male. Classical clinical correlates of neurobor-
reliosis (including meningitis, radiculitis,
cranial nerve palsy, ataxia, dizziness, en-
cephalitis) were present in 30 patients
and those of systemic Lyme borreliosis
(including erythema migrans, arthralgia,
myalgia, headache, malaise, nausea,
dizziness) were present in 20 patients. A
total of 50 patients had just a laboratory-
based diagnosis without typical clinical susceptibility to Borrelia seropositivity. Interestingly, both variants, located on
chromosomes 3 and 16, happen to be in
some context with spinocerebellar ataxia
(25,26). The SNP on chromosome 3,
rs41289586, represents the missense vari-
ant ANO10-R263H, encoded by the gene
ANO10. We provide here first evidence
of this variant modifying normal host
defense. The role of the variant on chro-
mosome 16, rs17850869, a synonymous
SNP in ZNF821 is presently less clear. Addressing the second objective of the
present study, that is, to potentially relate
Borrelia seropositivity to core phenotypes
of neuropsychiatric disorders, we ob-
tained a significantly higher symptom
load of seropositive versus seronegative
individuals in essentially all items of the
BSI (13) self-rating scale. signs and symptoms; 4 out of this total of
100 individuals carried the rs41289586
risk allele (T, ANO10-R263H variant) and
2 of 100 had the rs17850869 risk allele
(T); all were heterozygous (CT) for these
risk SNPs. Of the 6 (4 + 2) risk allele car-
riers, 5 had the diagnosis borreliosis
without typical clinical symptoms (only
laboratory signs of infection) in contrast
to 45 of 94 noncarriers (5 of 6 versus 45
of 94: Fisher exact P = 0.20). Moreover, 3
of 6 had cerebellar symptoms in contrast
to 28 of 94 (3 of 6 versus 28 of 94: Fisher
exact P = 0.37). Apart from these poten-
tially interesting hints that would need
to be consolidated in larger followup
studies, no prominent clinical differences
were detected. Phenotypes in Borreliosis Patients would differ in any respect from noncar-
riers. Specifically, due to the potential as-
sociation of both identified SNPs with
cerebellar ataxia, we searched for a po-
tential overrepresentation of cerebellar
ataxia-like symptoms that also have been
reported previously in cases of neurobor-
reliosis (43). To address this question, we
recruited prospectively 100 patients with
laboratory-confirmed diagnosis of borre-
liosis. Patients had a mean age of 56.3
years (standard deviation: 16.0 years,
range: 15 to 86 years), 58% were male. Classical clinical correlates of neurobor-
reliosis (including meningitis, radiculitis,
cranial nerve palsy, ataxia, dizziness, en-
cephalitis) were present in 30 patients
and those of systemic Lyme borreliosis
(including erythema migrans, arthralgia,
myalgia, headache, malaise, nausea,
dizziness) were present in 20 patients. A
total of 50 patients had just a laboratory-
based diagnosis without typical clinical DISCUSSION Macrophage function is essential for
eradication of Borrelia (41). We recently
found a role of anoctamin 6 (ANO6) for In the first GWAS on Borrelia antibody
serostatus, we identified two host ge-
nomic variants mediating differential M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 | H A M M E R E T A L . | 3 3 A N O C T A M I N 1 0 I N B O R R E L I A I N F E C T I O N ure 4. R263H inhibits volume regulation, IHypo and intracellular Ca2+ signaling in HEK293 cells. (A) Whole cell currents (voltage clamp
] = ± 100 mV) activated by cell swelling (IHypo, 33 % reduced extracellular osmolarity) in cells expressing ANO10 and R263H-ANO10
63H). (B) Current–voltage relationships for IHypo and inhibition of IHypo by removal of Cl– from the extracellular bath solution (5Cl–). Regulation of cell volume in the presence of Hypo (regulatory volume decrease, RVD) in cells expressing ANO10 or R263H (flow cy-
etry). (D) Effect of cell swelling on intracellular [Ca2+] in cells expressing ANO10 or R263H or mock transfected cells, as measured by
Ca2+ sensor GCAMP2. (E) Summary of the effects of cell swelling on [Ca2+]i (485/405 fluorescence emission ratio) in ANO10 and R263
ressing cells. (F) Collected recordings of the effects of cell swelling on [Ca2+]i, measured by Fura2. (G) Collected recordings of the ef
ts of ER-store emptying by cyclopiazonic acid (CPA; 10 μmol/L) on [Ca2+]i, measured by Fura2. (H) Confocal images of cells expressin
O10 or R263H suggesting weak membrane expression. (I) Live staining of ANO10-GFP (green) and ER (ER-tracker; red) suggesting ER
alization of ANO10. (J) Membrane biotinylation of cells expressing ANO10 or R263H, suggesting low membrane expression of ANO10,
ch is even reduced for R263H. Mean ± SEM (number of experiments); #significant difference when compared with mock (analysis of
ance [ANOVA]); §significant difference when compared with ANO10 (ANOVA). Bar = 20 μm. Numbers are given in the graph in pare
sis. z, z-scan, side view; DIC, differential interference contrast. Figure 4. R263H inhibits volume regulation, IHypo and intracellular Ca2+ signaling in HEK293 cells. (A) Whole cell currents (voltage clamp
[Vc] = ± 100 mV) activated by cell swelling (IHypo, 33 % reduced extracellular osmolarity) in cells expressing ANO10 and R263H-ANO10
(R263H). induced IHypo correspond well to those
described for VRAC (reviewed in
[44–46]). How does ANO10 control IHypo
and thereby affect RVD? It could be a induced IHypo correspond well to those
described for VRAC (reviewed in
[44–46]). How does ANO10 control IHypo
and thereby affect RVD? It could be a DISCUSSION (B) Current–voltage relationships for IHypo and inhibition of IHypo by removal of Cl– from the extracellular bath solution (5Cl–). (C) Regulation of cell volume in the presence of Hypo (regulatory volume decrease, RVD) in cells expressing ANO10 or R263H (flow cy-
tometry). (D) Effect of cell swelling on intracellular [Ca2+] in cells expressing ANO10 or R263H or mock transfected cells, as measured by
the Ca2+ sensor GCAMP2. (E) Summary of the effects of cell swelling on [Ca2+]i (485/405 fluorescence emission ratio) in ANO10 and R263H
expressing cells. (F) Collected recordings of the effects of cell swelling on [Ca2+]i, measured by Fura2. (G) Collected recordings of the ef-
fects of ER-store emptying by cyclopiazonic acid (CPA; 10 μmol/L) on [Ca2+]i, measured by Fura2. (H) Confocal images of cells expressing
ANO10 or R263H suggesting weak membrane expression. (I) Live staining of ANO10-GFP (green) and ER (ER-tracker; red) suggesting ER
localization of ANO10. (J) Membrane biotinylation of cells expressing ANO10 or R263H, suggesting low membrane expression of ANO10,
which is even reduced for R263H. Mean ± SEM (number of experiments); #significant difference when compared with mock (analysis of
variance [ANOVA]); §significant difference when compared with ANO10 (ANOVA). Bar = 20 μm. Numbers are given in the graph in paren-
thesis. z, z-scan, side view; DIC, differential interference contrast. immune functions of macrophages (17),
while volume regulation by anoctamins
has been reported earlier (18,33,34). We
therefore analyzed the role of ANO10 for volume regulation and found that IHypo
and RVD are depending on ANO10 in
oocytes, HEK293 cells, lymphocytes and
macrophages. The properties of ANO10- 3 4
| H A M M E R E T A L . | M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 R E S E A R C H A R T I C L E Figure 5. Role of ANO10 for volume regulation in macrophages. (A) RT-PCR analysis of anoctamin expression in THP-1 macrophages. (B) Western blot indicating knockdown of ANO10-expression by siRNA. (C) ANO10 (green) and peripheral actin (rhodamin-phalloidin) of
THP-1 cells suggesting dominant intracellular location of ANO10. (D) Summary trace for reshrinkage of cells exposed to hypotonic bath
solution (RVD), measured in single cells loaded with calcein. RVD was abolished after siRNA-knockdown of ANO10. (E) Summary of RVD
measured by absolute fluorescence change. (F) I/V curves indicating reduced IHypo in R263H-expressing cells. DISCUSSION (G) Migration assay in Boy-
den chambers. MCP-1 induced migration was inhibited by siRNA knockdown of ANO10 and anoctamin inhibitors TinhAO1 (20 μmol/L),
NPPB (50 μmol/L) or tannic acid (TA, 10 μmol/L). (H) THP-1 cells exposed to red-fluorescent cherry-labeled B. garinii. Accumulation of cy-
tosolic fluorescence, indicating progressing phagocytosis of Borrelia by THP-1 cells. (I) Increase in fluorescence intensity as a measure of
phagocytic activity. (J) Exposure of THP-1 cells to cherry-labeled B. garinii. (K) Release of TNFα upon exposure to B. garinii was not af-
fected by siRNA-knockdown of ANO10. Mean ± SEM (number of cells or assays). #Significant difference when compared with scrambled,
MCP-1 alone, mock or con (ANOVA); §significant increase in migration and phagocytosis, respectively (unpaired t test). M, marker; RT, re-
verse transcriptase; con, control; crbld: scrambled control RNA; siANO10: siRNA against ANO10. Bar = 20 μm. Figure 5. Role of ANO10 for volume regulation in macrophages. (A) RT-PCR analysis of anoctamin expression in THP-1 macrophages. (B) Western blot indicating knockdown of ANO10-expression by siRNA. (C) ANO10 (green) and peripheral actin (rhodamin-phalloidin) of
THP-1 cells suggesting dominant intracellular location of ANO10. (D) Summary trace for reshrinkage of cells exposed to hypotonic bath
solution (RVD), measured in single cells loaded with calcein. RVD was abolished after siRNA-knockdown of ANO10. (E) Summary of RVD
measured by absolute fluorescence change. (F) I/V curves indicating reduced IHypo in R263H-expressing cells. (G) Migration assay in Boy-
den chambers. MCP-1 induced migration was inhibited by siRNA knockdown of ANO10 and anoctamin inhibitors TinhAO1 (20 μmol/L),
NPPB (50 μmol/L) or tannic acid (TA, 10 μmol/L). (H) THP-1 cells exposed to red-fluorescent cherry-labeled B. garinii. Accumulation of cy-
tosolic fluorescence, indicating progressing phagocytosis of Borrelia by THP-1 cells. (I) Increase in fluorescence intensity as a measure of
phagocytic activity. (J) Exposure of THP-1 cells to cherry-labeled B. garinii. (K) Release of TNFα upon exposure to B. garinii was not af-
fected by siRNA-knockdown of ANO10. Mean ± SEM (number of cells or assays). #Significant difference when compared with scrambled,
MCP-1 alone, mock or con (ANOVA); §significant increase in migration and phagocytosis, respectively (unpaired t test). M, marker; RT, re-
verse transcriptase; con, control; crbld: scrambled control RNA; siANO10: siRNA against ANO10. Bar = 20 μm. olume regulation in macrophages. (A) RT-PCR analysis of anoctamin expression in THP-1 macrophages. LRR-motif for IHypo. DISCUSSION Moreover, LRCC8A-
D367stop inhibited ionomycin-activation
(1 μmol/L) of endogenous xANO1 cur-
rents by 43% ± 5.8% (n = 27) and abol-
ished IHypo in ANO10 expressing oocytes. binding partner of the essential VRAC
component LRRC8A (36,37), although we
did not find a potentiation of IHypo by co-
expression of ANO10 and LRRC8A in
oocytes and in HEK293 cells exogenous LRRC8A was even inhibitory on IHypo. In-
terestingly, no IHypo was found when we
expressed a LRRC8A mutant lacking the
leucine-rich repeat (LRCC8A-D367stop;
data not shown), suggesting a role of the M O L M E D 2 1 : 2 6 - 3 7 , 2 0 1 5 | H A M M E R E T A L . | 3 5 ACKNOWLEDGMENTS R263H-ANO10 had a dominant nega-
tive effect on this ANO10 function. Due
to the location of R263 close to the dimer
interface, the mutation could interfere
with dimerization of ANO10, thereby af-
fecting biosynthesis and/or protein func-
tion (32) (Supplementary Figures S8B, C). R263H compromised volume regulation,
migration and phagocytosis, thereby re-
ducing spirochete clearance. Interestingly
another member of the anoctamin family,
ANO9 (TMEM16J), is of potential rele-
vance for the defense against Mycobacte-
riae, because polymorphisms in the
PKP3-SIGIRR-TMEM16J gene region
were found to be associated with higher
susceptibility to tuberculosis (48). This work was supported by the Max
Planck Society and the Max Planck
Förderstiftung, as well as by the
DFGSFB699A12, DFG KU756/12-1,
Volkswagenstiftung AZ 87499 and the
Niedersachsen-Research Network on
Neuroinfectiology (N-RENNT) of the
Ministry of Science and Culture of Lower
Saxony. This work was also supported
by a Young Investigator Grant from the
Brain & Behavior Research Foundation,
as well as a postdoctoral scholarship
from the Daimler and Benz Foundation,
both awarded to C Hammer. We did not find any evidence for a
worse clinical outcome of schizophrenic
antibody carriers (independent of geno-
types) when compared with seronegative
schizophrenia patients with respect to
core symptoms of schizophrenia or to
neurological deficits, as assessed by
trained investigators. However, it is im-
portant to remember that antibody
seropositivity cannot simply be equated
with Lyme disease or neuroborreliosis. Nevertheless, when asked for self-
assessment of their overall condition em-
ploying the BSI (13) seropositive schizo-
phrenia patients rated more severe
symptoms throughout all inventory items
including the Global Severity Index (GSI). At this time, we cannot provide a reliable
interpretation of this data, but their non-
specific nature may reflect the reputation
of Borrelia as the “great imitator” (51) and
it is well known that subjective symp-
toms can persist after disappearance of
objective criteria (1). A N O C T A M I N 1 0 I N B O R R E L I A I N F E C T I O N bution to disease etiology, but rather as
disease-related deficits in personal hy-
giene or increased risk-taking behavior. Furthermore, considering the endemic
pattern of Borrelia infections (50), the dis-
tribution of patient recruitment centers
across Germany must also be taken into
consideration (8). In contrast to patients,
healthy volunteers were mainly from
Lower Saxony with a relatively low inci-
dence of borreliosis. This suggests a functional relationship
between LRRC8A and anoctamins. ANO10 also may control compartmental-
ized Ca2+ signals that have been shown
to be important for activation of IHypo
(40,47). This suggests a functional relationship
between LRRC8A and anoctamins. ANO10 also may control compartmental-
ized Ca2+ signals that have been shown
to be important for activation of IHypo
(40,47). shown recently for HIV (52) Progress in
the isolation and culture of Borrelia from
human serum might soon bring similar
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Identifikasi Tingkat Kesegaran Ikan Tongkol (Euthynnus sp.) Di Pasar Bumiayu, Kabupaten Brebes
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Journal of Marine Research (Universitas Diponegoro)
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DOI : 10.14710/jmr.v12i3.40444 DOI : 10.14710/jmr.v12i3.40444 Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518
EISSN: 2407-7690
https://ejournal3.undip.ac.id/index.php/jmr of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511- Identification of Freshness Level of Little Tuna (Euthynnus Sp) at Bumiayu Traditional
Market, Brebes Regency ABSTRACT: The level of fish freshness can affect the quality and nutrition of fish. The marketing
process of fresh little tuna which is marketed in Bumiayu Traditional Market, Brebes Regency has
less attention to fish handling and it can reduce the quality and nutrition of fresh eastern little tuna. The level of fish freshness can be identified or assessed through organoleptic test. The purpose of
this study was to determine the level of fish freshness of fresh little tuna at the Bumiayu Market
organoleptically. This research method was done by taking little tuna samples at 06.00am; 09.00am;
and 12.00pm WIB, then the freshness level was identified organoleptically and the pH of fresh fish
was tested as well. Sampling was carried out on September 14 and 28; also on October 12, 2022. The data analysis used descriptive analysis. The results of the study showed that tuna was still
categorized as fresh when the sampling was taken at 06.00 to 09.00; while the sampling taken at
12.00 had experienced a decrease in quality. The average organoleptic value is between 8.33 and
3.67. Based on SNI fresh fish SNI 2729:2013, the organoleptic value of fresh fish is 7. The pH of
little tuna meat is obtained from 5.56 to 6.11. pH is an indicator of the level of decrease in fish quality. The higher the pH value indicates that there is activity of decomposing microorganisms in fish body. Keywords: Freshness level 1; Little tuna 2; Bumiayu Traditional Market 3. Identifikasi Tingkat Kesegaran Ikan Tongkol (Euthynnus sp.)
di Pasar Bumiayu, Kabupaten Brebes M. Fajrun Najjah Al Fatich1, Aryanti Indah Setyastuti1*, Dewi Kresnasari1, Sarmin1
Program Studi Ilmu Perikanan, Fakultas Sains dan Teknologi, Universitas Nahdlatul Ulama Purwokerto
Jl. Sultan Agung No. 42, Karangklesem, Purwokerto Selatan, Kabupaten Banyumas, Jawa Tengah, Indonesia
*Corresponding author, e-mail: aryantiindahs@gmail.com M. Fajrun Najjah Al Fatich1, Aryanti Indah Setyastuti1*, Dewi Kresnasari1, Sarmin1
Program Studi Ilmu Perikanan, Fakultas Sains dan Teknologi, Universitas Nahdlatul Ulama Purwokerto
Jl. Sultan Agung No. 42, Karangklesem, Purwokerto Selatan, Kabupaten Banyumas, Jawa Tengah, Indonesia
*Corresponding author, e-mail: aryantiindahs@gmail.com ABSTRAK: Tingkat kesegaran ikan dapat mempengaruhi kualitas dan nutrisi ikan. Pemasaran ikan
tongkol segar yang dipasarkan di Pasar Bumiayu, Kabupaten Brebes kurang memperhatikan
penanganan, sehingga dapat menurunkan kualitas dan nutrisi ikan tongkol segar. Tingkat kesegaran
ikan dapat diidentifikasi atau dinilai secara organoleptik. Tujuan penelitian ini adalah untuk
mengetahui tingkat kesegaran ikan tongkol segar di Pasar Bumiayu secara organoleptik. Metode
penelitian ini dengan pengambilan sampel ikan tongkol pada pukul 06.00; 09.00; dan 12.00 WIB,
selanjutnya diidentifikasi kesegarannya secara organoleptik dan pengujian pH ikan segar. Pengambilan sampel dilakukan pada tanggal 14 dan 28 September 2022; serta tanggal 12 Oktober
2022. Analisa data menggunakan analisa deskriptif. Hasil penelitian selama 3 minggu, menunjukkan
bahwa ikan tongkol masih dikategorikan segar pada pengambilan sampel di pukul 06.00 hingga
09.00; sedangkan pada pukul 12.00 ikan sudah mengalami proses kemunduran mutu. Nilai
organoleptik rata-rata diantara 8,33 hingga 3,67. Berdasarkan SNI ikan segar SNI 2729:2013, nilai
organoleptik ikan segar adalah 7. pH daging ikan tongkol diperoleh sebesar 5,56 hingga 6,11. pH
merupakan indikator tingkat kemunduran mutu ikan. Semakin tinggi nilai pH menunjukkan bahwa
terjadi aktivitas mikroorganisme pembusuk dalam tubuh ikan. Kata kunci: Tingkat Kesegaran 1; Ikan Tongkol 2; Pasar Bumiayu 3. Identification of Freshness Level of Little Tuna (Euthynnus Sp) at Bumiayu Traditional
Market, Brebes Regency Keywords: Freshness level 1; Little tuna 2; Bumiayu Traditional Market 3. PENDAHULUAN g
Penelitian ini bertujuan untuk mengetahui tingkat kesegaran ikan tongkol di Pasar Induk Bumiayu
berdasarkan uji organoleptik. g
Penelitian ini bertujuan untuk mengetahui tingkat kesegaran ikan tongkol di Pasar Induk Bumiayu
berdasarkan uji organoleptik PENDAHULUAN Ikan memiliki sifat mudah mengalami pembusukan yang dipengaruhi oleh beberapa faktor
yaitu internal maupun eksternal. Adapun faktor internal antara lain jenis dan ukuran ikan, bakteri dan Diterima : 30-05-2023 ; Diterbitkan : 31-07-2023 ⧫512⧫ Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 enzim yang terdapat dalam ikan, oksidasi lemak dalam tubuh ikan. Sedangkan faktor eksternal
meliputi cara penangkapan, penanganan ikan selama transportasi dan pemasaran. Ikan tongkol
merupakan salah satu komoditas perikanan yang diminati oleh konsumen, karena kaya akan
kandungan protein, lemak, dan karbohidrat yang dapat memenuhi kebutuhan nutrisi tubuh (Sanger,
2020). Kandungan nutrisi protein maupun lemak yang relatif tinggi pada ikan tongkol, menyebabkan
ikan mudah mengalami oksidasi lemak yang dapat menurunkan nilai nutrisi ikan tongkol. Kesegaran
ikan dapat mempengaruhi kualitas ikan selama penanganan maupun pemasaran (Setyastuti et al.,
2021). Pemasaran ikan tongkol di Pasar Induk Bumiayu cukup tinggi mengingat ikan tongkol di
Bumiayu sangat diminati oleh masyarakat di Bumiayu dan sering diolah menjadi berbagai produk
olahan ikan. Komoditas perikanan di Pasar Bumiayu umumnya memiliki komposisi kimia yang tidak
stabil, mudah rusak akibat dari penanganan yang kurang baik yang dapat mempengaruhi tingkat
kesegaran ikan. Kesegaran ikan yang dipasarkan di Pasar Bumiayu kurang diperhatikan sehingga
ikan cepat mengalami pembusukan dan tidak layak untuk dikonsumsi. Tingkat kesegaran ikan dapat
dilihat secara organoleptik, dimana uji organoleptik adalah uji tingkat penerimaan konsumen yang
diperlukan dalam manajemen penanganan yang tepat agar tetap segar bagi konsumen. p
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Organoleptik merupakan salah satu metode uji produk pangan dengan mengamati organ tubuh
sebagai paramaternya berdasarkan penilaian subjektif oleh panelis (Puni et al., 2020). Uji
organoleptik dikenal juga dengan uji produk dengan panca indera pada manusia sebagai salah satu
alat uji (Nurmianto et al., 2018). Uji organoleptik ditujukan untuk mengetahui mutu ikan dengan
mengamati kenampakan (mata, insang, lendir permukaan badan), daging, bau dan tekstur (Vatria,
2020). Penanganan ikan dilakukan untuk mempertahankan kesegaran ikan setelah ditangkap dalam
jangka waktu tertentu sebelum dibeli oleh konsumen. Permasalahan yang diamati saat ini adalah
kondisi pedagang ikan di pasar yang tidak menerapkan kehati-hatian, prinsip penanganan yang
kurang cepat, dan bersih, ikan yang dijual oleh pedagang sering ditemukan tercemar oleh bahan
kimia, cemaran fisik, dan mikrobiologis sebelum sampai ke konsumen karena kurangnya
pengetahuan tentang metode penanganan ikan yang aman, sehingga ikan dapat mengalami
kemunduran mutu sebelum di tangan konsumen. Organoleptik Ikan Tongkol Segar g
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Uji organoleptik memiliki peran penting untuk mengetahui mutu suatu produk karena dapat
sebagai indikator awal kemunduran mutu dan kerusakan nutrisi ikan secara visual. Hasil penilaian
organoleptik ikan tongkol segar disajikan pada Tabel 1. Tabel 1 menunjukkan nilai masing-masing
spesifikasi yang digunakan dalam uji organoleptik, yakni mata, insang, lendir permukaan badan,
daging, bau, dan tekstur. Berdasarkan tabel tersebut dalam penelitian pertama pada tanggal 14
September 2022, bobot nilai untuk setiap spesifikasi penilaian memperoleh nilai yang cukup bagus
yakni rata-rata skor 8,67 untuk penelitian tahap pertama. Sampel dengan pengambilan ke-2 rata-
rata skor yang diperoleh adalah 7,00. Sementara untuk penelitian tahap ke-3 diperoleh rata-rata
5,67. Berdasarkan data tersebut maka rata-rata skor uji organoleptik pada penelitian pertama adalah
7 11 Penelitian kedua dilaksanakan pada tanggal 28 September 2022, perolehan nilai rata-rata uji
organoleptik pada penelitian tahap ke-1 sebesar 8,17. Perolehan skor pada penelitian tahap ke-2
sebesar 7,00 dan pada tahap ke-3 diperoleh rata-rata skor 5,17. Adapun rata-rata keseluruhan hasil
uji organoleptik pada penelitian kedua ini sebesar 6,78. Penelitian ketiga dilaksanakan pada 12
Oktober 2022, dengan perolehan rata-rata hasil uji organoleptik untuk penelitian tahap pertama
sebesar 7,83. Pada penelitian tahap ke-2 diperoleh rata-rata 6,83. Sementara untuk tahap ke-3
diperoleh skor rata-rata sebesar 4,67. Total rata-rata uji organoleptik pada penelitian ketiga ini
sebesar 6,44. Penelitian keempat atau yang terakhir dilaksanakan pada tanggal 28 Oktober 2022. Penelitian
ini memperoleh data skor rata-rata uji organoleptik pada tahap ke-1 sebesar 8. Pada penelitian tahap
ke-2 diperoleh rata-rata uji organoleptik sebesar 6,5. Kemudian, pada penelitian tahap ke-3
diperoleh rata-rata skor uji organoleptik sebesar 4,17. Total rata-rata keseluruhan uji organoleptik
pada penelitian keempat ini diperoleh angka sebesar 6,22. Dengan demikian. Berdasarkan
perolehan nilai tersebut ikan tongkol (Euthynnus sp) di pasar Induk Bumiayu tergolong segar dan
masih layak untuk dikonsumsi atau masih segar diantara pukul 6 hingga 9 pagi, akan tetapi diatas
pukul 12 siang ikan tongkol yang dipasarkan di Pasar Induk Bumiayu tidak dapat diterima oleh
konsumen atau telah mengalami kemunduran mutu ikan dengan ditunjukkan nilai rerata organoleptik
dibawah 6. Menurut SNI 2729: 2013 nilai organoleptik ikan segar adalah 7. Spesifikasi ikan segar berdasarkan hasil penelitian dari Lestari et al., (2015), mengenai tingkat
kesegaran ikan di Pasar Minggu Kota Bengkulu menunjukkan bahwa ikan pada pagi hari masih
tergolong segar, pada siang hari menunjukkan penurunan tingkat kesegaran ikan. MATERI DAN METODE Penelitian ini dilaksanakan pada bulan September-Oktober 2022. Pengambilan sampel ikan
tongkol (Euthynnus sp) dilaksanakan di Pasar Induk Bumiayu, Jl. Raya Bumiayu, Dukuh Bandung,
Bumiayu, Kabupaten Brebes, Jawa Tengah 52273, Indonesia. Pengambilan sampel dilakukan pada
jam 06.00 WIB; 09.00 WIB; dan 12.00 WIB dan dilakukan pengujian organoleptik dan pH. Analisa organoleptik sebagai alat untuk mengetahui tingkat kesegaran ikan melalui ciri fisik
yang mengacu pada Standar Mutu Ikan Segar (SNI 2729: 2013). Proses pengujian nilai organoleptik
dilihat dari ciri fisik ikan yang meliputi kenampakan mata, insang dan lendir permukaan; daging; bau;
tekstur. Metode uji organoleptik dalam penelitian ini diamati oleh 15 panelis semi terlatih dengan
menggunakan score sheet organoleptic test dengan interval skala 1-9, dan syarat mutu organoleptik
minimum adalah 7,0. Jika hasil uji menunjukkan nilai < 7, maka produk memiliki mutu rendah dan
tidak sesuai dengan standar, kemudian jika menunjukkan >7 maka mutu ikan tergolong baik dan
layak untuk di pasarkan. y
p
Analisa pH dalam penelitian ini berdasarkan manual prosedur dari alat pH. Prinsip kerja pH
meter adalah mengukur jumlah ion H3O+ dengan sensor probe berupa elektroda kaca di dalam
larutan. Perhitungan dilakukan dengan 10 gr sampel dihomogenkan dengan 10 ml aquadest,
selanjutnya diukur menggunakan pH meter. Elektorda dimasukkan dalam larutan sampel, tekan
tombol ukur dan biarkan elektroda mengukur nilai pH selama kurang lebih 1–2 menit atau angka
yang ditampilkan dalam pH meter konstan tidak berubah-ubah Analisa data dalam penelitian ini menggunakan analisis deskriptif dengan mendeskripsikan
kemunduran mutu ikan berdasarkan waktu pengambilan sampel secara organoleptik dan pH ikan
tongkol segar. Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) ⧫513⧫ Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 Kenampakan Ikan Karakteristik mutu ikan secara organoleptik, salah satunya dengan pengamatan kenampakan
pada mata, lendir, dan daging ikan. Pengamatan organoleptik kenampakan pada ikan tongkol segar
di Pasar Induk Bumiayu, dilakukan pada pukul 06.00 WIB; 09.00 WIB; dan 12.00 WIB. Adapun nilai
organoleptik kenampakan pada ikan tongkol segar dapat dilihat pada Gambar 1. Ikan segar memiliki karakteristik mata yang cerah, cemerlang, permukaan mata menonjol dan
kornea jernih. Hasil pengamatan organoleptik mata ikan tongkol segar yang dipasarkan di Pasar
Induk Bumiayu menunjukkan terjadi penurunan seiring dengan bertambahnya waktu pengambilan
sampel. Pada sampel ikan tongkol ikan yang diambil pada pukul 06.00 memberikan nilai
organoleptik 8,33 hingga 7,00, berdasarkan SNI Ikan segar menunjukkan bahwa bola mata rata,
kornea dan pupil jernih, agak mengkilap spesifik jenis ikan. Sedangkan pengambilan sampel pada
pukul 12.00, secara organoleptik menghasilkan nilai 5,67 hingga 5,33 dimana mata ikan tongkol
sudah tidak dapat diterima oleh konsumen, yang ditunjukkan dengan bola mata cekung, kornea
keruh, pupil keabu-abuan, dan tidak mengkilap. Mata pada ikan tongkol mengalami kerusakan
dengan cepat akibat dari proses handling yang kurang baik. Menurut Lestari et al., (2018), handling ikan yang kurang tepat akibat dari benturan selama
penangkapan maupun penyimpanan. Kerusakan fisik yang dialami ikan selama proses handling
dapat berupa memar, luka disebabkan oleh perlakuan yang kasar seperti membanting wadah
penyimpanan ikan sehingga menimbulkan hentakan dan tekanan fisik pada ikan, terutama bagian
mata. Insang merupakan alat bantu pernafasan ikan yang memiliki labirin untuk menyaring kotoran
air yang masuk ke dalam tubuh ikan. Insang merupakan salah satu indikator kemunduran ikan. Uji
organoleptik insang ikan tongkol, menunjukkan penurunan nilai selama dipasarkan dari pukul 06.00
hingga pukul 12.00 WIB. Ikan tongkol pada pukul 06.00 hingga 09.00 memiliki rata-rata nilai
organoleptik 7,00 sampai 8,00 masih dapat diterima konsumen, dimana memiliki insang dengan
karakteristik merah muda atau coklat muda dengan sedikit lendir agak keruh. Sedangkan lendir
permukaan ikan pada pukul 12.00 sudah menunjukkan penurunan kesegaran ikan, dimana lendir
permukaan badan agak tebal dan mulai berubah warna. Hasil ujii organoleptik insang dan lendir dari penelitian Patang (2014), menunjukkan nilai bobot
rata-rata diatas 50% sehingga dapat disimpulkan masih dalam kondisi yang dapat diterima
konsumen selama pemasaran di pelelangan ikan Rajawali, Kota Makasar. Pada uji scoring
organoleptik terhadap insang ikan, diuji berdasarkan skor 1-9. Skor 9 dengan warna insang
cemerlang, skor 8 insang berwarna merah kurang cemerlang, skor 7 warna merah insang agak
kusam tanpa lendir, skor 6 warna insang agak kusam sedikit lendir. Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 Tingkat kesegaran ikan merupakan indikator dari nilai nutrisi ikan, dimana ikan yang
mengalami penurunan nutrisi. Penanganan ikan yang kurang diperhatikan memberikan pengaruh
terhadap fisikokimia dan nutrisi. Ikan yang dipasarkan di Pasar Induk Bumiayu umumnya terkena
matahari secara langsung, hal ini dapat menyebabkan proses oksidasi lemak akibat peningkatan
suhu ikan yang dapat menurunkan nutrisi ikan. Perubahan nutrisi terjadi akibat dari denaturasi
protein, oksidasi lemak, dan sensory meliputi warna, tekstur dan kenampakan (Abraha et al., 2018). Kenampakan Ikan Skor 5 warna insang berubah
menjadi coklat, tanpa lendir, skor 3 warna merah insang agak kusam menjadi coklat lendir tebal,
skor 1 menjadi sangat pudar dengan kecoklatan sedikit putih dengan lendir tebal, tekstur sangat
lunak dan bau busuk (Kalista et al., 2018). Riyantono et al. (2009) menyatakan bahwa perubahan
warna insang pada ikan menjadi kecoklatan menunjukkan ikan telah mengalami kemunduran mutu
atau pembusukkan. Organoleptik Ikan Tongkol Segar Jenis kerusakan
fisik ikan rata-rata yaitu isi perut keluar, kepala lepas, terdapat memar yang dapat mempercepat
proses pembusukan ikan sehingga mempengaruhi tingkat penerimaan konsumen. Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.)
Tabel 1. Nilai Rerata Organoleptik Ikan Tongkol Segar
Tanggal
Waktu
Organoleptik
Mata
Insang
Lendir
Daging
Bau
Tekstur
14 Sep 22
06.00
7,00±0,00 7,00±0,00 7,00±0,00 7,00±0,00 8,00±1,00 8,67±0,57
09.00
7,00±0,00 7,00±0,00 7,00±0,00 7,33±0,57 5,67±0,58 5,33±0,57
12.00
5,33±0,57 5,33±0,57 3,67±1,15 3,67±1,15 5,00±0,00 5,67±0,57
28 Sep 22
06.00
8,33±0,57 7,67±0,57 7,33±0,57 8,00±0,00 7,33±0,57 7,33±0,57
09.00
7,33±0,57 6,67±0,57 7,33±0,57 7,00±0,00 6,67±0,57 5,67±1,15
12.00
5,67±0,57 5,33±0,57 3,67±1,15 5,33±0,57 3,67±1,15 5,33±0,57
12 Okt 22
06.00
7,33±0,57 8,00±0,00 7,33±0,57 7,33±0,57 7,00±0,00 7,00±0,00
09.00
6,67±0,57 5,67±0,57 6,33±1,15 5,67±0,57 5,33±0,57 6,33±0.57
12.00
5.67±0.57 3.67±1.15 4.33±1.15 5.67±0.57 3.67±1.15 3.67±1.15 Tabel 1. Nilai Rerata Organoleptik Ikan Tongkol Segar ⧫514⧫ Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 Daging g
g
Ikan tongkol segar memiliki karakteristik daging yang mampu mengikat air sehingga tekstur
daging ikan masih keras atau tidak lembek. Rerata nilai organoleptik daging ikan tongkol segar dapat
dilihat pada Gambar 2. Nilai organoleptik daging ikan tongkol selama pemasaran mengalami
penurunan dari 8,00 menjadi 3,67. Ikan tongkol dapat diperoleh dengan kategori segar apabila kita
membeli pada pukul 06.00 hingga 09.00. Penurunan nilai organoleptik daging disebabkan adanya Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 poses kemunduran mutu akibat aktivitas enzim proteolitik yang mendegradasi protein pada daging
ikan. Kenampakan daging ikan segar memiliki karakteristik daging ikan padat elastis, tidak mudah
lepas dari tulang belakangnya (Patang, 2014). poses kemunduran mutu akibat aktivitas enzim proteolitik yang mendegradasi protein pada daging
ikan. Kenampakan daging ikan segar memiliki karakteristik daging ikan padat elastis, tidak mudah
lepas dari tulang belakangnya (Patang, 2014). Organoleptik mata
Organoleptik insang
Organoleptik lendir
Gambar 1. Organoleptik Kenampakan Ikan Tongkol Segar
7,00
7,00
5,33
7,67
6,67
5,33
8,00
5,67
3,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
Waktu pengambilan sampel
14-Sep-22
28-Sep-22
12-okt-22
7,00
7,00
3,67
7,33
7,33
3,67
7,33
6,33
4,33
0,00
2,00
4,00
6,00
8,00
0
5
10
15
Waktu pengambilan sampel
14-Sep-22
28-Sep-22
12-okt-22
7,00
7,00
5,33
8,33
7,33
5,67
7,33
6,67
5,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
Waktu pengambilan sampel
14-Sep-22
28-Sep-22
12-okt-22 7,00
7,00
5,33
7,67
6,67
5,33
8,00
5,67
3,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
Waktu pengambilan sampel
7,00
7,00
5,33
8,33
7,33
5,67
7,33
6,67
5,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
Waktu pengambilan sampel ganoleptik mata
Organoleptik insang
Organoleptik lendir
Gambar 1. Organoleptik Kenampakan Ikan Tongkol Segar
14 Sep 22
28 Sep 22
12 o
7,00
7,00
3,67
7,33
7,33
3,67
7,33
6,33
4,33
0,00
2,00
4,00
6,00
8,00
0
5
10
15
Waktu pengambilan sampel
14-Sep-22
28-Sep-22
12-okt-22 Gambar 1. Organoleptik Kenampakan Ikan Tongkol Segar Gambar 2. Rerata Organoleptik Daging Ikan Tongkol
8,00
7,33
3,67
8,00
7,00
5,33
7,33
5,67
5,67
0,00
2,00
4,00
6,00
8,00
10,00
0
2
4
6
8
10
12
14
14-Sep-22
28-Sep-22
12-okt-22 Gambar 2. Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) Bau Bau
Proses kemunduran mutu ikan dapat menghasilkan baru atau aroma yang kurang diminati
oleh konsumen. Bau dapat ditimbulkan dari aktivitas mikroorganisme pembusuk maupun aktivitas
kimia seperti oksidasi lemak. Nilai organoleptik bau pada ikan tongkol dapat dilihat pada Gambar 3. Proses penanganan yang kurang tepat dapat menimbulkan aroma atau bau yang kurang
diminati oleh konsumen. Hasil organoleptik bau pada ikan tongkol segar menunjukkan penurunan
akibat adanya proses perombakan secara kimiawi menghasilkan senyawa volatil Ikan tongkol yang Proses kemunduran mutu ikan dapat menghasilkan baru atau aroma yang kurang diminati
oleh konsumen. Bau dapat ditimbulkan dari aktivitas mikroorganisme pembusuk maupun aktivitas
kimia seperti oksidasi lemak. Nilai organoleptik bau pada ikan tongkol dapat dilihat pada Gambar 3. g
g
Proses penanganan yang kurang tepat dapat menimbulkan aroma atau bau yang kurang
diminati oleh konsumen. Hasil organoleptik bau pada ikan tongkol segar menunjukkan penurunan
akibat adanya proses perombakan secara kimiawi menghasilkan senyawa volatil. Ikan tongkol yang
diambil pada pukul 12.00 memiliki nilai dibawah 5,00 sehingga dapat disimpulkan bahwa ikan
tongkol tersebut telah mengalami proses pembusukan dan menghasilkan senyawa amoniak. Pembusukkan dan lama waktu penyimpanan dapat meningkatkan pertumbuhan mikrobia
pembusuk. Mikroorganisme ini memproduksi kandungan volatil. Bau tidak enak atau rasa memiliki
penerimaan negatif oleh konsumen. Degradasi protein dan derivatnya akan membentuk basa volatil
yang mudah menguap yaitu amoniak, histamin dan H2S dan menimbulkan bau busuk (Kalista et al.,
2018). ⧫516⧫ Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) Daging Rerata Organoleptik Daging Ikan Tongkol
8,00
7,33
3,67
8,00
7,00
5,33
7,33
5,67
5,67
0,00
2,00
4,00
6,00
8,00
10,00
0
2
4
6
8
10
12
14
14-Sep-22
28-Sep-22
12-okt-22 Gambar 2. Rerata Organoleptik Daging Ikan Tongkol Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) ⧫516⧫ KESIMPULAN Tingkat kesegaran ikan di pasar induk Bumiayu berdasarkan Standar Nasional Indonesia
Nomor SNI 2729:2013 tentang Spesifikasi Ikan Segar menunjukkan bahwa ikan pada pagi hari
tergolong masih segar dan dapat diterima oleh konsumen, akan tetapi pada siang hari mengalami
penurunan tingkat kesegaran ikan sehingga mempengaruhi tingkat penerimaan konsumen. Nilai pH
ikan tongkol segar mengalami kenaikan pada pengamatan sampel di siang hari. Hal ini disebabkan
terjadinya aktivitas mikroorganisme yang menyebabkan suasana pH pada daging ikan menjadi
basa. Tekstur Tekstur merupakan gambaran karakteristik dari permukaan tubuh ikan yang dapat menjadi
indikator kesegaran ikan yang dapat diamati menggunakan indera peraba. Hasil analisa
organoleptik tekstur ikan tongkol segar menunjukkan penurunan selama pemasaran di Pasar Induk
Bumiayu. Nilai rata-rata tekstur ikan tongkol dapat dilihat pada Gambar 4. Gambar 3. Nilai Rerata Organoleptik Bau Ikan Tongkol Segar
8,67
5,67
5,00
7,33
6,67
3,67
7,00
7,00
3,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
14-Sep-22
28-Sep-22
12-okt-22 Gambar 3. Nilai Rerata Organoleptik Bau Ikan Tongkol Segar Gambar 4. Nilai Rerata Organoleptik Tekstur Ikan Tongkol Segar
8,67
5,33
5,67
7,33
5,67
5,33
7,00
6,33
3,67
0,00
2,00
4,00
6,00
8,00
10,00
0
5
10
15
14-Sep-22
28-Sep-22
12-okt-22 Gambar 4. Nilai Rerata Organoleptik Tekstur Ikan Tongkol Segar Identifikasi Tingkat Kesegaran Ikan Tongkol (M.F.N. Al Fatich et al.) ⧫517⧫ Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 pH p
Analisa pH merupakan salah satu indikator tingkat kesegaran ikan. Hasil analisa pH ikan
tongkol segar di Pasar Induk Bumiayu dapat dilihat pada Tabel 2. Berdasarkan uji pH ikan tongkol
segar, menunjukkan nilai pH tertinggi diperoleh pada sampel yang diambil pada waktu pukul 09.00
dan 12.00 yaitu 6,11. Hasil penelitian dari Suprayitno (2020), nilai pH ikan segar yang diambil dari
pasar tradisional dan modern memiliki nilai rata-rata pH sebesar 6,12. Perubahan nilai pH dimana
menjadi semakin tinggi disebabkan akibat adanya aktivitas bakteri asam laktat yang menghasilkan
suasana basa. Nilai pH adalah salah satu indikator untuk menentukan tingkat kesegaran ikan. Pada
proses pembusukan ikan, perubahan pH daging ikan sangat besar peranannya karena
mempengaruhi proses autolisis dan aktivitas bakteri. Pada proses glikolisis, enzim sangat berperan
sampai terbentuknya asam laktat. Hal ini menyebabkan akumulasi asam laktat berjalan lebih lambat
sehingga penurunan pH ikan juga berlangsung lebih lambat. Selain itu, proses penguraian protein
menjadi senyawa-senyawa yang bersifat basa oleh bakteri juga terhambat sehingga peningkatan
pH ikan berlangsung lebih lambat (Munandar et al., 2009). Tabel 2. Rerata pH ikan Tongkol Segar
Tanggal
Waktu
06.00
09.00
12.00
14 Sept 2022
5,56±0,02
5,65±0,02
5,76±0,00
28 Sept 2022
5,57±0,02
6,11±0,00
5,97±0,01
12 Okt 2022
5,90±0,00
5,90±0,01
6,11±0,01
KESIMPULAN Tabel 2. Rerata pH ikan Tongkol Segar Tabel 2. Rerata pH ikan Tongkol Segar Journal of Marine Research Vol 12, No. 3 Agustus 2023, pp. 511-518 Berdasarkan hasil analisa organoleptik ikan tongkol segar, nilai tekstur yang masih sesuai
dengan SNI ikan segar adalah ikan tongkol yang diambil pada pukul 06.00 dengan karakteristik
padat, kompak, dan elastis. Tekstur merupakan salah satu indikator kualitas mutu ikan dalam
pengujian organoleptik. Hasil pengujian organoleptik tekstur yang dilakukan di TPI Mayangan,
Probolinggo pada ikan kurisi diperoleh nilai 7,36±0,30 dan 8,11±0,38 pada ikan swanggi. SNI
2729:2013 menetapkan batas minimum dari nilai organoleptik tekstur sebesar 7. Ikan yang memiliki
tekstur yang tidak kompak disebabkan oleh aktivitas bakteri yang mengontaminasi ikan tersebut. Hal tersebut disebabkan ikan merupakan media yang baik dalam pertumbuhan bakteri karena ikan
memiliki kandungan air yang tinggi (Wati dan Hafiludin, 2023). Abraha, B., Habtamu A., Abdu M., Negasi T., Xia WS., & Yang F. 2018. Effect of Processing Methods
on Nutritional and Physico-chemical Composition of Fish: A Review. MOJ Food Processing
Technology, 6(4): 78-382 Abraha, B., Habtamu A., Abdu M., Negasi T., Xia WS., & Yang F. 2018. Effect of Processing Methods
on Nutritional and Physico-chemical Composition of Fish: A Review. MOJ Food Processing
Technology, 6(4): 78-382
Badan Standarisasi Nasional. 2013. SNI Ikan Segar. SNI 2729:2013.
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Lestari N., Yuwana., & Zulman E. 2015. Identifikasi Tingkat Kesegaran dan Kerusakan Fisik Ikan di DAFTAR PUSTAKA Abraha, B., Habtamu A., Abdu M., Negasi T., Xia WS., & Yang F. 2018. Effect of Processing Methods
on Nutritional and Physico-chemical Composition of Fish: A Review. MOJ Food Processing
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( )
Badan Standarisasi Nasional. 2013. SNI Ikan Segar. SNI 2729:2013. gy,
( )
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25-38.
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https://openalex.org/W2942972298
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https://centaur.reading.ac.uk/83383/11/rsos.190026.pdf
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English
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Scenario-led modelling of broadleaf forest expansion in Wales
|
Royal Society open science
| 2,019
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cc-by
| 10,884
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To link to this article DOI: http://dx.doi.org/10.1098/rsos.190026 To link to this article DOI: http://dx.doi.org/10.1098/rsos.190026 To link to this article DOI: http://dx.doi.org/10.1098/rsos.190026 To link to this article DOI: http://dx.doi.org/10.1098/rsos.190026 Publisher: The Royal Society All outputs in CentAUR are protected by Intellectual Property Rights law,
including copyright law. Copyright and IPR is retained by the creators or other
copyright holders. Terms and conditions for use of this material are defined in
the End User Agreement
. www.reading.ac.uk/centaur Open access Manzoor, S. A., Griffiths, G., Latham, J. and Lukac, M. ORCID:
https://orcid.org/0000-0002-8535-6334 (2019) Scenario-led
modelling of broadleaf forest expansion in Wales. Royal
Society Open Science, 6 (5). 190026. ISSN 2054-5703 doi:
https://doi.org/10.1098/rsos.190026 Available at
https://centaur.reading.ac.uk/83383/ Article Published Version
Creative Commons: Attribution 4.0 (CC-BY) Creative Commons: Attribution 4.0 (CC-BY) It is advisable to refer to the publisher’s version if you intend to cite from the
work. See Guidance on citing
. It is advisable to refer to the publisher’s version if you intend to cite from the
work. See Guidance on citing
. CentAUR Central Archive at the University of Reading royalsocietypublishing.org/journal/rsos Author for correspondence:
Syed Amir Manzoor
e-mail: s.a.manzoor@pgr.reading.ac.uk Author for correspondence:
Syed Amir Manzoor
e-mail: s.a.manzoor@pgr.reading.ac.uk SAM, 0000-0002-2203-4696 Received: 11 January 2019
Accepted: 12 April 2019 Significant changes in the composition and extent of the UK
forest cover are likely to take place in the coming decades. Current policy targets an increase in forest area, for example, the
Welsh Government aims for forest expansion by 2030, and a
purposeful
shift
from
non-native
conifers
to
broadleaved
tree
species,
as
identified
by
the
UK
Forestry
Standard
Guidelines on Biodiversity. Using the example of Wales, we aim
to generate an evidence-based projection of the impact of
contrasting policy scenarios on the state of forests in the near
future, with the view of stimulating debate and aiding decisions
concerning
plausible
outcomes
of
different
policies. We
quantified changes in different land use and land cover (LULC)
classes in Wales between 2007 and 2015 and used a multi-layer
perceptron–Markov chain ensemble modelling approach to
project the state of Welsh forests in 2030 under the current and
an alternative policy scenario. The current level of expansion and
restoration of broadleaf forest in Wales is sufficient to deliver on
existing policy goals. We also show effects of a more ambitious
afforestation policy on the Welsh landscape. In a key finding, the
highest intensity of broadleaf expansion is likely to shift from
southeastern to more central areas of Wales. The study identifies
the key predictors of LULC change in Wales. High-resolution
future land cover simulation maps using these predictors offer an
evidence-based tool for forest managers and government officials
to test the effects of existing and alternative policy scenarios. Subject Category:
Earth science
Subject Areas:
ecology/environmental science
Keywords:
habitat restoration, land use change,
Markov chain, multi-layer perceptron,
broadleaf expansion Subject Category:
Earth science
Subject Areas:
ecology/environmental science Research Cite this article: Manzoor SA, Griffiths G,
Latham J, Lukac M. 2019 Scenario-led modelling
of broadleaf forest expansion in Wales. R. Soc. open sci. 6: 190026. http://dx doi org/10 1098/rsos 190026 1School of Agriculture, Policy and Development, University of Reading, Reading, UK
2Department of Forestry and Range Management, Bahauddin Zakariya University,
Bosan Road, Multan, Pakistan
3Department of Geography and Environmental Sciences, University of Reading, Reading, UK
4Natural Resources Wales, Wales, UK
5Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague,
Czech Republic 5Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague,
Czech Republic SAM, 0000-0002-2203-4696 Keywords: habitat restoration, land use change,
Markov chain, multi-layer perceptron,
broadleaf expansion 1School of Agriculture, Policy and Development, University of Reading, Reading, UK
2Department of Forestry and Range Management, Bahauddin Zakariya University,
Bosan Road, Multan, Pakistan
3Department of Geography and Environmental Sciences, University of Reading, Reading, UK
4Natural Resources Wales, Wales, UK
5Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague,
Czech Republic 1. Introduction Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.figshare. c.4496072. Global forest cover has receded rapidly in the recent past, largely
due to the conversion of forests into pasture and croplands [1]. & 2019 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited. In contrast to many other parts of the world where forest cover is still declining, Europe has reversed this
deforestation trend and forest cover here is increasing as a consequence of land abandonment and
subsequent secondary succession [2] and as a result of deliberate planting [3]. Afforestation of Great
Britain is a good example of such reversal: forest cover stood at 5% in the 1920s but increased to 13% by
2013 [4]. Early twentieth-century forestry policy in Britain focused on planting fast-growing non-native
conifer tree species to boost domestic timber supply [5], nearly tripling forest cover in the process. However, extensive plantations of single-species non-native conifers resulted in the loss of important
habitats [6,7], with a direct negative effect on species diversity [8]. In the latter part of the twentieth
century, British forestry policy gradually changed to address a wider set of objectives to complement
timber production, resulting in a broad focus on the expansion of native tree species cover [5]. 2 At present, non-native conifers constitute 36% of the total forest area in Britain. Typically represented by
Sitka spruce (Picea sitchensis Bong.), the conversion of these woodlands to native species, where appropriate,
is an aspiration stated in the UK Forestry Standard Guidelines on Biodiversity (UKFS) [9]. Non-native
plantations often consist of even-aged and often single-species conifers mostly present in the uplands. UKFS guidelines on biodiversity encourage the large-scale expansion of native woodlands, primarily by
replacing non-native species by native broadleaves or Scots pine (Pinus sylvestris L.). ‘Forest conversion’
in this context thus refers to the silvicultural process of changing forest stands dominated by non-native
conifers into forests composed of native tree species [9]. There are several reasons why this type of forest conversion should become an important tool of forestry
policy in Britain. 1. Introduction When compared to a conifer plantation, broadleaf tree species tend to increase soil pH [10],
improve growth condition for ground vegetation and soil fauna [11–13], enhance nesting opportunities and
seasonal availability of food to bird communities [14] and decrease the prevalence of insect pests [15] and
plant pathogens [16]. Further, and no less important in densely populated landscapes, forest conversion to
native broadleaves impacts on landscape aesthetics—an attribute keenly perceived and appreciated by the
public [17]. The conversion of conifer stands to broadleaf woodland is also supported by forestry policy in
Wales, specifically the rules governing the restoration of planted ancient woodland sites (PAWS) that have
historically been afforested with conifers [18]. This strategy is also supported by the objectives of the UK
Biodiversity Action Plan. There is strong political interest to enhance forest cover of the UK (e.g. [19]), with the Welsh Government
committed to increasing the overall forest area in Wales as its contribution to sustainable development. In 2010, the Welsh Assembly Government’s Climate Change Strategy [20] recommended that woodland
establishment rates be increased to 5000 ha per year for 20 years as an option for meeting Wales’ carbon
emission reduction targets. This figure was subsequently adopted by Welsh Government as a policy
target in the form of planting 100 000 ha of new, primarily broadleaved, woodland by 2030. However,
actual planting levels over the following years were insufficient to meet this. The Welsh Government
then regressively reduced the target, first to 50 000 ha, and eventually to the current commitment to
plant at least 2000 ha of woodland a year between 2020 and 2030 [19]. Despite these reductions in
aspiration, the broad commitment to increasing woodland cover has been bolstered by recent legislation
and policy development. The Environment (Wales) Act (2016) requires the Welsh Government’s
principal advisor on the environment and natural resources, Natural Resources Wales, to periodically
produce a State of Natural Resources Report that makes an assessment of the state of natural resources in
Wales and the extent to which they are sustainably managed. The first such report highlighted the need
for the increased woodland cover to deliver multiple benefits [21]. In this context, maintaining a balance
between conservation and sustainable development is seen as a challenge in policy making and
landscape management in Wales. Where possible, forest management policies should be evidence-based [22,23]. 1. Introduction One tool that can be
used to generate evidence and test policy scenarios is landscape modelling which can project the
spatial and temporal implications of policies under consideration [24]. Spatially explicit land use and
land cover (LULC) models can project land use change patterns according to given policy objectives
and generate alternative scenarios [25,26], thus establishing a link between policy and implications on
the ground [27]. The LULC change models offer an evidence-based approach to visualize, analyse and
quantify LULC changes in what-if future scenarios, thus providing useful insights to policy makers
and relevant stakeholders to set priorities and reasonable goals for sustainable forest management [28]. To date, several spatially explicit modelling environments have been developed by incorporating remote
sensing and geographical information system tools to project future LULC change scenarios. Some of the
most widely used models are based on Markov chain, logistic regression, artificial neural network and
cellular automata models [29]. Integrated use of these models is often advised to overcome limitations of
individual models and generate robust LULC change simulations [30]. In this study, we employed an integrated multi-layer perceptron–Markov chain analysis (MLP–MCA) method to quantify historic LULC
change and to model future scenarios of LULC change in Wales. The MLP–MCA is a robust and well-
documented approach for modelling spatial and temporal LULC changes [31–33]. To develop realistic
future scenarios of LULC change, it is critical to understand the spatial patterns of current LULC and to
develop an understanding of the potential impacts of current and future policies affecting LULC change
and, specifically, native woodland expansion. A key assumption underlying most LULC modelling is that
socio-economic drivers of change remain stable over time; this is, however, unlikely in reality [34]. To
meet this challenge, we considered the following key questions prior to embarking on the modelling exercise:
royalsocietypublishing
3 3 1. Under recent policies, what would the state (type and area) of forests in Wales be in 2030? p
yp
2. What are the implications of an alternative policy scenario designed to stimulate new woodland
creation while considering other objectives, such as the conservation value of non-woodland habitats? 2. What are the implications of an alternative policy scenario designed to stimulate new woodland
creation while considering other objectives, such as the conservation value of non-woodland habitats? 2.1. Study area Wales is a country with an area of nearly 21 000 km2 and a population of over 3 million, most of which live in
rural communities [35]. The population is unequally distributed, with most people living in coastal areas in
the northeast and south Wales. The country is characterized by a wide variety of landscapes, reflecting both
its rugged topography and a long history of agricultural settlement and industrialization. Significant areas
of land (approx. 6000 km2) are at an altitude above 300 m. Welsh countryside contains a range of important
habitats, including woodlands, semi-natural grasslands, heathland, fens, bogs, coastal ecosystems including
sand dunes and saltmarshes, and a diverse range of upland and montane habitats [6,36]. Only a small
proportion—6%—of the country is occupied by arable agriculture, while the major land use types are
grazing (77%) and forestry (15%) [37–39]. 2.2. Modelling methodology We made use of TerrSet Geospatial Monitoring and Modelling System (version 18.31, Clark Labs, Clark
University, USA) [40]. Specifically, we used the ‘Land Change Modeler’ (LCM) tool in TerrSet to generate
two future LULC change scenarios: business-as-usual and an alternative scenario. 1. Introduction Thus, this paper presents an analysis of the current situation and likely future trends, together with an
indication of policy requirements necessary to achieve the stated afforestation goal for Wales. We
mapped historical patterns of LULC and LULC change in Wales and developed two contrasting
future LULC scenarios based on (i) current trends and (ii) an alternative policy. We also discuss the
usefulness of the resulting future LULC maps of Wales for habitat, biodiversity and ecosystem
services analysis. 2.2.2.1. Rationale for the choice of explanatory variables Land use change modelling is complex, and a wide range of factors is likely to affect future decisions of
landowners [42]. Therefore, the kind and number of explanatory factors for future land use change can
vary considerably. Spatial and temporal scale of the study also affects the choice of explanatory variables
[42]. Given that the spatial focus of this study is regional (i.e. Wales) and temporal scale is only 15 years
(2015–2030), we applied the following rationale to the choice of explanatory variables: — Biophysical variables. Biophysical factors such as topography or soil type influence land use and
allocation decisions. For example, expansion of arable land may be limited by slope incline and/or
altitude, planting new woodlands could be driven by soil quality or land parcel accessibility. Biophysical variables, especially at a fine spatial resolution of 25 m, are very strong proxies of
climatic variables which are otherwise only available at a spatial resolution of 1 km or higher and
thus cannot be used in fine-scale land use change modelling (e.g. altitude is a strong proxy for
temperature, wind speed, etc.) [42,43]. — Proximate variables. At a regional scale, variables such as distance to markets or roads are strong
determinants of landowners’ decision-making [42,44]. For example, areas closer to roads or green
spaces often have a higher market price. Areas in close proximities to major road junctions are
more likely to experience change [45]. Furthermore, we used variables such as distance to exiting
the broadleaf forest and arable land because the expansion of a habitat is very likely to be in the
near vicinity of the already existing patches of that habitat [24,46]. y
y
g p
— Evidence likelihood of land use transition. We mapped transitions from all land cover classes to the
broadleaf forest and vice versa during 2007–2015 and used the evidence likelihood function to
convert the patterns of these transitions into usable continuous variables. Evidence likelihood is an
empirical probability of change for a qualitative map [47] and describes the relative frequency with
which different LULC categories occurred within areas that transitioned between 2007 and 2015. These variables thus represent the likelihood of finding a specific LULC at the pixel in question, if
the pixel covers an area suitable for the transition. 2.2.1. Analysis of past change The first step of this type of analysis generates the spatial pattern of changes that are discernible from the
comparison of historical LULC maps. A minimum of two maps is required (describing two different
points in time), the comparison seeking to understand the nature of LULC change and to generate
samples of transitions to be projected [40]. We used high-resolution (25 m) LULC maps of the country
from 2007 and 2015, generated from satellite imagery by the Centre for Ecology & Hydrology, UK
(https://digimap.edina.ac.uk) [41]. Both LULC maps had the same legend and spatial resolution. By
comparing the two maps, LCM evaluates LULC change and generates a visual representation of net
change, persistence, losses and gains and transitions between different LULC categories covered by the
two raster maps. In this study, we excluded LULC categories that showed a negligible transition
between 2007 and 2015 and thus are not expected to change over the time horizon under consideration
(e.g. urban areas) and those not relevant to our objectives (e.g. water bodies or coastal areas). The final
list of LULC categories selected for our modelling exercise is presented in the electronic supplementary
material, table S1. We then generated change analysis maps indicating which LULC classes changed and
the spatial pattern of changes across the Welsh landscape. This analysis of LULC change in Wales
between 2007 and 2015 resulted in a total of 28 transition types between different LULC classes
(electronic supplementary material, table S1). 2.2.2. Explanatory variables 2.2.2.1. Rationale for the choice of explanatory variables Since the decision of change on a land parcel is
strongly influenced by the decisions of the neighbouring land parcel [48–50], these variables can
have important information. — Evidence likelihood of land use transition. We mapped transitions from all land cover classes to the
broadleaf forest and vice versa during 2007–2015 and used the evidence likelihood function to
convert the patterns of these transitions into usable continuous variables. Evidence likelihood is an
empirical probability of change for a qualitative map [47] and describes the relative frequency with
which different LULC categories occurred within areas that transitioned between 2007 and 2015. These variables thus represent the likelihood of finding a specific LULC at the pixel in question, if
the pixel covers an area suitable for the transition. Since the decision of change on a land parcel is
strongly influenced by the decisions of the neighbouring land parcel [48–50], these variables can
have important information. 2.2.2.2. Explanatory variables used in the study royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026 2.2.2.1. Rationale for the choice of explanatory variables 2.2.2. Explanatory variables 4 2.2.2.2. Explanatory variables used in the study As shown in table 1, we considered 20 variables in total as having the potential to explain LULC
transitions occurring in Wales between 2007 and 2015. We collected a range of explanatory variables
such as topography, soil factors or distance from key biophysical features such as water bodies and
existing forests [33,51,52]. Six variables—distances between each of the six LULC classes—were
dynamic, while the rest were static. Values of a dynamic variable change at each time step of the
model run and thus need to be recalculated (e.g. the distance from the broadleaf forest as these forests
expand). By contrast, static variables remain constant over time (e.g. altitude, slope, soil type). Furthermore, we used the ‘Evidence Likelihood Transformation’ tool in the LCM to convert
categorical variables to continuous (Clark Labs, 2015). The following transformations were made: (1) We generated two Boolean images: first, change from all LULC types to Broadleaf Forest (All to
Broadleaf) and change from Broadleaf Forest to all other LULC types (Broadleaf to All). In these
images, 0 represents no change while 1 represents the indicated change. The Boolean images were
then used in the Evidence Likelihood Transformation process to generate two continuous
variables: ‘Evidence Likelihood of change from Broadleaf Forest to All Other Classes’ and
‘Evidence Likelihood of change from All Other Classes to Broadleaf Forest’. (2) Welsh soil type data used in this research consisted of 27 soil classes. We used the ‘Evidence Likelihood
Transformation’ option to generate the following two continuous variables from this categorical
variable: ‘Evidence Likelihood of change from Broadleaf Forest to All Other Classes based on Soil
type’ and ‘Evidence Likelihood of change from All Other Classes to Broadleaf Forest based on Soil type’. The potential of each of the 20 variables to explain observed LULC change was tested by calculating
Cramer’s V [40]. Six variables were dropped from the final list on the basis of low Cramer’s V value
(V , 0.15) [52]. The potential of each of the 20 variables to explain observed LULC change was tested by calculating
Cramer’s V [40]. Six variables were dropped from the final list on the basis of low Cramer’s V value
(V , 0.15) [52]. Table 1. List of explanatory variables considered in the study. Variables in italics were chosen in the final model based on
Cramer’s V values (V . 0.15). 2.2.3. Transition sub-models (training and validation) LCM enlists all shortlisted transitions between the two LULC maps, each represented by a transition sub-
model. Explanatory variables which have Cramer’s V . 0.15 were used to explain the observed
transitions; the accuracy rates of the transition sub-models are given in electronic supplementary material,
table S2. MLP procedure in LCM was then used to run the transition sub-models to empirically model
future LULC. MLP used a backwards stepwise variable selection in which all variables are tested
individually and in pairs for their impact on model accuracy and finally the likelihood of model overfitting
is reduced by selecting an optimum number of variables to be included in the final model. When training
and validating a transition sub-model, MLP makes use of sample pixels that have undergone a transition
between the two time periods. By default, MLP takes 10 000 randomly selected pixels for running each
transition sub-model. One half of these pixels is used to train the model, while the other half is used for
model validation. At the end of each model training run, MLP generates accuracy results for each
transition sub-model. The details of transition sub-models considered in this study and their respective
accuracy are given in the electronic supplementary material, table S2. To project future changes, we
generated a projected potential map—a map of the study area showing the potential of each pixel across
the landscape to undergo each of the LULC transitions. The potential map was subsequently used to
project LULC change to desired future date (2030). 2.2.2.2. Explanatory variables used in the study 5 explanatory variables
Cramer’s V
type of variable
1
altitude
0.345
static
2
aspect
0.077
static
3
slope
0.168
static
4
hillshade
0.135
static
5
distance from access points
0.224
static
6
distance from green spaces
0.223
static
7
distance from water channels
0.011
static
8
distance from roads
0.254
static
9
distance from hydronodes
0.065
static
10
distance from motorway junctions
0.161
static
11
distance from broadleaf forest
0.155
dynamic
12
distance from conifer forest
0.081
dynamic
13
distance from arable land
0.21
dynamic
14
distance from improved grassland
0.155
dynamic
15
distance from semi-natural grassland
0.161
dynamic
16
distance from mountain, heath and bog
0.013
dynamic
17
evidence likelihood of change from broadleaf forest to all other classes
based on earlier land use transition
0.249
static
18
evidence likelihood of change from all other classes to broadleaf forest
based on earlier land use transition
0.533
static
19
evidence likelihood of change from broadleaf forest to all other classes
based on soil type
0.251
static
20
evidence likelihood of change from all other classes to broadleaf forest
based on soil type
0.256
static
oyalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026 2.2.4.1. The business-as-usual scenario 2.2.4.1. The business-as-usual scenario This is the default scenario created in the Markov chain probability matrix. The business-as-usual (B-a-U)
scenario represents a linear projection of current trends to 2030; the trends were identified and modelled
based on changes observed between 2007 and 2015. 2.2.5. Step 5: change projection In the final step, we generated future LULC maps based on the transition probability matrices of the two
scenarios considered in this study. 2.2.4.2. Ecosystem conservation scenario y
By manipulating the transition probability matrix generated by the Change Demand Modelling panel,
we created an ecosystem conservation (EC) scenario reflecting forestry and environmental policies
currently in place: (a) Conversion of conifer to broadleaf woodland in Wales would deliver a range of environmental benefits
and is specifically required to deliver policies aimed at restoring PAWS. To accommodate these
considerations in the EC scenario, we assumed that the probability of Conifer-to-Broadleaf Forest
conversion would increase by 50% as compared to the current trend (B-a-U scenario). (b) Conservation of ecologically important non-forest LULC categories such as Mountain, Heath and
Bog is an integral part of Welsh forestry policy, for reasons of climate change in addition to
biodiversity [53]. For this reason, we assumed that in 2015–2030, the probability of the LULC
class ‘Mountain, Heath and Bog’ persisting itself will increase by 50% as compared to the current
trend (B-a-U scenario). (c) The Climate Change Strategy for Wales [20] inspired the Welsh Government to set a target of
expanding existing Welsh woodland by 100 000 ha by 2030, although subsequently reduced to a
minimum of 20 000 ha for this period [54]. Such expansion reflects wider aspirations for woodland
expansion in the UK, e.g. in the ‘UK 25 years environmental plan’ [19]. We assumed, therefore, that
the current trend of LULC classes such as Semi-Natural Grassland and Improved Grassland
converting to Broadleaf Forest would continue, but Broadleaf Forest will not be converted to any
other LULC category during 2015–2030. 2.2.4. Change demand modelling In this step, we used the ‘Change Demand Modelling’ procedure in LCM to determine the amount of
change that is likely to occur in selected LULC categories at some point in the future. By default, LCM uses a Markov chain prediction process which calculates the amount of change based on historical
observations and determines the area of land expected to undergo such transition in the future. At this
stage, LCM generates a transition potential file in the form of a matrix which shows the probability of
each LULC category to change to every other category. This potential file matrix generated by Markov
chain showing probabilities of all transitions for the year 2030 is shown in table 2. LCM allows the user
to manipulate these transition probabilities to create different modelling scenarios in the future. In this
study, the following two scenarios for LULC transitions by 2030 were considered. royalsocietypublishi
6 6 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026 3.1. LULC changes (2007–2015) We computed LULC transitions between different LULC classes that have occurred in Wales, UK between
2007 and 2015 by applying the cross-tabulation module in LCM (figure 1a). The predictor variables
considered in this study explained the LULC transitions between 2007 and 2015 well; the average
accuracy of all transition sub-models considered in this study was 79% (electronic supplementary
material, table S2). An accuracy rate of 75% or above is considered indicative of good model performance
(Clark Labs, 2015). Gains and losses between different LULC types and net change calculation (figure 1a)
show that ‘Improved Grassland’ and ‘Broadleaf Forest’ experienced the biggest expansion (148 521 and
39875 ha, respectively). Moreover, an analysis of contributions to the net change of individual LULC
types (electronic supplementary material, figure S1) suggests that the biggest contributor to ‘Broadleaf
Forest’ during 2007–2015 is the ‘Semi-Natural Grassland’ category (19 589 ha). On the other hand, around
6458 ha of ‘Broadleaf Forest’ was lost to ‘Coniferous Forest’ during this time. Of the other major LULC
changes in this period, around 80 778 ha of ‘Semi-Natural Grassland’ and 69 688 ha of ‘Arable Land’ were ov chain transition probability matrix (business-as-usual scenario, ecosystem conservation scenario) showing probability of change to 2030 in Wales, UK. probability of changing to
broadleaf forest
conifer forest
arable land
improved grassland
semi-natural grassland
mountain, heath and bog
sual scenario
est
0.570
0.142
0.019
0.191
0.062
0.013
0.188
0.754
0.003
0.029
0.020
0.004
0.052
0.009
0.098
0.752
0.079
0.007
ssland
0.040
0.008
0.057
0.784
0.103
0.006
grassland
0.073
0.049
0.034
0.411
0.381
0.051
eath and bog
0.061
0.088
0.019
0.167
0.452
0.211
nservation scenario
est
1
0
0
0
0
0
0.282
0.659
0.003
0.029
0.020
0.004
0.052
0.009
0.098
0.752
0.079
0.007
ssland
0.040
0.008
0.057
0.784
0.103
0.006
grassland
0.073
0.049
0.034
0.411
0.381
0.051
eath and bog
0.061
0.088
0.019
0.167
0.347
0.316 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
7 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
7 nario, ecosystem conservation scenario) showing probability of change to 2030 in Wales, UK. 3.1. LULC changes (2007–2015) mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
–300
300
200
100
0
–100
–200
–300
300
200
100
0
–100
–200
–300
300
200
100
0
(thousand hectares)
–100
–200
gain
loss
net change
mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
(a)
(b)
(c)
Figure 1. In Wales, UK, gains, losses and net changes between different LULC classes (hectares) during (a) 2007–2015, (b) 2015–
2030 (B-a-U scenario) and (c) 2015–2030 (EC scenario). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
8 mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
–300
300
200
100
0
–100
–200
(a) 0
–300
300
200
100
0
–100
–200
mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
(b)
y
yp
g
g royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
8 8 (b) (a) (c) –300
300
200
100
0
(thousand hectares)
–100
–200
gain
loss
net change
mountain, heath and bog
semi-natural grassland
improved grassland
arable land
conifer forest
broadleaf forest
(c) net change Figure 1. In Wales, UK, gains, losses and net changes between different LULC classes (hectares) during (a) 2007–2015, (b) 2015–
2030 (B-a-U scenario) and (c) 2015–2030 (EC scenario). broadleaf forest
conifer forest
arable land
improved grassland
semi-natural grassland
mountain, heath, bog
(a) 2015
(b) 2030 (business-as-usual scenario)
(c) 2030 (ecosystem conservation scenario)
N
E
S
W
0
37.5
75
150 km
Figure 2. Current (a) and projected land use map of Wales, UK for the year 2030 under B-a-U (b) and EC (c) modelling scenarios. (b) 2030 (business-as-usual scenario) (b) 2030 (business-as-usual scenario) (a) 2015 Figure 2. Current (a) and projected land use map of Wales, UK for the year 2030 under B-a-U (b) and EC (c) mode converted to ‘Improved Grassland’. A visual overview of historical and projected spatial distribution of
LULC transitions in Wales is presented in electronic supplementary material, figure S2. converted to ‘Improved Grassland’. A visual overview of historical and projected spatial distribution of
LULC transitions in Wales is presented in electronic supplementary material, figure S2. 3.2.2. The ecosystem conservation scenario Projected transition potential map for the second scenario considered in this study is shown in electronic
supplementary material, figure S3. An analysis of the LULC transition under this modelling scenario
reveals that ‘Broadleaf Forest’ LULC category is expected to gain 127 129 ha of the area between 2015
and 2030 (figure 1c). Most of the expansion in the area of ‘Broadleaf Forest’ under this modelling
scenario is expected to come at the cost of ‘Coniferous Forest’ (45 322 ha), ‘Improved Grassland’
(39 494 ha) and ‘Semi-Natural Grassland’ (31 318 ha). 3.2.1. The business-as-usual scenario Based on LULC change observed over the period between 2007 and 2015, we generated a map showing
projected LULC map of Wales for the year 2030 under the B-a-U scenario (figure 2) which is based on the
projected potential for transition in Wales (electronic supplementary material, table S2 and figure S3). An analysis of the B-a-U scenario shows that the ‘Broadleaf Forest’ is likely to experience a net rate of conversion
value
high
low
N
E
S
W
0
37.5
75
150 km
(a)
(b)
(c)
Figure 3. Spatial trend of conifer to broadleaf forest conversion in Wales, UK during 2007–2015 (a), 2015–2030 B-a-U scenario (b)
and 2015–2030 EC scenario (c). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
9 9 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
9 E (c) (a) /rsos
R. Soc. open sci. 6: 190026 Figure 3. Spatial trend of conifer to broadleaf forest conversion in Wales, UK during 2007–2015 (a), 2015–2030 B-a-U scenario (b)
and 2015–2030 EC scenario (c). increase of 43 366 ha of area (figure 1b), most of which is likely to come at the expense of ‘Semi-Natural
Grassland’ (21 321 ha), ‘Improved Grassland’ (8815 ha) and ‘Coniferous Forest’ (7480 ha). increase of 43 366 ha of area (figure 1b), most of which is likely to come at the expense of ‘Semi-Natural
Grassland’ (21 321 ha), ‘Improved Grassland’ (8815 ha) and ‘Coniferous Forest’ (7480 ha). 3.3. State of forests in Wales in 2030 Compared to the baseline of 159 951 ha in 2015, the ‘Broadleaf Forest’ category is likely to expand to 203 317
and 287 080 ha under the B-a-U and EC modelling scenarios, respectively. Existing conifer woodlands are
expected to experience a conversion to broadleaf of 19% and 28% under the B-a-U and EC scenarios,
respectively. The total forest area in Wales (combining broadleaf and conifer forests) is expected to
increase by 66 358 and 112 280 ha under the B-a-U and EC scenarios, as compared to the total area
under forests in 2015. Historically (2007–2015), most of the conversion took place in the southeastern
part of Wales. A slight modification of the geographical distribution of conversion is expected in
both future scenarios modelled in this study: the contour map of conversion intensity indicates a
westward and northward shift (figure 3). As an example, showing projected change of the area of the
broadleaf forest against topographic detail, we show the detail of an area of Snowdonia National Park
in Wales in figure 4. 4. Discussion Forests covered nearly 15% of the total area of Wales in 2015. Most Welsh forests were privately owned
(59%), the remainder was owned by the Welsh Government Woodland Estate [55]. The forestry sector
makes a significant contribution to the Welsh economy. Recent data (2015–2016) indicate that this
sector contributes a gross value added of £528.6 million, supports around 700 businesses and 0
37.5
75
150 km
N
E
S
W
8 km
4
2
0
Figure 4. Projected conifer to broadleaf forest conversion in Wales during 2015 and 2030 under the EC modelling scenario. Broadleaf
forest expansion in Snowdonia National Park is enlarged for detailed view (OS Crown copyright Edina Digimap). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
10 10 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026 /rsos
R. Soc. open sci. 6: 190026 8 km Figure 4. Projected conifer to broadleaf forest conversion in Wales during 2015 and 2030 under the EC modelling scenario. Broadleaf
forest expansion in Snowdonia National Park is enlarged for detailed view (OS Crown copyright Edina Digimap). provides over 10 000 jobs. Overall, the value of publicly owned forest in Wales in 2015 was estimated to
be £642 million [55]. Besides their economic value, Welsh woodlands provide a range of ecosystem
services by sequestering 1 419 000 tonnes of carbon dioxide equivalent per year and by playing a
crucial role in soil and water management by reducing nutrient runoff, diffusing pollution, reducing
flood risk and improving water quality [18]. Recent forest planting in Wales, however, has changed its
focus from the twentieth century, the planting of broadleaf tree species increasing in comparison to
conifers. Since 2001, the estimated area under conifer forests has decreased by 18 000 ha, while the
estimated area under broadleaf species has increased by 35 000 ha [56]. The forested land area in Wales is very low for a European country, the average forest cover in Europe
being 37% [18]. In order to increase the amount of land under forests, with the main motivation being
ecosystem service provision, the Climate Change Strategy for Wales prompted the Welsh Government
to set a target of increasing forest from 15% up to 19%; because of perceived the low levels of
planting this target has been progressively reduced, and currently is the equivalent 20 000 ha by 2030
[54]. 4. Discussion At the same time, the conversion of non-native conifers to native broadleaf species, primarily in
the PAWS, is an aim of the UKFS [57]. In our B-a-U scenario, we found that if the current rate of
change continues, the total forest area in Wales by 2030 is likely to increase from current 15 to 17%. However, in the EC scenario where the rates of afforestation and conversion to broadleaf are
prioritized, future forest cover could reach the original target: 19% of the total Welsh area. This is
considerably in excess of Welsh Government’s revised target. This disparity is interesting and
highlights the need for more informed projections of forest cover to help set policy targets. Historical
conifer-to-broadleaf conversion rate projected to 2030 indicates that about 19% of existing conifers will
undergo conversion, the EC scenario increases the rate by half to 28%. In standard forestry practice in
Wales, where the average rotation of non-native conifers in Wales is about 60 years, the expectation is
to harvest one-sixth of these woodlands every decade. The modelled B-a-U conversion rate of 19% is
thus very close to the natural rotation harvest and replanting. Aiming for a higher conversion rate by
2030 implies shortening the rotation in some forests. Although the major policy driver of conifer-to-broadleaf woodland conversion in Wales is PAWS, there
are other factors that may motivate this type of conversion. For instance, the UKFS encourages diversifying
forested areas in a way that a forest management unit should have at least 5% of native broadleaf trees species and should not contain more than 75% of a single species [57]. The UKFS guidelines also
advocate large-scale conversion in areas with the potential to enhance existing ancient semi-natural
woodlands and on sites which are sufficiently large to overcome edge effects [57]. In a survey of
private woodland managers in England, Scotland and Wales tasked with managing planted non-native
conifer woodlands (but excluding PAWS), the managers were interviewed about their intentions
regarding conversion to broadleaves and the reasons behind their plans. The results suggested that
woodland managers are considering a conversion of anywhere between 5 and 95% of their woodlands
from conifers to native broadleaf forest, even if not required to do so by environmental policy. royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
11 In the recent past, considerable research has been carried out to improve understanding of costs
and benefit of converting PAWS to the native broadleaf forest; however, no dedicated effort has been
made on the conversion of non-PAWS, leaving a potential research gap [9]. Since the conversion
needs to be gradual and well planned [9,58], our study presents a useful spatially explicit decision-
making aid that pinpoints sites most suitable for conifer-to-broadleaf conversion across Wales. Projected maps of conifer-to-broadleaf conversion may also prove useful for forest managers who are
hesitant of conversion by indicating site suitability on the basis of past experience. Concerns such as
suitability of site and distance to already existing broadleaf seed source have been indicated by forest
managers [9]; our projected LULC models address these concerns as we incorporate past LULC
changes, soil factors, slope, water availability, road access and distance to the broadleaf forest as
predictor variables in the models. Analysis of the conifer-to-broadleaf forest conversion between 2007 and 2015 indicated that altitude is
the most influential factor, altitude alone resulting in an accuracy of 80% of the ‘conifer forest to broadleaf
forest’ sub-model. Most of the transitions from conifer plantation to broadleaf forests in Wales took place
between altitudes of 100 and 250 m. Conversion was minimal close to sealevel and above 275 m. Most of
the land between 0 and 100 m of altitude is dedicated to agriculture and grasslands, while forests are
less likely to occur above 275 m in Wales as land use above this altitude predominantly belongs to the
Mountain, Heath and Bog category. In addition to altitude, the distance from roads made a notable
contribution in explaining the conifer-to-broadleaf conversion between 2007 and 2015. Most of the
conifers converted to broadleaves were located in areas 200–300 m from a road. Habitat expansion or
restoration activity is more likely to be within 1 km of the road network as easy and frequent access is
generally possible in this range [52]. One of the motivations for designing the EC scenario in this study was conservation of the ‘Mountain,
Heath and Bog’ habitat. Heaths and peat bogs include all inland and coastal, dry and wet heaths and
mires [59]. These are some of Britain’s most scarce habitats having a unique ecological value as these
habitat types support a range of animals, insects and plants and provide food and shelter to
migrating birds [60]. royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026
11 Moreover, heath and peat bogs—which can be thousands of years old—contain a
wealth of historical data on climate, landscape and biodiversity. A study in 1995 had indicated that
around 20% of upland heather moorland present in England and Wales in the mid-1940s was lost by
1990, high grazing pressure being one of the chief reasons for this loss [60]. A recent study modelled species richness in heath and peat bog habitat in the UK and warned of major
declines in this habitat by 2030 [59]. Our data show that 70% of Welsh heather moorland present in 1990s is
‘at risk of change’ in future. During 2007–2015, 40% of ‘Mountain, Heath and Bog’ was lost, three-quarters
of the loss due to the conversion to ‘Semi-Natural Grassland’ category. Under the B-a-U scenario, 47% of the
2015 area under this LULC class is likely to be lost by 2030. Under the EC scenario—where we increased the
probability of ‘Mountain, Heath and Bog’ to persist by 50%—we still saw a 38% loss. This indication of
serious threats to ‘Mountain, Heath and Bog’ habitat suggests that major interventions are required to
conserve this highly valuable and scarce habitat. 4. Discussion Those
willing to convert indicated biodiversity conservation, improved resilience and recreation as the chief
factors behind their intention, while those unwilling to make large-scale conversions mentioned timber
production, cost of conversion and—crucially—lack of guidance and advice [9]. y
yp
g
g j
1 11 5.2. Predicting carbon storage and sequestration The Welsh Government has a commitment to reduce annual carbon equivalent emissions by 3% per annum
[64,65]. In this regard, monitoring the rates of carbon emissions and sequestration is a challenge. Carbon
sequestration models are being used to estimate future carbon stocks and rates of sequestration [66]. The
future LULC maps of Wales can be used to estimate future amounts of carbon stored in the landscape
and sequestered over time. Current and future LULC maps along with rates of wood harvest,
degradation rates of harvested products and carbon stocks in the current LULC classes can be used to
calculate carbon storage and sequestration. Additionally, these maps can also be used to calculate the
market value of sequestered carbon in current and future land cover of Wales [47]. 5.4. Invasive species distribution modelling in Wales Invasive species are major drivers of ecosystem degradation in Wales costing the Welsh economy
approximately £7 million per annum [68]. Predicting the future distribution of invasive species is key to
effective invasive species management and planning [69]. Future distribution of invasive species is often
governed by land cover type. Evidence suggests that in Wales the spread of invasive species such as
Rhododendron ponticum is more sensitive to the land cover type than any other biophysical or climatic
factor [69]. The projected future maps of our study can be used to run species distribution models of
different invasive as well as any other species of interest. Contrasting the future scenarios may help
understand how the different policy approaches are likely to affect the course of invasive species
distribution in Wales. 5.1. Modelling the abundance of crop pollinators 12 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190026 It is estimated that 20% of agricultural crops in the UK depend on pollinators; the economic value of
pollinators to UK agriculture is estimated to be £690 million per year [61]. The National Ecosystem
Assessment showed that the abundance of wild pollinators in the UK has declined in the last 30 years
and this trend is likely to continue [62]. In Wales, the declining abundance of pollinators is an increasingly
important issue and several action plans have been proposed in the recent past to conserve pollinators
[61,63]. The future LULC maps of Wales can be used to evaluate the likely effects of contrasting LULC
scenarios on the future abundance of wild bees as pollinators for agricultural crops. The abundance of
wild bees largely depends on factors like nesting site availability and flight range, and models based on
current and future LULC maps can be used to estimate the number of wild bees visiting agricultural sites
and identifying areas that are likely to gain the most benefits from wild pollination [47]. 5.3. Future landscape change process analysis As stated in the State of Nature (2016) report for Wales, major changes in ecosystem services and abundance
of species have occurred due to degradation and fragmentation of habitat in Wales [67]. Current and
projected future LULC maps of Wales can be used to measure the nature of the change underway within
each land use class under different policy scenarios of broadleaf forest expansion in Wales. In spatial
analysis environments such as TerrSet, it is done by using a decision tree method that compares the land
cover patches in each LULC category between the two time periods and calculates the changes in the
perimeter and area of the corresponding patches [47]. The output map helps visualizing where in future
a given LULC category is likely to experience persistence, fragmentation or aggregation of patches. In the
context of Wales where there is a major drive to conserve and repair rare natural habitat such as bogs,
this could be an important analysis as it would allow one to visualize the nature of landscape change
that might occur under different future land use change scenarios. 5. Applications of future land use and land cover maps for ecosystem
services and biodiversity analysis in Wales In addition to the utility as a decision support tool for broadleaf forest expansion in Wales, the output
LULC maps of this study can be used for a range of habitat, ecosystem services and biodiversity
analysis, some of which are discussed in the following. 7. Conclusion This study reveals the changes in LULC in Wales from 2007 to 2015 by using a combined approach of GIS
and land change prediction models. An integrated MLP–MCA method was applied to improve
understanding of the scale and location of probable LULC changes under linear projection (B-a-U) and a
policy-based future scenario (EC) up to 2030. Broadleaf forest expansion is likely to reach the targets set
by the Welsh Government under the EC scenario. The study shows the potential of LULC predictions to
test alternative policy aims and to generate evidence at a scale useful to local decision makers. This type
of tool can contribute to sustainable development by providing an evidence-based spatial framework to
support restoration and conservation of ecologically important habitats in Wales. Since land use and
land cover change is a highly complex phenomenon affected by a range of ecological, political and socio-
economic factors, we contend that models incorporating the widest range of factors be used to test future
LULC scenarios. Data accessibility. The land cover maps of Wales used in this study were downloaded from Edina Digimap Environment
Products https://digimap.edina.ac.uk/environment. The explanatory variables used in the study were downloaded
from the Ordnance Survey https://digimap.edina.ac.uk/os. The software used for land use change modelling was
acquired from Clark Labs https://clarklabs.org/terrset/. Authors’ contributions. S.A.M. and G.G. carried out the spatial analysis and participated in land use change modelling. M.L. and J.L. designed and discussed the contrasting future scenarios for the land use change modelling in Wales
in the light of Welsh forest policy. S.A.M., G.G., M.L. and J.L. conceived the study, designed the study, coordinated
the study and helped draft the manuscript. All authors gave final approval for publication. Competing interests W d
l
h
ti
i t
t Competing interests. We declare we have no competing interests. Funding. This PhD study was funded by the Commonwealth Scholarship Commission (2016-86). Acknowledgements. We thank Dr Kotaro Iizuka (University of Tokyo) for his advice on land use change modelling. 6. Limitations of the study Explanatory variables used in land use change modelling studies are generally divided into three categories:
biophysical, proximate and socio-economic variables [42]. In this study, we did not include socio-economic
variables owing to the coarse resolution of available datasets. Instead, we preserved the fine spatial
resolution of biophysical and proximate variables which are likely to be stronger determinants of land
use change and reasonable proxies for the socio-economic variables. However, it is advisable to bear in mind that the resulting future projection does not directly represent the socio-economic landscape. Availability of fine-scale socio-economic and climatic variables may improve the modelling outputs in
the future. Moreover, we used LULC maps generated by the Centre for Ecology & Hydrology, UK [41]
which, to the best of our knowledge, are currently the most accurate, verified and finest-resolution LULC
temporally repeated maps covering the UK. The fact that we used only two time points is a limitation of
our study; we did not have a map of LULC after 2015 that could have been used for verification of the
future projection. Although we adopted published protocols for future LULC projections [70,71], we
suggest that the use of three or more historical LULC time points be considered for verification of
projections. As we gather more archived LULC data, this approach should become the norm. Finally, a
key limitation of this type of analysis is its ‘blindness’ to major shifts of socio-economic landscape and
hence its inability to factor these into projections. A case in point is Brexit, where a set of self-imposed
trade sanctions threatens a severe adjustment of existing drivers of land use. royalsocietypublishing.org/journal/rso
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Snowdonia. 65. Committee for Climate Change. 2017 Building a
low-carbon economy in Wales. Setting Welsh
carbon targets. See https://www.theccc.org.uk/
wp-content/uploads/2017/12/CCC-Building-a-
low-carbon-economy-in-Wales-Setting-Welsh-
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https://openalex.org/W2523342415
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https://bmcpregnancychildbirth.biomedcentral.com/track/pdf/10.1186/s12884-016-1061-2
|
English
| null |
Predicting stillbirth in a low resource setting
|
BMC pregnancy and childbirth
| 2,016
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cc-by
| 6,080
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Abstract Background: Stillbirth is a major contributor to perinatal mortality and it is particularly common in low- and
middle-income countries, where annually about three million stillbirths occur in the third trimester. This study aims
to develop a prediction model for early detection of pregnancies at high risk of stillbirth. Methods: This retrospective cohort study examined 6,573 pregnant women who delivered at Federal Medical
Centre Bida, a tertiary level of healthcare in Nigeria from January 2010 to December 2013. Descriptive statistics were
performed and missing data imputed. Multivariable logistic regression was applied to examine the associations
between selected candidate predictors and stillbirth. Discrimination and calibration were used to assess the model’s
performance. The prediction model was validated internally and over-optimism was corrected. Results: We developed a prediction model for stillbirth that comprised maternal comorbidity, place of residence,
maternal occupation, parity, bleeding in pregnancy, and fetal presentation. As a secondary analysis, we extended
the model by including fetal growth rate as a predictor, to examine how beneficial ultrasound parameters would
be for the predictive performance of the model. After internal validation, both calibration and discriminative
performance of both the basic and extended model were excellent (i.e. C-statistic basic model = 0.80 (95 % CI
0.78–0.83) and extended model = 0.82 (95 % CI 0.80–0.83)). Conclusion: We developed a simple but informative prediction model for early detection of pregnancies with a
high risk of stillbirth for early intervention in a low resource setting. Future research should focus on external
validation of the performance of this promising model. Keywords: Predicting, Stillbirth, Low-resource setting Keywords: Predicting, Stillbirth, Low-resource setting Keywords: Predicting, Stillbirth, Low-resource setting occur after 22 and before 28 weeks of gestation [13],
the stillbirth rate has been underestimated. Studies
have examined the associations between stillbirths
and clinical [14–19] and non-clinical characteristics
[20–22] of pregnant women but the knowledge ge-
nerated is yet to have any positive impact on intra-
uterine survival in LMIC [23]. This indicates limited
application of research findings to clinical settings,
notably in low-resource settings, due to the inability
of healthcare providers to combine these multiple
predictors of stillbirth accurately to identify pregnan-
cies with a high risk of stillbirth for early interven-
tions [5, 6]. Background
Stillbirth is a major but silent contributor to perinatal
mortality [1], and about 3 million third-trimester
stillbirths [2, 3] occur annually, mainly (98 %) in low-
and middle-income countries (LMICs) [4]. * Correspondence: g.a.kayode@umcutrecht.nl
1Julius Global Health, Julius Center for Health Sciences and Primary
Care|University Medical Centre Utrecht, P.O. Box 855003508, GA, Utrecht, The
Netherlands
Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274
DOI 10.1186/s12884-016-1061-2 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274
DOI 10.1186/s12884-016-1061-2 Predicting stillbirth in a low resource
setting Gbenga A. Kayode1*, Diederick E. Grobbee1,5, Mary Amoakoh-Coleman1, Ibrahim Taiwo Adeleke2, Evelyn Ansah3,
Joris A. H. de Groot1 and Kerstin Klipstein-Grobusch1,4,5 Abstract Despite
several calls for action to reduce the rate of stillbirth
[1, 4–8], stillbirths are yet to be addressed in the
Global Burden of Disease metrics [9, 10], and Sus-
tainable Development Goals [11]. Given that neither
vital registration nor national stillbirth registers are
adequately provided in LMIC [2, 12], together with
the frequent omission from records of stillbirths that Therefore, it is important to develop an easy-to-apply
clinical decision making tool for early detection of preg-
nancies with a high risk of stillbirth as recommended by Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 2 of 8 Page 2 of 8 of previous pregnancies carried beyond viability i.e. up to 28 weeks gestational age), maternal education
(woman who can read and write), maternal occupation,
ethnicity, place of residence, previous fetal loss (number
of previous pregnancy losses), bleeding in pregnancy
(whether the woman had any complaint of vaginal
bleeding during the index pregnancy), maternal height,
number
of
previous
caesarean
sections,
maternal
weight, multiple gestation, sex, fetal presentation (part
of the fetus closest the pelvic inlet, was categorized as
cephalic, breech, and others), fetal growth rate (birth
weight divided by gestational age at birth), and number
of comorbid conditions. The following medical condi-
tions, diagnosed by a physician were considered to gen-
erate a number of comorbid conditions: hypertension
(defined as blood pressure of 140/90 mmHg and above)
[28], pre-eclampsia (presence of hypertension and pro-
teinuria) [28], diabetes (Diabetes is defined as Fasting
Blood Sugar (FBS) > 7 mmol/L or 2-h Blood Sugar
(RBS) > 11.1 mmol/L; Impaired Glucose tolerance is de-
fined as Fasting Blood Sugar (FBS) 6.1–6.9 mmol/L or
2-h Blood Sugar (RBS) > 7.8–11 mmol/L) [29], sickle
cell disease (presence of HbSS, HbSC or HbS β-
thalassemia), renal disease (presence of clinical features,
ultrasound findings, and elevated serum urea and
creatinine), thyroid disease (presence of clinical mani-
festations
and
elevated
serum
free
thyroxine
and
triiodothyroxine concentration) [29], syphilis (diagnosed
using Venereal Disease Research Laboratory test) and
pelvic inflammatory disease. All candidate predictors
were selected based on availability, clinical experience
and medical literature. experts in maternal and child health [12]. To date, only
few attempts have been made to develop a decision
making tool for early detection of pregnancies with a
high risk of stillbirth but these models cannot be ap-
plied to low-resource settings. Data analysis
Descriptive statistics The outcome of the study was stillbirth, defined as
fetal death that occurred after 20 completed weeks
of gestation. Data were inspected and descriptive analyses performed
using the complete dataset. Categorical data were described
in terms of numbers and percentages while numerical
data were expressed as median and interquartile range;
the percentage of missing data in each potential pre-
dictor was determined. Sample size calculation We expected 2,000 deliveries per year and the incidence
of stillbirth was assumed to be 4 % [30, 31]. Thus, 320
cases of stillbirths were expected to have occurred
among 8,000 pregnant women who delivered at the
hospital from 2010 to 2013. We planned to recruit all
the 8,000 pregnant women who delivered at the hospital
retrospectively. Given that at least 10 events to a poten-
tial predictor will be adequate to build a prediction
model [32], we expected to have a sufficient number of
events to build a robust prediction model. Data collection
P
b
d h Paper-based health records of all the included patients
were retrieved from the Department of Health Infor-
mation, Federal Medical Center Bida. Information was
collected on clinical and non-clinical profile of the
participants by the use of data extraction form in an
anonymous format. Information on data extraction
forms was transmitted to an electronic database using
double data entry. Abstract For example a prediction
model for both stillbirth and neonatal death was devel-
oped in the United Kingdom [24] and subsequently
validated in the United Kingdom and the Netherlands
[25, 26]. This model predicts a different outcome
(stillbirth and neonatal death in very preterm babies)
and availability of routine data to validate it would be
a great challenge in low-resource settings. Likewise,
the prediction model developed by Akolekar et al. [27]
contains some parameters such as Maternal Serum
Pregnancy-Associated Plasma Protein-A and Reversed
A-Wave in Ductus Venosus, that are not routinely
assessed in low resource settings [27]. In this study we
aimed to develop a prediction model to be applied in
the second trimester of a pregnancy to identify preg-
nancies at high risk of stillbirth using routine clinical
and non-clinical profiles of pregnant women who re-
ceived care at a tertiary hospital in a low resource
setting. Study population A retrospective cohort of 6,573 pregnant women that
delivered at Federal Medical Centre Bida, a tertiary
hospital in Niger state, Nigeria, from January 2010 to
December 2013 was utilized to develop a prediction
model for stillbirth. Only those women who delivered at
the hospital after 20 completed weeks of gestation and
gave birth to babies with no life-threatening congenital
malformation were recruited. Model shrinkage All potential predictors were entered into a multivariable
logistic regression model and significant predictors were
identified using stepwise backward selection with the
Akaike Information Criterion (AIC) stopping rule. Pre-
dictors that were consistently retained in the model were
selected and entered into a multivariable logistic regres-
sion. The best model was identified based on AIC and
the results from each imputed dataset were pooled using
Rubin’s rule [35]. Eventually, a prediction model for still-
birth was developed which we called the basic model. Subsequently, the basic model was extended with the
variable fetal growth rate to become the extended model. The extended model was developed for those patients
who had information on obstetric ultrasound, a proced-
ure that is not routinely done in low-resource settings. The shrinkage factor was used to adjust for over-optimism
in each of the original models and the adjusted regression
coefficients were calculated. The predictive performance
of the final models was then re-assessed. All analyses were
performed in R statistical software package [38]. Candidate predictors For prediction modelling, the following candidate pre-
dictors were considered: maternal age, parity (number Page 3 of 8 Page 3 of 8 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 3 of 8 Page 3 of 8 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Internal validation Multiple imputation technique using fully conditional
specification was applied to impute missing data [33, 34]. A bootstrap re-sampling technique was applied to the
whole data to generate 200 testing datasets. The original
models were re-fitted in the testing datasets and their
shrinkage factors were estimated. Patient characteristics Of the 6,808 pregnant women who were recorded to have
given birth in the delivery register; 6,573 (96.5 %) of them
were recruited into this study based on the inclusion
criteria as shown in Fig. 1. A total of 6,956 newborns were
delivered, 443 of them were lifeless at birth meaning that
six in 100 newborns delivered at this center were lifeless
at birth. Table 1 shows the descriptive characteristics of
the study population and percentage of missing data in
each characteristic of the patients. The median age of
women who delivered at the center was 27 years with an
average parity of two. About two-thirds of the women had
at least primary education; half of them were unemployed. The Nupe ethnic group accounted for 72 % of the women
and they dwelled mainly in the urban areas (89 %). Almost
90 % of the babies delivered were singleton fetuses in
cephalic presentation and 51 % were male. The median
birth weight was 3.1 kg and the mean gestational age at
birth was 39 weeks. Average percentage of missing data Performance of the model The predictive performance of the final models was
assessed by evaluating calibration and discrimination. Calibration determines the level of agreement between
the observed events and model’s prediction and was
presented by the calibration plot [36]. Discrimination
examines how well the model can differentiate between
participants with or without event and was expressed as
C-statistic (which is equivalent to the area under the
receiver operator curve) [37]. Fig. 1 Follow up of study participants Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 4 of 8 per potential predictor was 11 %. About 23 % of the
women have co-existing medical conditions, 30 % of them
were nulliparous while one quarter of them have had fetal
loss (Additional file 1: Table S1). final model comprised maternal comorbidity, place
residence, maternal occupation, parity, bleeding in pre
nancy, and fetal presentation as independent predicto
of stillbirth. Performance of the model For every morbid condition co-existi
h
h
l k l h
d
f
llb
h
Table 1 General characteristics of the study population
Characteristics
All women
Live infant (6,513)
Stillbirth (443)
Missing data
Maternal age (years)
27 (24; 30)
26 (24; 30)
27 (25; 30)
0.5
Maternal height (centimeters)
156(153; 160)
156 (152; 160)
156 (153; 161)
32.1
Maternal weight (Kg)
65 (57; 75)
66.0 (57; 75)
66.0 (54.5; 75.5)
30.3
Parity
2(0; 3)
1 (0; 3)
3 (0; 4)
1.6
Number of previous fetal loss
0 (0; 1)
0 (0; 1)
0 (0; 1)
1.8
Number of previous caesarean section
0 (0; 0)
0 (0; 0)
0 (0; 0)
9.0
Maternal comorbidity
0 (0; 0)
0 (0; 0)
0 (0; 0)
1.5
Birth weight (Kg)
3.1 (2.7; 3.4)
3.1 (2.8; 3.4)
2.8 (2.0; 3.2)
4.1
Gestational age at birth (days)
265 (137; 276)
266 (137; 276)
225 (140; 254)
30.8
Maternal education (Educated)
3,284 [63.8]
3,171 [96.6]
113 [3.4]
26.0
Maternal education (Not educated)
1,866 [36.2]
1,747 [96.6]
119 [6.4]
Male infant
3,506 [51.4]
3,287 [93.6]
219 [6.3]
2.0
Female infant
3,310 [48.6]
3,113 [94.0]
197 [6.0]
Bleeding in pregnancy (Yes)
341 [5.1]
220 [64.5]
121 [35.5]
3.0
Bleeding in pregnancy (No)
6,406 [94.9]
6,107 [95.3]
299 [4.8]
Maternal occupation
16.2
Not employed
2,894 [49.6]
2,650 [91.6]
244 [8.4]
Self-employed
1,969 [33.8]
1,884 [95.7]
85 [4.3]
Private/public employee
968 [16.7]
930 [96.1]
38 [3.9]
Ethnicity
7.7
Nupe
4,611 [71.9]
4,297 [93.2]
314 [6.8]
Hausa / Fulani
246 [3.8]
220 [89.4]
26 [10.6]
Yoruba
790 [12.3]
758 [95.9]
32 [4.1]
Igbo
395 [6.2]
378 [95.7]
17 [4.3]
Gwari
19 [0.3]
17 [89.5]
2 [10.5]
Others
356 [5.6]
342 [96.1]
14 [3.9]
Place of residence (Urban)
5,707 [89.1]
5,449 [95.5]
258 [4.5]
7.9
Place of residence (Rural)
700 [10.9]
552 [78.9]
148 [21.1]
Multiple gestation
<0.01
Singleton
6,201 [89.2]
5,813 [93.7]
388 [6.3]
Twins
719 [10.3]
665 [92.5]
54 [7.5]
Triplets
35 [0.5]
34 [97.1]
1[2.86]
Fetal presentation
<0.01
Cephalic
6,506 [93.7]
6,159 [94.7]
347 [5.3]
Breech
334[4.8]
280 [83.8]
54 [16.2]
Others
100[1.4]
62 [62.0]
38 [38.0]
Median (interquartile range); number [percentage] Median (interquartile range); number [percentage] final model comprised maternal comorbidity, place of
residence, maternal occupation, parity, bleeding in preg-
nancy, and fetal presentation as independent predictors
of stillbirth. For every morbid condition co-existing
with pregnancy the likelihood of stillbirth increased. Performance of the model Being an unemployed, rural-dwelling woman with a
positive history of bleeding in pregnancy increased
risk of stillbirth. As parity increased risk of stillbirth per potential predictor was 11 %. About 23 % of the
women have co-existing medical conditions, 30 % of them
were nulliparous while one quarter of them have had fetal
loss (Additional file 1: Table S1). Multivariable prediction model The results of the multivariable prediction model for
stillbirth (i.e. the basic model) are shown in Table 2. The Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 5 of 8 Table 2 Multivariable prediction model for stillbirth (Basic model)
Predictors
Unadjusted β coef. Standard error
P-value
Adjusted β coef. Maternal comorbidity
0.71
0.097
<0.001
0.71
Place of residence (rural)
1.31
0.129
<0.001
1.30
Maternal occupation
Self employed
−0.30
0.144
0.035
−0.30
Employee
−0.38
0.182
0.037
−0.38
Maternal parity
0.08
0.024
0.001
0.08
Bleeding (yes)
2.18
0.139
<0.001
2.16
Fetal presentation
Breech
0.96
0.182
<0.001
0.96
Others
2.12
0.240
<0.001
2.06
Unadjusted β coef. denotes β coefficient before penalization; Adjusted β coef. denotes β coefficient after penalization
C-statistic before and after penalization 0.80 (95 % CI 0.78–0.83)
Risk of stillbirth ¼ 1 = 1 þ exp ‐ ‐3:6486 þ 0:7077 comorbidity
ð
Þ þ
ð
ð
1:3047 rural
ð
Þ –0:3022 self‐employed
ð
Þ‐0:3788 employee
ð
Þ þ 0:0797 parity
ð
Þ
þ 2:1579 bleeding in pregnancy
ð
Þ þ 0:9616 breech presentation
ð
Þ þ 2:0588 other presentations
ð
ÞÞ
For example the risk of a para-7, unemployed, hypertensive, diabetic pregnant woman in compound presentation with a positive history of vaginal bleeding in
pregnancy, dwelling in a rural area is
Risk of stillbirth ¼ 1 = 1 þ exp ‐ ‐3:6486 þ 0:7077 2
ð Þ þ 1:3047 1
ð Þ – 0:3022 1
ð Þ – 0:3788 0
ð Þ þ 0:0797 7
ð Þ þ 2:1579 1
ð Þ
ð
þ 0:9616 0
ð Þ þ 2:0588 1
ð ÞÞ
ð
¼ 1=1 þ
exp ‐3:5439
ð
Þ
Risk of stillbirth ¼ 0:97 Risk of stillbirth ¼ 0:97 Risk of stillbirth ¼ 0:97 adjusted β coef. denotes β coefficient before penalization; Adjusted β coef. denotes β coefficient after penalization
atistic before and after penalization 0.82 (95 % CI 0.80–0.85) p
(
)
stillbirth ¼ 1 = 1 þ expð‐ð‐1:7035 þ 0:5965 comorbidity
ð
Þ
þ 1:2603 rural
ð
Þ – 0:2647 self‐employed
ð
Þ –0:3265 employee
ð
Þ þ C statistic before and after penalization 0.82 (95 % CI 0.80 0.85)
Risk of stillbirth ¼ 1 = 1 þ expð‐ð‐1:7035 þ 0:5965 comorbidity
ð
Þ
þ 1:2603 rural
ð
Þ – 0:2647 self‐employed
ð
Þ –0:3265 employee
ð
Þ þ 0:0959 parity
ð
Þ 0.82 (95 % CI 0.80 0.85)
035 þ 0:5965 comorbidity
ð
Þ
þ 1:2603 rural
ð
Þ – 0:2647 self‐employed
ð
Þ –0:3265 employee
ð
Þ þ 0:0959 parity
ð
Þ þ 2:0149 bleeding in pregnancy
ð
Þ þ 0:8342 breech presentation
ð
Þ þ 2:0677 other presentations
ð
Þ – 0:1810 fetal growth rate
ð
Þ For example the risk of a para-five, unemployed, hypertensive pregnant woman in breech presentation with a positive history of vaginal bleeding in pregnancy,
dwelling in a rural area and the estimated fetal weight by obstetric scan at 22 weeks was 650 g g
g
y
g
tillbirth ¼ 1 = 1 þ exp ‐ ‐1:7035 þ 0:5965 1
ð Þ þ 1:2603 1
ð Þ – 0:2647 1
ð Þ – 0:3265 0
ð Þ þ 0:0959 5
ð Þ þ 2:0149 1
ð Þþ
ð
0:8342 1
ð Þ þ 2:0677 0
ð Þ – 0
ð Performance of the model increased. Pregnancies in cephalic presentation lowered
the risk of stillbirth. Subsequently, the basic model was
extended by the variable fetal growth rate and the results
of the multivariable prediction model (i.e. the extended
model) are shown in Table 3. All predictors in the
extended model showed similar associations as observed
in the basic model. For fetal growth rate, the likelihood of
stillbirth decreased as growth rate increased. increased. Pregnancies in cephalic presentation lowered
the risk of stillbirth. Subsequently, the basic model was
extended by the variable fetal growth rate and the results
of the multivariable prediction model (i.e. the extended
model) are shown in Table 3. All predictors in the
extended model showed similar associations as observed
in the basic model. For fetal growth rate, the likelihood of
stillbirth decreased as growth rate increased. The discriminative performance of the final basic model
was very good with a C-statistic of 0.80 (95 % CI 0.78–
0.83). The extended model (i.e. with obstetric ultra-
sound variable growth rate added) showed a slightly
improved discriminative performance of 0.82 (95 % CI
0.80–0.85). Calibration for both models was good
(Figs. 2 and 3). Table 3 Extended multivariable prediction model for stillbirth (Extended model)
Predictors
Unadjusted β coef. Standard error
P-value
Adjusted β coef. Maternal comorbidity
0.60
0.100
<0.001
0.60
Place of residence (rural)
1.27
0.129
<0.001
1.26
Maternal occupation
Self employed
−0.27
0.143
0.07
−0.26
Employee
−0.33
0.183
0.07
−0.33
Maternal parity
0.10
0.024
<0.001
0.10
Bleeding (yes)
2.04
0.142
<0.001
2.01
Fetal presentation
Breech
0.83
0.181
<0.001
0.83
Others
2.15
0.241
<0.001
2.07
Growth rate
−0.18
0.026
<0.001
−0.18 For example the risk of a para-five, unemployed, hypertensive pregnant woman in breech presentation with a positive history of vaginal bleeding in pregnancy,
dwelling in a rural area and the estimated fetal weight by obstetric scan at 22 weeks was 650 g
k f
llb
h
=
ð Þ
ð Þ
ð Þ
ð Þ
ð Þ
ð Þ
ð
ð Þ
ð Þ
=
ð
ÞÞ
ð ¼ 1=1 þ exp ‐2:4532
ð
Þ Risk of stillbirth ¼ 0:92 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 6 of 8 Fig. 2 Calibration plot of the basic model Internal validation gestational age) to see whether this not routinely measured
variable (obstetric ultrasound) would improve predictions. This study was reported based on the TRIPOD (Transpar-
ent reporting of a multivariable prediction model for
individual prognosis or diagnosis) guidelines [39] and to the
best of our knowledge, these are the first prediction models
for stillbirth that can easily be applied in the second trimes-
ter of pregnancy in low-resource settings where 98 % of
third-trimester stillbirths occur [4]. This study reaffirms
stillbirth as an important public health issue; 6 in every 100 Both models were penalized but the discriminative
performance of both models remained unchanged while
their calibration improved (Figs. 2 and 3). Conclusion It allows for early
detection of pregnancies at high risk of stillbirth for timely
allocation of targeted interventions and to benefit from
closer monitoring throughout the pregnancy. Prioritization
of care allocation is particularly relevant in low resource
settings. Interventions to improve neonatal, intrauterine
and maternal survival have been identified and inte-
grated as a continuum of care because they are related
[5, 6]; thus, it is expected that this prediction model
may not only improve prevention of stillbirth but may
also have a positive collateral effect on maternal and We developed a simple but informative prediction model
for early detection of pregnancies at high risk of stillbirth
for timely intervention in low resource settings. It is
important for future studies to conduct an external valid-
ation of this prediction model at all levels of care using
prospectively collected data and include information on
maternal HIV status. Funding
h
d This study was funded by the Netherlands Organization for Scientific Research /
WOTRO Science for Development (Grant Number: 07.45.102.00). The funder did
not play any role in the design of the study and collection, analysis, and
interpretation of data and in writing the manuscript. Acknowledgements The authors appreciate Federal Medical Centre, Bida, Nigeria for permitting us to
conduct this study. We express our gratitude to all the staff of the Department
of Health Information for their support. We also gratefully acknowledge the
financial support from the Netherlands Organization for Scientific Research /
WOTRO Science for Development (Grant Number: 07.45.102.00) towards the
completion of this study. Gbenga A Kayode is a recipient of a University Medical
Center Utrecht Global Health Support Fellowship. Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 7 of 8 Page 7 of 8 Page 7 of 8 Ethical approval and consent to participate Ethical approval to conduct this research was obtained from the Ethical
Review Committee of Federal Medical Centre Bida, Nigeria. Informed consent
was obtained from the Head of Department of Obstetrics and Gynaecology
before the study commenced. Ethical approval to conduct this research was obtained from the Ethical
Review Committee of Federal Medical Centre Bida, Nigeria. Informed consent
was obtained from the Head of Department of Obstetrics and Gynaecology
before the study commenced. Availability of data and materials Based on the ethical approval obtained, the authors were not allowed to
release the data to public domain. However, individuals can request for the
data and it will be made available. Abbreviations Abbreviations
AIC: Akaike information criterion; FBS: Fasting blood sugar; LMICs: Low- and
middle-income countries; RBS: Random blood sugar; TRIPOD: Transparent
reporting of a multivariable prediction model for individual prognosis or
diagnosis AIC: Akaike information criterion; FBS: Fasting blood sugar; LMICs: Low- and
middle-income countries; RBS: Random blood sugar; TRIPOD: Transparent
reporting of a multivariable prediction model for individual prognosis or
diagnosis Conclusion newborns delivered at the center were lifeless at birth, justi-
fying the clinical relevance of an easy to use prediction
model to detect high risk pregnancies at an early stage (i.e. the 2nd trimester). The basic prediction model comprised
six easy-to-measure, readily available, inexpensive parame-
ters, promoting its easy use during antenatal visits in low-
resource settings. A previous model [27] included more
predictors, but also used Pregnancy-Associated Plasma
Protein-A and Reversed Flow of A-wave in Ductus Venosus
that are not routinely measured in low-resource settings. Age restriction was not included in the eligibility criteria so
as to broaden its application among pregnant women. A
large cohort was used to develop the model to increase the
power of the study and lower the possibility of overfitting. The predictive performance of the model in terms of
discrimination and calibration was very good also after
internal validation. As a secondary analysis we generated
fetal growth rate using birth weight and gestational age at
birth. This proxy predictor was included in the extended
model (Table 3) instead of using ultrasound estimated fetal
weight and gestational age, because up to 60 % of the
women did not undergo obstetric ultrasound investigation
during antenatal care due to various reasons. To acknow-
ledge the importance of monitoring intrauterine growth
restriction in stillbirth, fetal growth rate was included in the
extended multivariable model. We preferred to generate
fetal growth rate from birth weight and gestational weight
at birth instead of using obstetric ultrasound information
because based on our knowledge of these data some of the
reasons why obstetric ultrasound was not done might be
related to the outcome e.g. antenatal visit. Missing data was
observed in some of our predictors and multiple imput-
ation was applied to address it instead of performing a
complete case analysis which may give biased results. Studies have shown repeatedly that multiple imputation
reduces the possibility of bias in the estimates compared
to complete case analysis [40–42]. It is important to
emphasize that this prediction model has not undergone
external validation, and this is planned to be done in a
future study; but its predictive performance remained
unchanged after internal validation. Experts have expressed
the need to develop a prediction model for stillbirth
because of its clinical importance [12]. Authors’ contributions GAK, DEG, and KKG designed the study. GAK carried out data collection and
literature review. JAHG and GAK performed data analysis; GAK drafted the
first version of the manuscript. All authors (GAK, KKG, DEG, JAHG, ITA, EA,
and MAC) reviewed and approved the final version of the manuscript. Additional file Additional file 1: Table S1. Describes characteristics of the women. (PDF 117 kb) Competing interests p
g
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. Discussion In this study we developed an easy to use clinical prediction
model to identify pregnancies at high risk of stillbirth for
timely interventions. We also extended this basic model
with the variable fetal growth rate (fetal weight divided by Fig. 3 Calibration plot of the basic model Fig. 3 Calibration plot of the basic model References Stillbirths: how can health
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1 1Julius Global Health, Julius Center for Health Sciences and Primary
Care|University Medical Centre Utrecht, P.O. Box 855003508, GA, Utrecht, The
Netherlands. 2Department of Health Information, Federal Medical Centre
Bida, Bida, Nigeria. 3Ghana Health Service, Greater Accra Region, Accra,
Ghana. 4Division of Epidemiology and Biostatistics, School of Public Health,
Faculty of Health Science, University of Witwatersrand, Johannesburg, South
Africa. 5Global Geo and Health Data Center, Utrecht University, Utrecht,
Netherlands. Page 8 of 8 Page 8 of 8 Page 8 of 8 Kayode et al. BMC Pregnancy and Childbirth (2016) 16:274 Page 8 of 8 Submit your next manuscript to BioMed Central
and we will help you at every step: 18. Andrade LG, Amorim MM, Cunha AS, Leite SR, Vital SA. Factors associated
with stillbirth in a school maternity in Pernambuco: a case control study. Rev Bras Ginecol Obstet. 2009;31(6):285–92. • We accept pre-submission inquiries
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and we will help you at every step: • We accept pre-submission inquiries 19. Sharma PP, Salihu HM, Kirby RS. Stillbirth recurrence in a population of
relatively low-risk mothers. Paediatr Perinat Epidemiol. 2007;21 Suppl 1:24–30. 20. Little RE, Weinberg CR. Risk factors for antepartum and intrapartum stillbirth. Am J Epidemiol. 1993;137(11):1177–89. 21. Romero Gutierrez G, Martinez Ceja CA, Ponce Ponce de Leon AL, Abrego
Olvira E. Risk factors for stillbirth. Ginecol Obstet Mex. 2004;72:109–15. 22. Gardosi J, Madurasinghe V, Williams M, Malik A, Francis A. Maternal and fetal
risk factors for stillbirth: population based study. BMJ. 2013;346:f108. 22. Gardosi J, Madurasinghe V, Williams M, Malik A, Francis A. Maternal and fetal
risk factors for stillbirth: population based study. BMJ. 2013;346:f108. 23. Cnattingius S, Stephansson O. The challenges of reducing risk factors for
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stillbirths. Lancet. 2011;377(9774):1294–5.
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English
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The association between residual neuromuscular blockade (RNMB) and critical respiratory events: a prospective cohort study
|
Perioperative medicine
| 2,021
|
cc-by
| 6,610
|
THE ASSOCIATION BETWEEN RESIDUAL
NEUROMUSCULAR BLOCKADE (RNMB) AND
CRITICAL RESPIRATORY EVENTS: A PROSPECTIVE
COHORT STUDY
Faraj Alenezi
(
farajalenezi@gmail.com
)
King Saud bin Abdulaziz University for Health Sciences
https://orcid.org/0000-0001-8596-1983
Khalid Alnababtah
Birmingham City University
Mohammed Alqahtani
King Saud bin Abdulaziz University for Health Sciences
Mohammed Al Harbi
King Saud bin Abdulaziz University for Health Sciences
Lafi Olayan
King Saud bin Abdulaziz University for Health Sciences
Research
Keywords: Neuromuscular blocking drugs, Residual neuromuscular blockade, Critical respiratory events. Posted Date: July 9th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-24919/v2
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at Perioperative Medicine on May 4th, 2021. See the published version at https://doi.org/10.1186/s13741-021-00183-7. THE ASSOCIATION BETWEEN RESIDUAL
NEUROMUSCULAR BLOCKADE (RNMB) AND
CRITICAL RESPIRATORY EVENTS: A PROSPECTIVE
COHORT STUDY Faraj Alenezi
(
farajalenezi@gmail.com
) Version of Record: A version of this preprint was published at Perioperative Medicine on May 4th, 2021.
See the published version at https://doi.org/10.1186/s13741-021-00183-7. Research Version of Record: A version of this preprint was published at Perioperative Medicine on May 4th, 2021. See the published version at https://doi.org/10.1186/s13741-021-00183-7. Page 1/16 Abstract Background: Inadequate neuromuscular recovery might impair pulmonary function among adult patients
who undergo general anaesthesia and might thus contribute to critical respiratory events in the post-
anaesthesia care unit (PACU). The aim of this study to investigate whether critical respiratory events
(CREs) that described in a modified Murphy’s criteria are significantly associated with residual
neuromuscular blockade (RNMB). Method: A prospective cohort study from January to March 2017 from a tertiary hospital from Saudi
Arabia for thirty adult patients over 18 years old scheduled for elective surgery under general anaesthesia
with neuromuscular blocking drugs (NMBDs) were enrolled in the study. The Mann-Whitney U tests, Chi-
Square tests and Independent-Samples T tests were used. The Train-of-four (TOF) ratios were measured
upon arrival in the PACU by using Acceleromyography with TOF-Scan. Subjects’ demographics,
perioperative data, and the occurrence of postoperative CREs in the PACU were recorded. Results: 26 (86.7%) patients out of thirty in the study has received Rocuronium as NMBDs while
Neostigmine as a reversal drug with only 23 (76.7%). The incidence of RNMB (TOF ratio < 0.9) was in 16
patients (53.3%). The incidence of RNMB was significantly higher in female patients (p= 0.033), in
patients who had not undergone quantitative neuromuscular monitoring before extubation (p= 0.046)
and in patients with shorter duration of surgery (p= 0.001). Postoperative CREs occurred in twenty
patients (66.7%), and there were significantly more of these CREs among patients with RNMB (P= 0.001). In addition, a statistically significant difference was observed in the occurrence of CREs according to
body mass index (P= 0.047). Conclusion: This research showed that RNMB is a significant contributing factor to the development of
critical respiratory events during PACU stay. Therefore, routine quantitative neuromuscular monitoring is
recommended to reduce the incidence of RNMB. Introduction Many patients are at high risk of adverse events during the early postoperative period, which might be
caused by anaesthetic or surgical procedures. These adverse events include cardiovascular or respiratory
complications (Buhre and Rossaint 2003). Therefore, areas known as post-anaesthesia care units (PACU)
have been established in many hospitals worldwide, which are intended to provide specialised care and
minimise morbidity and mortality through the timely detection or prevention of adverse events (Kiekkas et
al. 2014). In clinical practice, neuromuscular blocking drugs (NMBDs) are commonly used by
anaesthesiologists during general anaesthesia in order to maintain optimal surgical conditions by
maintaining the deep muscle relaxation and to facilitate tracheal intubation (Claudius et al. 2009). However, their effects might continue after extubation: this is called residual neuromuscular blockade
(RNMB). According to Murphy et al. (2008a), between 33% and 64% of patients have evidence of
incomplete neuromuscular recovery or RNMB during their stay in the PACU, despite the use of techniques Page 2/16 Page 2/16 proven to limit the degree of RNMB, such as reversal drugs. The incidence of RNMB ranges between 26%
and 88%, based on the type of NMBDs, reversal drugs, or neuromuscular monitoring used (Cammu et al. 2012; Fortier et al. 2015). The RNMB can be reliably detected through the use of neuromuscular
monitoring such as Train-of-Four (TOF) monitoring, which is according to Murphy et al. (2011) is
considered one of the most commonly used approaches in clinical practice. This method however, is
used during recovery from the application of NMBDs to objectively decide how well a patient's muscles
are able to function, which includes the application of electrical stimulation to nerves and the recording of
muscle responses, called the TOF ratio (Butterworth 2013). The TOF ratio when it is ≥ 0.90 is considered
the gold standard for defining adequate neuromuscular recovery, whereas RNMB is present when the
TOF ratio < 0.90 (Murphy 2006). Accordingly, The RNMB might place patients at an increased risk of
critical respiratory events (CREs) in the PACU. Evidence from observational studies suggests that TOF
ratios when it is less than 0.90 are usually associated with the incidence of CREs that include inadequate
recovery of pulmonary function, upper airway obstruction, impaired pharyngeal reflexes, decreased
muscle coordination, impaired hypoxic ventilatory response and increased risk of aspiration (Cammu et
al. 2012; Herbstreit et al. 2009; Murphy et al. 2008a). Introduction To date, the incidence of RNMB and its association with CREs during the PACU stay has not been
investigated in Saudi Arabia. In addition, the issue of whether or not neuromuscular monitoring is usually
used has not been investigated. This study aims to examine whether RNMB is significantly associated
with postoperative CREs by determining the incidence of RNMB and postoperative CREs and the
associated risk factors during the PACU stay in a tertiary hospital in Saudi Arabia. Study Design A pilot prospective cohort design was used in this study which is part of a student project at Cardiff
University. The study was conducted in the post anaesthesia care unit (PACU) at the National Guard
Hospital, King Abdulaziz Medical City in Riyadh, Saudi Arabia. The study has been approved by the King
Abdullah International Medical Research Centre (KAIMRC) at King Abdulaziz Medical City. Informed
written consent was obtained from all patients before entry into the study. Participants were enrolled
between 21st January 2017 and 21st March 2017. The following baseline demographic variables were collected: age, gender, height, weight, body mass
index (BMI), Operative variables collected included type and duration of the operation. The
anaesthesiologists were blinded to the patients’ participation in the study and the NMBDs were
supervised according to the standards of patient care in PACU and whether these patients required
oxygen following their extuabation in operating theatre. On arrival at the PACU, type and dose of neuromuscular blockade drugs (NMBDs), reversal drugs and
duration of surgery are recorded by qualified nurses in PACU. They also measured the following Page 3/16 postoperative outcomes: peripheral oxygen saturation (SpO2), respiratory rate and TOF ratio during the
first thirty minutes of arrival into the PACU in which all of them were obtained by the researcher. Research
assistant was obtained TOF ratio via acceleromyography (quantitative neuromuscular monitoring) at the
adductor pollicis of the thumb (TOF-Scan™, IdMed, Marseille, France). When the patients experienced any
of CREs based on the modified Murphy’s criteria within the first thirty minutes in admission to the PACU,
the nurses have directly informed the researcher about these changes in the patients’ condition as shown
in (Table 2). Table 2: Types of Critical Respiratory Events (Modified Murphy’s Criteria) Table 2: Types of Critical Respiratory Events (Modified Murphy’s Criteria) 1. Upper airway obstruction: if the patient required an intervention such as: oral airway jaw thrust 2. Mild-moderate hypoxemia (SpO2 of 93%–90%); 7. Patient requiring re-intubation in the PACU; and 8. Clinical evidence or suspicion of pulmonary aspiration after tracheal extubation (gastric contents observed
in the oropharynx and hypoxemia). 8. Clinical evidence or suspicion of pulmonary aspiration after tracheal extubation (gastric contents observed
in the oropharynx and hypoxemia). Results A total of 120 patients were screened for their eligibility based on the inclusion and exclusion criteria at a
pre-anaesthesia clinic over a period of 4 weeks (Table 1). 19 patients with Chronic obstructive pulmonary
disease (COPD), 22 with abdominal emergency surgery, and 49 undergone elective surgery that lasted
more than four hours (240 minutes) were excluded. A total of 30 patients, consisting of 17 males and 13
females, were recruited for analysis (Fig. 1). Table 1: inclusion and exclusion criteria Table 1: inclusion and exclusion criteria Table 1: inclusion and exclusion criteria
Inclusion
1. Patients aged over 18 years. 2. Patients who had undergone elective surgical procedures. 3. Patients who received general anaesthesia only
4. American Society of Anaesthesiologists (ASA) from 1-3
Exclusion
Patients who were undergoing cardiac, thoracic or neurological surgeries, as well as
patients who were undergoing abdominal emergency surgery
1. Patients who are having history of pulmonary disease or cardiovascular comorbidities
2. Patients undergoing elective surgery that lasted more than four hours (240 minutes)
3. American Society of Anaesthesiologists (ASA) from 4 and 5 Table 1: inclusion and exclusion criteria Table 1: inclusion and exclusion criteria
Inclusion
1. Patients aged over 18 years. 2. Patients who had undergone elective surgical procedures. 3. Patients who received general anaesthesia only
4. American Society of Anaesthesiologists (ASA) from 1-3
Exclusion
Patients who were undergoing cardiac, thoracic or neurological surgeries, as well as
patients who were undergoing abdominal emergency surgery
1. Patients who are having history of pulmonary disease or cardiovascular comorbidities
2. Patients undergoing elective surgery that lasted more than four hours (240 minutes)
3. American Society of Anaesthesiologists (ASA) from 4 and 5 2. Patients who had undergone elective surgical procedures. 3. Patients who received general anaesthesia only 4. American Society of Anaesthesiologists (ASA) from 1-3 1. Patients who are having history of pulmonary disease or cardiovascular comorbidities
2. Patients undergoing elective surgery that lasted more than four hours (240 minutes)
3. American Society of Anaesthesiologists (ASA) from 4 and 5 3. American Society of Anaesthesiologists (ASA) from 4 and 5 Demographic data of the population, surgical and anaesthesia details are summarized in Table 3. The
median age of participants was 35 years. The majority of these participants were male (56.7%), while
female patients represented 43.3% of the sample. Based on Fortier et al. (2015) and Murphy et al. Statistical Analysis All Statistical analysis for the current study was performed by using the Statistical Package for the Social
Sciences (version 23.0). Categorical data were presented as percentages or frequencies and Chi-Square
test was used for comparison. For non-normally distributed data, Mann–Whitney U test, median and Page 4/16 Page 4/16 interquartile range (IQR) were used, while the mean, standard deviation (SD) and Independent-Samples T
tests were used if normally distributed. A p value of less than 0.05 was considered for the significance
level. interquartile range (IQR) were used, while the mean, standard deviation (SD) and Independent-Samples T
tests were used if normally distributed. A p value of less than 0.05 was considered for the significance
level. Results Variables
Findings (n=30)
Age (years), median (IQR)
Gender, n (%)
Male
Female
ASA-PS Class, n (%)
I
II
III
BMI (kg/m2), mean ± SD
Type of surgery, n (%)
General
Urology
ENT
Plastic
OB/GYN
NMBD used, n (%)
Succinylcholine
Rocuronium
Cisatracurium
Reversal used, n (%)
None
Neostigmine
35 (27-53)
17(56.7%)
13(43.3%)
10 (33.3%)
17 (56.7%)
3 (10%)
29.45 ± 5.56
13 (43.3%)
4 (13.3%)
10 (33.3%)
2 (6.7%)
1 (3.3%)
1 (3.3%)
26 (86.7%)
3 (10%)
7 (23.3%)
23 (76.7%)
tage); IQR= Interquartile Range; ± SD= ± standard deviation; ASA-PS Class=
siologists Physical Status class; BMI= Body Mass Index; kg/m2= Kilogram per Variables
Findings (n=30)
Age (years), median (IQR)
Gender, n (%)
Male
Female
ASA-PS Class, n (%)
I
II
III
BMI (kg/m2), mean ± SD
Type of surgery, n (%)
General
Urology
ENT
Plastic
OB/GYN
NMBD used, n (%)
Succinylcholine
Rocuronium
Cisatracurium
Reversal used, n (%)
None
Neostigmine
35 (27-53)
17(56.7%)
13(43.3%)
10 (33.3%)
17 (56.7%)
3 (10%)
29.45 ± 5.56
13 (43.3%)
4 (13.3%)
10 (33.3%)
2 (6.7%)
1 (3.3%)
1 (3.3%)
26 (86.7%)
3 (10%)
7 (23.3%)
23 (76.7%) Key: n (%) = number (percentage); IQR= Interquartile Range; ± SD= ± standard deviation; ASA-PS Class=
American Society of Anaesthesiologists Physical Status class; BMI= Body Mass Index; kg/m2= Kilogram per
meter square ENT= Ear, Nose and Throat; OB/GYN= Obstetrics and Gynaecology; NMBD = neuromuscular
blocking drug. The incidence of RNMB (TOF ratio < 0.90) was 53.3% (16/30). In comparing the preoperative variables
among patients who had RNMB on PACU admission, the Chi-Square test clearly showed that there were
no significant differences between the incidence of RNMB according to the age of patients (18 – 50 vs. ≥
51 years) or ASA-PS Class, with P=0.0544 and P=0.103, respectively. The same test demonstrated that
the incidence of the RNMB was significantly higher in female gender (76.9% vs. 35.3%, P=0.033) (Fig. 2),
in patients who had not had neuromuscular monitoring before extubation (66.7% vs. 22.2%, P=0.046),
and in shorter duration of surgery (81.8% vs. 21.4%, P=0.001). Independent-Samples T tests
demonstrated no significant differences in RNMB in relation to BMI (P=0.294). In comparing patients who
had RNMB upon arrival in the PACU with those who did not, the Mann-Whitney U test demonstrated a
significant relationship between the incidence of RNMB and both oxygen desaturation and respiratory
rate, at P=0.034 and P=0.025, respectively. Results (2015),
patients’ age was categorized into two age groups: younger patients (age 18 to 50 years) and elderly
patients (age ≥ 51 years). More than half of the studied patients had ASA PS class II (56.7%), followed by
ASA PS class I (33.3%). The majority of the cohort was undergoing general surgeries (43.3%) and ENT
surgeries (33.3%), followed by urology surgeries (13.3%). The most frequently used NMBD during the
current study was rocuronium with initial dose 50 mg (86.7%), while the most frequently used reversal
drug was Neostigmine (76.7%). The mean of BMI among the participants was 29.45 ± 5.56 kg/m2. Table 3: Demographic Data Page 5/16 Variables
Findings (n=30)
Age (years), median (IQR)
Gender, n (%)
Male
Female
ASA-PS Class, n (%)
I
II
III
BMI (kg/m2), mean ± SD
Type of surgery, n (%)
General
Urology
ENT
Plastic
OB/GYN
NMBD used, n (%)
Succinylcholine
Rocuronium
Cisatracurium
Reversal used, n (%)
None
Neostigmine
35 (27-53)
17(56.7%)
13(43.3%)
10 (33.3%)
17 (56.7%)
3 (10%)
29.45 ± 5.56
13 (43.3%)
4 (13.3%)
10 (33.3%)
2 (6.7%)
1 (3.3%)
1 (3.3%)
26 (86.7%)
3 (10%)
7 (23.3%)
23 (76.7%)
Key: n (%) = number (percentage); IQR= Interquartile Range; ± SD= ± standard deviation; ASA-PS Class=
American Society of Anaesthesiologists Physical Status class; BMI= Body Mass Index; kg/m2= Kilogram per
meter square ENT= Ear, Nose and Throat; OB/GYN= Obstetrics and Gynaecology; NMBD = neuromuscular
blocking drug. Key: n (%) = number (percentage); IQR= Interquartile Range; ± SD= ± standard deviation; ASA-PS Class=
American Society of Anaesthesiologists Physical Status class; BMI= Body Mass Index; kg/m2= Kilogram per
meter square ENT= Ear, Nose and Throat; OB/GYN= Obstetrics and Gynaecology; NMBD = neuromuscular
blocking drug. Results Overall, 66.7% of patients who underwent elective surgeries under general anaesthesia experienced
postoperative CREs, compared with 33.3% who did not (Table 5). The Mann-Whitney U test revealed that
there was no significant difference in postoperative CREs in the PACU between age groups and groups
with long vs. short surgery times. The median of the TOF ratio upon arrival in the PACU was 0.88 in Page 6/16 Page 6/16 patients with postoperative CREs. The Chi-Square test demonstrated that RNMB (TOF ratio <0.90) was
more frequent among those participants with postoperative CREs (75% vs. 10%, P=0.001). The Mann-
Whitney U test demonstrated no significant association between patients’ temperature and the incidence
of CREs. The Independent-Samples T tests showed a significant difference in postoperative CREs
between the two BMI groups (31.23 ± 6.1 vs. 26.78 ± 3.94, P= 0.047). Table 5: Comparison between the variables with presence or absence of CREs upon arrival in the PACU. Variables
CRE (n=20)
No CREs (n=10)
P value
Age (years), median (IQR)
BMI (kg/m2), mean ± SD
Duration of surgery (min), median (IQR)
Temperature (Co), median (IQR)
TOF ratio upon arrival in the PACU, median (IQR)
Incidence of RNMB
(TOF ratio <0.90), n (%)
35 (27-51)
31.23± 6.17
82(75-161)
36.50(36.40-36.80)
0.88 (0.72-0.89)
15 (75%)
35 (30-53)
26.78±3.94
106(86-158)
36.65(36.40-36.90)
0.96 (0.91-1.00)
1(10%)
0.62
0.047
0.403
0.689
0.001
0.001
Key: n (%) = number (percentage); IQR = Interquartile Range; ± SD= ± Standard Deviation; CREs= Critical
Respiratory Events; BMI= Body Mass Index; kg/m2= Kilogram per meter square; min = minutes; TOF= Train-of-
Four; PACU= Post-Anaesthesia Care Unit; RNMB= Residual Neuromuscular Blockade. Table 5: Comparison between the variables with presence or absence of CREs u ween the variables with presence or absence of CREs upon arrival in the PACU. Key: n (%) = number (percentage); IQR = Interquartile Range; ± SD= ± Standard Deviation; CREs= Critical
Respiratory Events; BMI= Body Mass Index; kg/m2= Kilogram per meter square; min = minutes; TOF= Train-of-
Four; PACU= Post-Anaesthesia Care Unit; RNMB= Residual Neuromuscular Blockade. Key: n (%) = number (percentage); IQR = Interquartile Range; ± SD= ± Standard Deviation; CREs= Critical
Respiratory Events; BMI= Body Mass Index; kg/m2= Kilogram per meter square; min = minutes; TOF= Train-of-
Four; PACU= Post-Anaesthesia Care Unit; RNMB= Residual Neuromuscular Blockade. During the present study, the incidence of critical respiratory events in the PACU was defined according to
the described criteria by Murphy et al. (2008a). Results Based on these criteria, twenty patients experienced
postoperative CREs during the first thirty minutes in the PACU in this current study. The one or two signs
of CREs commonly observed in the sample of this study with respiratory distress (50%), mild to moderate
hypoxemia (30%), and upper airway obstruction (16.7%). The other CREs recorded were severe
hypoxemia (13.3), upper airway weakness (10%) and inability to breathe deeply (6.7%) as in table 6. None
of the patients had pulmonary aspiratory and none of them required re-intubation in the PACU stay. Page 7/16
Table 6: Incidence of CREs in the PACU
Variable
Incidence
Upper airway obstruction
Mild-moderate hypoxemia
Severe hypoxemia
Signs of respiratory distress
Inability to breathe deeply
Upper airway weakness
5 (16.7%)
9 (30%)
4 (13.3%)
15 (50%)
2 (6.7%)
3 (10 %) Table 6: Incidence of CREs in the PACU
Variable
Incidence
Upper airway obstruction
Mild-moderate hypoxemia
Severe hypoxemia
Signs of respiratory distress
Inability to breathe deeply
Upper airway weakness
5 (16.7%)
9 (30%)
4 (13.3%)
15 (50%)
2 (6.7%)
3 (10 %) Discussion The aim this pilot study was to investigate whether RNMB is significantly associated with postoperative
CREs by determining the incidence of RNMB and postoperative CREs and the associated risk factors
during the PACU stay in a tertiary hospital in Saudi Arabia. The incidence of RNMB in our cohort was
53.3% and postoperative CREs were observed among 66.7% of our cohort. The findings show that there
was a statistically significant association between the incidence of RNMB and postoperative CREs (75%
vs 10%, P=0.001). Based on the patients’ demographic data and preoperative variables, which are presented in table 4, the
results of the current study do not show that female patients were significantly more likely to experience
RNMB in the PACU than males (P=0.033) as shown in table 4. These findings are consistent with other
studies conducted by Aytac et al. (2016), Pietraszewski and Gaszyński (2013), and Kaan et al. (2012). This could be to that women are more sensitive to rocuronium, the onset time of rocuronium is shorter in
women and its effective period is prolonged (Xue et al, 1997 and Adamus et al., 2008). They also added
that the variability of sensitivity and action duration of NMBDs between women and men is believed to be
associated with physiological differences in body structures. Whilst, Aytac et al. (2016) also found that
female patients undergoing anaesthesia of short duration are more likely to have RNMB in the PACU. Therefore, both Xue et al. (1997) and Adamus et al. (2008) suggest that the dose of rocuronium routinely
used in females could be reduced. Therefore, both Xue et al. (1997) and Adamus et al. (2008) suggest that the dose of rocuronium routinely
used in females could be reduced. Page 8/16
Table 4: Patient characteristics and the incidence of RNMB
Variables
RNMB
(TOF ratio <0.90),
n= 16
No RNMB
(TOF ratio ≥0.90),
n= 14
P
value
Age, n (%)
18 – 50 vs. ≥ 51 years
Gender, n (%)
Male/Female
ASA-PS Class, n (%)
I
II
III
BMI (kg/m2), mean ± SD
Neuromuscular monitoring before extubation, n (%)
Yes/No
Duration of the surgery,
< 90 min vs. ≥ 90 min
SpO2, upon arrival in the PACU, median (IQR)
RR(breaths/min), upon arrival in the PACU, median
(IQR)
11(50%) vs. 5 (62.5%)
6(35.3%)/10(76.9%)
7(43.8%)
9(56.3%)
0(0.0%)
30.82 ± 5.29
2(22.2%) / 14(66.7%)
13 (81.8%) vs. 3
(21.4%)
91(90-93)
22(19-25)
11(50%) vs. Discussion 3 (37.5%)
11(64.7%)/ 3(23.1%)
3(21.4%)
8(57.1%)
3(21.4%)
28.53 ± 6.41
7(77.8%) / 7(33.3%)
3 (18.8%) vs. 11
(78.6%)
96(91-100)
20(16-21)
0.0544
0.033
0.103
0.294
0.046
0.001
0.034
0.025 Table 4: Patient characteristics and the incidence of RNMB
Variables Table 4: Patient characteristics and the incidence of RNMB Duration of the surgery,
< 90 min vs. ≥ 90 min
SpO2, upon arrival in the PACU, median (IQR)
RR(breaths/min), upon arrival in the PACU, median
(IQR)
2(22.2%) / 14(66.7%)
13 (81.8%) vs. 3
(21.4%)
91(90-93)
22(19-25)
7(77.8%) / 7(33.3%)
3 (18.8%) vs. 11
(78.6%)
96(91-100)
20(16-21)
0.046
0.001
0.034
0.025 Key: n (%) = number (percentage); IQR = Interquartile Range; ± SD= ± Standard Deviation; RNMB= Residual
Neuromuscular Blockade; ASA-PS Class = American Society of Anaesthesiologists Physical Status class; BMI=
Body Mass Index; kg/m2= Kilogram per meter square SpO2 = Oxygen Saturation; RR = Respiratory Rate; min =
minutes; TOF= Train-of-Four; PACU= Post-Anaesthesia Care Unit. Key: n (%) = number (percentage); IQR = Interquartile Range; ± SD= ± Standard Deviation; RNMB= Residual
Neuromuscular Blockade; ASA-PS Class = American Society of Anaesthesiologists Physical Status class; BMI=
Body Mass Index; kg/m2= Kilogram per meter square SpO2 = Oxygen Saturation; RR = Respiratory Rate; min =
minutes; TOF= Train-of-Four; PACU= Post-Anaesthesia Care Unit. Other significant findings in the current studies that the incidence of RNMB is significantly more frequent
among patients for whom neuromuscular monitoring (acceleromyography) has not been used prior to
extubation (P=0.046). Several observational studies conducted in different countries (Fuchs-Buder et al. 2003; Grayling and Sweeney 2007; Sorgenfrei et al. 2005) have reported similar to this, but they cannot be
directly compared because they used different techniques for neuromuscular monitoring. And this was
also supported by Murphy et al. (2008b) who found that RNMB was more common in the conventional
monitoring group than that in the quantitative monitoring group (p<0.001). The findings of this study suggest that there was a significant difference in the incidence of RNMB in
relation to the duration of surgery whether short or long (<90 minutes compared with ≥90 minutes ;
P=0.001). Stewart et al. (2016) also showed agreement with the findings as the RNMB was significantly
more likely to be associated with shorter operations (P=0.001). Discussion There were no significant differences between the age of patients and the incidence of postoperative
CREs (P=0.62) in the current study. However, Cedborg et al. (2014) found that there was an intense
negative effect on airway integrity in elderly patients with increased prevalence of pharyngeal
dysfunction: from 37% in patients without RNMB to 71% during the presence of RNMB. Pedersen (1994)
examined the risk factors associated with postoperative complications following general anaesthesia,
and found that postoperative pulmonary complications were associated with elderly patients. Therefore,
the differences in the complications among the previous studies and the current study might be impacted
by the age of their samples included (Sieber and Barnett 2011). The results of this study seem to suggest a significant association between BMI and postoperative CREs
during the early PACU stay. BMI (31.23 ± 6.1 kg/m2) was significantly greater (P= 0.047) among patients
who experienced CREs compared to those who did not (26.78 ± 3.94 kg/m2). According to the World
Health Organisation (2016), subjects with BMI equal to or greater than 30 kg/m2 are considered obese. Obesity has physiological effects on the respiratory system’s compliance and lung volumes and that BMI
was significantly associated with severe hypoxemia (SpO2 < 90%) during the PACU stay (P=0.01)
(Hodgson et al. 2015; Cammu et al. (2012). The findings reported here can improve the current knowledge and clinical practice of perioperative
management, specifically the management of NMBDs. The first step for improving NMBDs’ management
and postoperative outcomes is basically through following the evidence-based medical management of
NMBDs, which can help to reduce complications initiated by unsuitable perioperative management. Therefore, based on the current literature and AAGBI guidelines (2016), the researcher recommends that
neuromuscular monitoring in the operating theatre before extubation is required for all patients who have
received NMBDs. Quantitative neuromuscular monitoring (acceleromyography) is essential to accurately
assess the TOF ratio, which can help to diminish the incidence of RNMB. This will also help to prevent the
postoperative complications associated with RNMB, and that will reduce the incidence of postoperative
critical respiratory events. The current pilot study has some limitations that need to be mentioned. The convenience sampling
method and time frame sample size were applicable and appropriate for this study. The researcher was
limited by resources and time boundaries, hence the sample employed was small, which might reduce the
external validity of the findings of the current study (LoBiondo-Wood and Haber 2014). Discussion The agreement among the conclusions of
these two studies could be referred to the fact that sufficient time is required for neostigmine to provide
effective reversal, or as Miller (2011) for spontaneous recovery from neuromuscular blockade to a TOF
ratio ≥ 0.90, which may not be allowed during shorter surgery times. Despite advances in the neuromuscular monitoring methods and pharmacological agents used in
anaesthesia practice, the findings of this study showed that postoperative CREs were significantly
associated with RNMB (P=0.001), which was consistent with the previous studies (Grosse-Sundrup et al. 2012; Murphy et al. 2008a; Murphy et al. 2008b; Norton et al. 2013; Sauer et al. 2011) as shown in table 5. Murphy et al. (2008b) in their study which has examined the effectiveness of quantitative
(acceleromyography) and qualitative neuromuscular monitoring in reducing the incidence of RNMB after
tracheal extubation. They found that RNMB (TOF ratio < 0.90) was more frequent in the conventional
group (30%) compared with the acceleromyography group (4.5%; P<0.001). In addition, the authors
revealed that mild hypoxaemia (SpO2 90–93%) was significantly more frequent in the conventional
group (43.3% vs. 6.7%) and severe hypoxaemia (SpO2 < 90%) was also more common in the
conventional group during the first 30 minutes of their PACU stay (21.1%; all P < 0.001). The result of
Murphy et al. (2008b) suggested that RNMB is significantly associated with hypoxaemia, which was
similar to the findings of the current study. Postoperative CREs occurred in twenty patients (66.7%), and there were significantly more of these CREs
among patients with RNMB (P= 0.001). Xará et al. (2015) examined 340 patients and found that CREs
happened in 67 of them (19.7%) and CREs were more frequent in patients with RNMB (P<.001). Moreover,
among the 599 patients studied by Stewart et al. (2016), 97 participants experienced one or more CREs in
the PACU, and these CREs were more frequent in patients with RNMB (P=0.033). In addition, Stewart et al. (2016) observed some CREs that were not observed by Xará et al. (2015) or by the current study, such as Page 9/16 Page 9/16 pulmonary aspiration after tracheal extubation or the need for assisted ventilation. However, the
differences in findings among these studies could be impacted by either the sample size or the type of
patients recruited during the studies. Discussion However, lack of
generalizability and sampling bias are the main disadvantages of this sampling method (Polit 2014). The
study was carried out only on elective surgical patients in a single tertiary hospital and therefore, the
results might be difficult to generalize to other surgical populations or to different settings (Greenhalgh
2014). Moreover, the present study was a prospective, observational study: no interventions were made in
anaesthetic practice during the pre-operative, intra-operative or post-operative period, and all treatments Page 10/16 Page 10/16 were applied and managed according to the standards of the clinician who was in charge of each patient
that day. Further research into the association between the incidence of RNMB and postoperative CREs following
general anaesthesia should address the limitations of this pilot study. This study suggests the need for
multicentre, randomised trials in Saudi Arabia to provide better findings and more conclusive results. Enrolment of large samples is necessary for the appropriate detection of associations with the incidence
of RNMB and postoperative CREs. Conclusion This is the first study conducted in Saudi Arabia that has investigated the association between the
incidence of RNMB and postoperative critical respiratory events among patients undergoing elective
surgery under general anaesthesia. This study observed evidence that the majority of patients with RNMB
(TOF ratio < 0.90) experienced critical respiratory events in the PACU. In this study, the analysis
demonstrated that high BMI (31.23 ± 6.1 kg/m2) can also contribute to critical respiratory events during
the early PACU stay. Through further descriptive analysis of the sample characteristics, it was shown that
female gender, shorter duration of surgery and absence of intraoperative quantitative neuromuscular
monitoring were the variables that were most likely to lead to the occurrence of RNMB (TOF ratio < 0.90). In agreement with previous researches, the current study confirms the continued high incidence of RNMB
upon PACU arrival during regular clinical practice, despite the use of neostigmine and qualitative TOF
monitoring. Routine quantitative neuromuscular monitoring is recommended to enhance patient safety. Therefore, these results should provoke a re-evaluation of current practice and techniques used for the
monitoring and reversal of NMBDs in anaesthesia practice in Saudi Arabia. The limitations of this
research, as highlighted above, along with the suggested recommendations for future practice, will
provide further evidence to supplement the current research and the extant literature, which is essential in
improving the quality of anaesthesia practice. Authors' contributions: Faraj Alenezi, Principal Investigator: research idea, study design, data collection, data analysis,
interpretation of the results, writing manuscript. Faraj Alenezi, Principal Investigator: research idea, study design, data collection, data analysis,
interpretation of the results, writing manuscript. Khalid Alnababtah: study design, ethical approvals, interpretation of the results. Mohammed M. Alqahtani: interpretation of the data. Mohammed M. Alqahtani: interpretation of the data. Lafi Olayan: interpretation of the data. Lafi Olayan: interpretation of the data. Mohammed Al Harbi: co-author: participants recruitment, data collection. Competing interests: none College of Applied Medical Sciences King Saud bin Abdulaziz University for Health Sciences,
King Abdullah International Medical Research Center (KAIMRC), King Abdullaziz Medical City,
National Guard Health Affairs, Riyadh, Saudi Arabia. College of Applied Medical Sciences King Saud bin Abdulaziz University for Health Sciences, College of Applied Medical Sciences King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Center (KAIMRC), King Abdullaziz Medical City, King Abdullah International Medical Research Center (KAIMRC), King Abdullaziz Medical City,
National Guard Health Affairs, Riyadh, Saudi Arabia. King Abdullah International Medical Research Center (KAIMRC), King A National Guard Health Affairs, Riyadh, Saudi Arabia. National Guard Health Affairs, Riyadh, Saudi Arabia. List Of Abbreviations List Of Abbreviations Page 11/16 Abbreviations
Definition
PACU
Post-anaesthesia care units
NMBDs
Neuromuscular blocking drugs
NMBD
Neuromuscular blocking drug
RNMB
Residual neuromuscular blockade
TOF
Train-of-Four
CREs
Critical respiratory events
BMI
Body Mass Index
ASA-PS
American Society of Anaesthesiologists Physical Status
SpO2
Oxygen Saturation
RR
Respiratory Rate Ethics approval and consent to participate: obtained Ethics approval and consent to participate: obtained Consent for publication: obtained Consent for publication: obtained Availability of data and material: available Availability of data and material: available Competing interests: none Acknowledgements: We would like to thank anaesthesia department, King Abdulaziz Medical City, for their cooperation
during data collection. Page 12/16 Page 12/16 References Adamus, M. et al. 2008. Influence of gender on the course of neuromuscular block following a single
bolus dose of cisatracurium or rocuronium. European Journal of Anaesthesiology 25(7), pp. 589-595. Adamus, M. et al. 2008. Influence of gender on the course of neuromuscular block following a single
bolus dose of cisatracurium or rocuronium. European Journal of Anaesthesiology 25(7), pp. 589-595. Aytac, I. et al. 2016. Survey of postoperative residual curarization, acute respiratory events and approach
of anesthesiologists. Brazilian Journal of Anesthesiology (English edition) 66(1), pp. 55-62. Aytac, I. et al. 2016. Survey of postoperative residual curarization, acute respiratory events and approach
of anesthesiologists. Brazilian Journal of Anesthesiology (English edition) 66(1), pp. 55-62. Buhre, W. and Rossaint, R. 2003. Perioperative management and monitoring in anaesthesia. The Lancet
362(9398), pp. 1839-1846. Butterworth, J. F. 2013. Morgan & Mikhail's clinical anesthesiology. 5th ed. ed. New York: New York :
McGraw-Hill. Cammu, G. et al. 2012. A prospective, observational study comparing postoperative residual curarisation
and early adverse respiratory events in patients reversed with neostigmine or sugammadex or after
apparent spontaneous recovery. Anaesthesia and intensive care 40(6), p. 999. Cedborg, H. A. I. et al. 2014. Pharyngeal Function and Breathing Pattern during Partial Neuromuscular
Block in the Elderly: Effects on Airway Protection. Anesthesiology 120(2), pp. 312-325. Claudius, C. et al. 2009. The undesirable effects of neuromuscular blocking drugs. Anaesthesia 64(s1),
pp. 10-21. Fortier, L.-P. et al. 2015. The RECITE study: a Canadian prospective, multicenter study of the incidence and
severity of residual neuromuscular blockade. Anesthesia and Analgesia 121(2), pp. 366-372. Fuchs-Buder, T. et al. 2003. Einsatz des neuromuskulären Monitorings in Deutschland. Der Anaesthesist
52(6), pp. 522-526. Grayling, M. and Sweeney, B. 2007. Recovery from neuromuscular blockade: a survey of practice. Anaesthesia 62(8), pp. 806-809. Greenhalgh, T. 2014. How to read a paper : the basics of evidence-based medicine. Fifth edition. ed. Chichester, West Sussex : John Wiley & Sons Inc. Grosse-Sundrup, M. et al. 2012. Intermediate acting non- depolarizing neuromuscular blocking agents
and risk of postoperative respiratory complications: prospective propensity score matched cohort study. British Medical Journal 345, pp. 1-14. Herbstreit, F. et al. 2009. Impaired Upper Airway Integrity by Residual Neuromuscular Blockade Increased
Airway Collapsibility and Blunted Genioglossus Muscle Activity in Response to Negative Pharyngeal
Pressure. Anesthesiology 110(6), pp. 1253-1260. Page 13/16 Page 13/16 Kaan, N. et al. 2012. The incidence of residual neuromuscular blockade associated with single dose of
intermediate-acting neuromuscular blocking drugs. References Middle East Journal of Anesthesiology 21(4), pp. 535-
542. Kiekkas, P. et al. 2014. Residual neuromuscular blockade and postoperative critical respiratory events:
literature review. Journal of clinical nursing 23(21-22), pp. 3025-3035. Kiekkas, P. et al. 2014. Residual neuromuscular blockade and postoperative critical respiratory events:
literature review. Journal of clinical nursing 23(21-22), pp. 3025-3035. Kopman, A. 2016. Residual Neuromuscular Blockade and Adverse Postoperative Outcomes: An Update. Curr Anesthesiol Rep 6(2), pp. 178-184. LoBiondo-Wood, G. and Haber, J. 2014. Nursing research : methods and critical appraisal for evidence
based practice. 8th ed. ed. St. Louis, MO: St. Louis, MO : Elsevier Mosby. McCaul, C. et al. 2002. Atracurium is associated with postoperative residual curarization. British Journal
of Anaesthesia 89(5), pp. 766-769. Miller, R. D. 2011. Basics of anesthesia. 6th ed Philadelphia: 6th ed. Miller, R. D. 2011. Basics of anesthesia. 6th ed Philadelphia: 6th ed. Moreno, C. et al. 2013. Residual neuromuscular block and critical respiratory events in a post‐anaesthesia
care unit: 5AP2‐11. European Journal of Anaesthesiology (EJA) 30, pp. 86-86. Murphy, G. S. 2006. Residual neuromuscular blockade: incidence, assessment, and relevance in the
postoperative period. Minerva Anestesiol 72(3), pp. 97-109. Murphy, G. S. et al. 2011. Intraoperative Acceleromyography Monitoring Reduces Symptoms of Muscle
Weakness and Improves Quality of Recovery in the Early Postoperative Period. Anesthesiology 115(5), pp. 946-954. Murphy, G. S. et al. 2013. Postoperative residual neuromuscular blockade is associated with impaired
clinical recovery. Anesthesia and Analgesia 117(1), pp. 133-141. Murphy, G. S. et al. 2015. Residual neuromuscular block in the elderly: Incidence and clinical implications. Anesthesiology 123(6), pp. 1322-1326. Murphy, G. S. et al. 2008a. Residual neuromuscular blockade and critical respiratory events in the
postanesthesia care unit. Anesthesia and Analgesia 107(1), pp. 130-137. Murphy, G. S. et al. 2008b. Intraoperative Acceleromyographic Monitoring Reduces the Risk of Residual
Meeting Abstracts and Adverse Respiratory Events in the Postanesthesia Care Unit. The Journal of the
American Society of Anesthesiologists 109(3), pp. 389-398. Naguib, M. et al. 2010. A survey of current management of neuromuscular block in the United States and
Europe. Anesthesia & Analgesia 111(1), pp. 110-119. Page 14/16 Page 14/16 Nava-Ocampo, A. et al. 2002. Preferences of Mexican anesthesiologists for vecuronium, rocuronium, or
other neuromuscular blocking agents: a survey. BMC anesthesiology 2(1), p. 1. Newell, M. A. et al. 2007. Body Mass Index and Outcomes in Critically Injured Blunt Trauma Patients:
Weighing the Impact. Figures Figures Figures Figure 1 Figure 1 Flow diagram of participants’ enrolment Flow diagram of participants’ enrolment Figure 2 References Journal of the American College of Surgeons 204(5), pp. 1056-1061. Norton, M. et al. 2013. Residual neuromuscular block as a risk factor for critical respiratory events in the
post anesthesia care unit. Revista Española de Anestesiología y Reanimación 60(4), pp. 190-196. Pedersen, T. 1994. Complications and death following anaesthesia. A prospective study with special
reference to the influence of patient-, anaesthesia-, and surgery- related risk factors. Danish Medical
Bulletin 41(3), pp. 319-331. Pietraszewski, P. and Gaszyński, T. 2013. Residual neuromuscular block in elderly patients after surgical
procedures under general anaesthesia with rocuronium. Anaesthesiology Intensive Therapy 45(2), pp. 77-
81. Polit, D. F. 2014. Essentials of nursing research : appraising evidence for nursing practice. 8th ed. ed. Philadelphia ; London: Philadelphia ; London : Wolters Kluwer Health/Lippincott Williams & Wilkins. Polit, D. F. 2014. Essentials of nursing research : appraising evidence for nursing practice. 8th ed. ed. Philadelphia ; London: Philadelphia ; London : Wolters Kluwer Health/Lippincott Williams & Wilkins. Sauer, M. et al. 2011. The influence of residual neuromuscular block on the incidence of critical
respiratory events. A randomised, prospective, placebo-controlled trial. European Journal of
Anaesthesiology (EJA) 28(12), pp. 842-848. Sieber, F. E. and Barnett, S. R. 2011. Preventing postoperative complications in the elderly. Anesthesiology
clinics 29(1), pp. 83-97. Sorgenfrei, I. et al. 2005. [Does evidence lead to a change in clinical practice? Danish anaesthetists' and
nurse anesthetists' clinical practice and knowledge of postoperative residual curarization]. Ugeskrift for
laeger 167(41), pp. 3878-3882. Stewart, P. A. et al. 2016. The Impact of Residual Neuromuscular Blockade, Oversedation, and
Hypothermia on Adverse Respiratory Events in a Postanesthetic Care Unit: A Prospective Study of
Prevalence, Predictors, and Outcomes. Anesthesia & Analgesia 123(4), pp. 859-868. Stewart, P. A. et al. 2016. The Impact of Residual Neuromuscular Blockade, Oversedation, and Tsai, C.-C. et al. 2008. Postoperative residual curarization: clinical observation in the post-anesthesia care
unit. Chang Gung Med J 31(4), pp. 364-368. World Health Organization. 2016. WHO | Obesity and overweight [Online]. [Online]. Available at:
http://www.who.int/mediacentre/factsheets/fs311/en/ [Accessed: 05/05/2017]. Xará, D. et al. 2015. Adverse Respiratory Events in a Post- Anesthesia Care Unit. Archivos de
Bronconeumología (English Edition) 51(2), pp. 69-75. Page 15/16 Xue, F. S. et al. 1997. Dose- Response and Time Course of Effect of Rocuronium in Male and Female
Anesthetized Patients. Anesthesia and Analgesia 85(3), pp. 667-671. Xue, F. S. et al. 1997. References Dose- Response and Time Course of Effect of Rocuronium in Male and Female
Anesthetized Patients. Anesthesia and Analgesia 85(3), pp. 667-671. Xue, F. S. et al. 1997. Dose- Response and Time Course of Effect of Rocuronium in Male and Female
Anesthetized Patients. Anesthesia and Analgesia 85(3), pp. 667-671. Page 16/16
Figures
Figure 1
Flow diagram of participants’ enrolment
Figure 2
The incidence of RNMB among the sample based on their gender Figure 2 Page 16/16
The incidence of RNMB among the sample based on their gender
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https://openalex.org/W4388633432
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https://proceedings-szmc.org.pk/index.php/szmc/article/download/435/216
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English
| null |
Relationship Of Burnout with Screen Time In Under Graduate Medical Students
|
Proceedings
| 2,023
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cc-by
| 7,625
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INTRODUCTION
p
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Burnout can cause serious consequences related to
mental and physical health and can negatively affect
personal and work relationships. Given the
aforementioned consequences, its is imperative that
indicators of burnout be proactively identified and
addressed in a timely manner utilizing various
approaches such as taking time off, implementing
self-care
strategies
and
seeking
professional
assistance3.A variety of factors can contribute
towards burnout such as lack of control, unclear
expectations
and
lack
of
social
support4,5. Individuals exposed to prolonged high-pressure
environments are susceptible to burnout, and
healthcare professionals are particularly vulnerable
and morbidity due to the overactive inflammatory
cascade
brought
on
by
the
release
of
endotoxins5.Clinical evidence is used to identify
peritonitis. Diagnosis can be achieved via upright
plain x-ray of the abdomen, USG, or CT scan. This
is often done through diagnostic laparoscopy
nowadays6.Resuscitation,
diagnosis,
prompt
exploration, treatment of the underlying cause, and
extensive surgical peritoneal lavage have always
been the cornerstones of peritonitis therapy
regimens (IOPL)7,8. Regular IOPL is performed to
lessen bacterial contamination and burden. Even
though large volumes of normal saline are used in
IOPL, the rates of sepsis, wound infection, and
mortality remain alarmingly high. Proceedings S.Z.M.C. Vol: 37(4): pp. 15-20, 2023.
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Keywords: Burnout, screen-time, BCSQ-12-SS
metronidazole group was comparable to mean
significant (p=0 067) Male/female ratio in Metro
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Muhammad Akra
Academy Islam
8PDLU.KDQ0
HFWXUHV
VVDLQ 5D]D =D Material & Methods: Consecutive sampling strategy followed by a randomized controlled trial were used to induct and
provide intervention to 90 patients aged 15-65 years with peritonitis caused by hollow viscus perforation. The patients
were subdivided into 2 groups A& B(n=45 each).Two liters of normal saline were used for peritoneal lavage in group A,
while two liters of normal saline were combined with 200 mL of metronidazole solution and administered to group B. intraoperatively.Baseline physiological parameters such as age, sex, BMI , intra operative surgical parameters as
duration of operation and post-operative course were recorded till discharge. On 10th POD, patients returned to OPD for
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L Conclusion: The present study reveals a significant correlation between screen time among medical students and
burnout, highlighting the potential impact on both physical and mental health. further monitoring. An infection was diagnosed if the patient had post-operative symptoms such as a high temperature,
increased TLC, wound discharge, redness, or pain. Data was entered and analyzed using SPSS version 23. ABSTRACT
Muhammad Akra
Academy Islam
8PDLU.KDQ0
HFWXUHV
VVDLQ 5D]D =D Results: A majority (54 44%) of the patients were young adults Mean age of 37 33 ± 10 53 years of patients in the
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5HVXOWV $ WRWDO RI UHVSRQVHV ZHUH UHFHLYHG RI ZKLFK UHVSRQVHV ZHUH FRPSOHWH 2I ZKLFK ZHUH IURP VW\HDU ABSTRACT
Muhammad Akra
Academy Islam
8PDLU.KDQ0
HFWXUHV
VVDLQ 5D]D =D The students who provided written informed consent were enrolled in the study.These
participants completed a structured questionnaire, which included various demographic variables and information
regarding students' screen time, specifically for academic purposes and leisure activities. To evaluate burnout, the study
utilized the "Burnout Clinical Subtype Questionnaire" (BCSQ), which has been adapted for student use (BCSQ-12-
SS).The data was entered & analyzed using SPSS version 23, a p-value of 0.05 was considered significant.
mortality. Largely peritonitis is caused by a gastrointestinal perforation or anastomotic leak. In peritonitis, anaerobes &
gram-negative organisms are mostly responsible for sepsis and morbidity due to the overactive inflammatory cascade by
endotoxins which is amenable to timely intervention. Aims & Objectives: The study's aim was to evaluate whether using normal saline or metronidazole solution during
intraoperative peritoneal lavage (IOPL), results in a lower rate of postoperative wound infection. Place and Duration of Study: This study was undertaken at the South Surgical Ward, Mayo Hospital Lahore for 6
months from February 2nd 2021 to August 1st 2021
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W Results: The mean age of participants of the study was 20.56±1.73 years. The correlation analysis showed a significant
correlation of neglect, lack of development and total burnout score with screen time for non-educational purposes in
medical students with a p-value of 0.022, 0.009 and 0.009 respectively. Screen time for educational purposes hada
significant correlation with the overload domain of burnout; r=0.157 (p=0.039). Students with self-perception of bad
health had a significant correlation of screen time for non-educational purposes with total burnout score;
r=0.234(p=0.04). Scores of total burnout and subtypes of overload, neglect and lack of development were significantly
lower in students with self-perception of good health. months from February 2nd, 2021, to August 1st, 2021. ABSTRACT
Muhammad Akra
Academy Islam
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Muhammad Akra
Academy Islam
8PDLU.KDQ0
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often accompanied by a diminished sense of personal accomplishment. Burnout may adversely affect the well-being and
motivation of medical students consequently resulting in compromised academic performance. Department of Urology, Chaudhary Muhammad Akram Hospital, Lahore
4Department of Plastic Surgery, ANMC / Chaudhary Muhammad Akram Hospital, Lahore, Pakistan
ABSTRACT
2Department of Management Sciences, Riphah Int University, Islamabad. 3Department of Medicine, IHITC, Islamabad. 4Department of Rheumatology, PIMS, Islamabad. 2Department of Orthopedics, LGH, Lahore
3Department of Orthopedics, THQ, Ferozwala
4Department of Orthopedics, CMH, Lahore
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1Department of Anatomy, UCMD, University of Lahore, Lahore
2Department of Anatomy, Punjab Medical College, Faisalabad Medical University, Faisalabad
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motivation of medical students consequently resulting in compromised academic performance. Aim and Objectives: The present study aims to explore the correlation of screen time with burnout and its subtypes
including overload, neglect and lack of development.
ABSTRACT
Introduction: Globall
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5Department of Hand & Upper Limb Surgery, CMH, Lahore
6Department of Orthopedics, Al-Rehmat Benevolent Trust
2Department of Anatomy, Punjab Medical College, Faisalabad Medical University, Faisalabad
3Department of Medical Education, Pak Red Crescent Medical and Dental College, Lahore q
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Aim and Objectives: The present study aims to explore the correlation of screen time with burnout and its subtypes
including overload, neglect and lack of development. ABSTRACT
Introduction: Globally acute generalized peritonitis ranks among the top surgical emergencies Different studies have
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6Department of Orthopedics, Al-Rehmat Benevolent Trust
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Place and Duration of Study: The present cross-sectional study was conducted at Shifa College of Medicine from
September 2019-February 2021. Introduction: Globally, acute generalized peritonitis ranks among the top surgical emergencies. ABSTRACT
Muhammad Akra
Academy Islam
8PDLU.KDQ0
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were subdivided into 2 groups A& B(n=45 each).Two liters of normal saline were used for peritoneal lavage in group A,
while two liters of normal saline were combined with 200 mL of metronidazole solution and administered to group B. intraoperatively.Baseline physiological parameters such as age, sex, BMI , intra operative surgical parameters as
duration of operation and post-operative course were recorded till discharge. On 10th POD, patients returned to OPD for
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convenience purposive sampling. ABSTRACT
Muhammad Akra
Academy Islam
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been conducted to show the amplitude of peritonitis worldwide eliciting a huge impact on overall patient morbidity and
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Material & Methods: The present study included a total of 284 students from the first year to the final year using
convenience purposive sampling. The students who provided written informed consent were enrolled in the study.These
participants completed a structured questionnaire, which included various demographic variables and information
regarding students' screen time, specifically for academic purposes and leisure activities. To evaluate burnout, the study
utilized the "Burnout Clinical Subtype Questionnaire" (BCSQ), which has been adapted for student use (BCSQ-12-
SS).The data was entered & analyzed using SPSS version 23, a p-value of 0.05 was considered significant.
Results: The mean age of participants of the study was 20.56±1.73 years. The correlation analysis showed a significant
correlation of neglect, lack of development and total burnout score with screen time for non-educational purposes in
medical students with a p-value of 0.022, 0.009 and 0.009 respectively. Screen time for educational purposes hada
significant correlation with the overload domain of burnout; r=0.157 (p=0.039). Students with self-perception of bad
health had a significant correlation of screen time for non-educational purposes with total burnout score;
r=0.234(p=0.04). Scores of total burnout and subtypes of overload, neglect and lack of development were significantly
lower in students with self-perception of good health. mortality. Largely peritonitis is caused by a gastrointestinal perforation or anastomotic leak. In peritonitis, anaerobes &
gram-negative organisms are mostly responsible for sepsis and morbidity due to the overactive inflammatory cascade by
endotoxins which is amenable to timely intervention. Aims & Objectives: The study's aim was to evaluate whether using normal saline or metronidazole solution during
intraoperative peritoneal lavage (IOPL), results in a lower rate of postoperative wound infection. Place and Duration of Study: This study was undertaken at the South Surgical Ward, Mayo Hospital Lahore for 6
months from February 2nd, 2021, to August 1st, 2021. Material & Methods: Consecutive sampling strategy followed by a randomized controlled trial were used to induct and
provide intervention to 90 patients aged 15-65 years with peritonitis caused by hollow viscus perforation. Relationship Of Burnout with Screen Time In Under
Graduate Medical Students
Intraoperative Peritoneal Lavage in Peritonitis: Norm
Saline vs Metronidazole
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or other activities, decrease in job satisfaction and
motivation, loss of energy, changes in sleep patterns
and physical symptoms in extreme cases2. solution did so (p 0.0001). metronidazole solution for intraoperative peritoneal lavage
ve wound infection. erative peritoneal lavage
and morbidity due to the overactive inflammatory
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Keywords: peritonitis, postoperative wound infection, intr
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Islamabad
2.2Department of Pharmacology, Shifa College of Dentistry, STMU,
Islamabad
3.3Department of Physiology, Shifa College of Medicine, STMU,
Islamabad
4.4Department of Pharmacology, Shifa College of Medicine, STMU,
Islamabad
5.5Department of Psychiatry, Shifa College of Medicine, STMU,
Islamabad
6.6Medical Students, Shifa College of Medicine, STMU, Islamabad
Correspondance:
Dr. Fahad Azam, Professor, Department of Pharmacology &
Therapeutics, Shifa College of Dentistry, Shifa Tameer-e-Millat
University, Islamabad. Email: fahad.scm@stmu.edu.pk
Submission Date: 17th August 2023
1st Revision Date: 7th September 2023
Acceptance Date: 1st November 2023
INTRODUCTION
Globally, acute generalized peritonitis ranks among
the top surgical emergencies1. It is more common in
Third World nations. The prevalence of perforation
is low (0.6% - 4.9%) in developed nations but high
(33% - 63%) in West Africa2. 554 persons were
discovered to have peritonitis in a study that took
place over three years in India3. Researchers in
Pakistan have conducted studies with similar
methods, with one study reporting 650 cases in a
just 9 months4. Most cases of peritonitis are caused
by a gastrointestinal perforation or anastomotic
leak5. Proceedings S.Z.M.C. Vol: 37(4): pp. 15-20, 2023.
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RIKHDOWKUHODWHGILHOGVZKLFKLVWDXJKWWUDGLWLRQDOO\ 1.1Department of Physiology, Shifa College of Dentistry, STMU,
Islamabad
2.2Department of Pharmacology, Shifa College of Dentistry, STMU,
Islamabad
3.3Department of Physiology, Shifa College of Medicine, STMU,
Islamabad
4.4Department of Pharmacology, Shifa College of Medicine, STMU,
Islamabad
5.5Department of Psychiatry, Shifa College of Medicine, STMU,
Islamabad
6.6Medical Students, Shifa College of Medicine, STMU, Islamabad
Correspondance:
Dr. Fahad Azam, Professor, Department of Pharmacology &
Therapeutics, Shifa College of Dentistry, Shifa Tameer-e-Millat
University, Islamabad. Email: fahad.scm@stmu.edu.pk
Submission Date: 17th August 2023
1st Revision Date: 7th September 2023
Acceptance Date: 1st November 2023
INTRODUCTION
Globally, acute generalized peritonitis ranks among
the top surgical emergencies1. It is more common in
Third World nations. The prevalence of perforation
is low (0.6% - 4.9%) in developed nations but high
(33% - 63%) in West Africa2. 554 persons were
discovered to have peritonitis in a study that took
place over three years in India3. Researchers in
Pakistan have conducted studies with similar
methods, with one study reporting 650 cases in a
just 9 months4. Most cases of peritonitis are caused
by a gastrointestinal perforation or anastomotic
leak5. INTRODUCTION
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DSSURDFKHV WR $QDWRP\ 7R FRQWLQXH PHGLFDO 2.2Department of Pharmacology, Shifa College of Dentistry, STMU,
Islamabad
Globally, acute generalized peritonitis ranks among
the top surgical emergencies1 It is more common in
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Islamabad
the top surgical emergencies1. It is more common in
Third World nations. The prevalence of perforation
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Islamabad
p
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(33%
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Islamabad
(33% - 63%) in West Africa2. MATERIAL AND METHODS The present cross-sectional survey was conducted at
Shifa College of Medicine (SCM) from September
2019-February 2021 with 284 medical students after
obtaining approval from the Institutional Review
Board and Ethics Committee (IRB& EC #002-492-
2019). The data was collected after getting informed
consent from the study participants. SCM follows an
integrated modular system and curricular contents
are delivered utilizing problem-based learning
(PBL), small-group discussions (SGD),large group
interactive sessions (LGIS), self-directed learning
(SDL), junior clerkships in year fourth and senior
clerkships in the final year.The present study
included 284 medical students from all five years
using convenience sampling after obtaining written
informed consent. A single proportion formula was
utilized to calculate the sample size, assuming a
50% prevalence of burnout, a 95% confidence level,
5% margin of error, and accounting for a 10% non-
response rate. To maximize the sample size, a
prevalence of 0.5 was used because there had been
no prior research at Shifa College of Medicine on
burnout prevalence. Additionally, the modified
Kish-Leslie formula was employed to calculate the
estimated sample size from the overall study
population. A structured questionnaire including
demographic variables such as gender, academic
year, marital status and place of residence was
provided to all participants. The screen time of
students for academic purposes and leisure activities
was also assessed by the questionnaire.The study
utilized avail dated Burnout Clinical Subtype
Questionnaire" (BCSQ) to evaluate burnout, which
has been adapted for student use (BCSQ-12-SS)16. This questionnaire encompasses a comprehensive
exploration of burnout, encompassing various
dimensions and subtypes. Specifically, it examines
the frenetic subtype, characterized by stress in
ambitious and overworked individuals; the under-
challenged subtype, which pertains to stress arising
from tasks lacking intellectual stimulation and a
sense of personal growth; and the worn-out subtype,
associated with feelings of under-appreciation and
perception
of
decreased
control
influencing
outcomes, resulting in a diminished focus on work
responsibilities.11By utilizing the BCSQ-12-SS, the
study aimed to explore the prevalence of burnout
across these different subtypes among medical
students Burnout syndrome encompasses two significant
elements
of
emotional
exhaustion
and
depersonalization. Emotional exhaustion indicates a
state of diminished emotional sensitivity which is
often accompanied by a perception of reduced
personal accomplishment. On the other hand,
depersonalization is characterized by reduced
empathy and motivation and a heightened sense of
isolation8. INTRODUCTION
p
g
(6.67% of those who rec
sed on the results of this
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iscovered to have peritonitis in a study that to
lace over three years in India3
Researchers
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LQFOXG 1586701 Relationship Of Burnout with Screen Time In Undergraduate Medical Students due to their sustained exposure to demanding
situations2. burnout and its subtypes including overload, neglect
and lack of development in undergraduate medical
students. Besides medical professionals, medical students are
also considered a vulnerable group for developing
burnout
because
of
intensive
coursework
requirements in addition to the constant social
interaction with patients in high-pressure hospital
environments2.In recent times, excessive screen
time and consequently social isolation is becoming a
significant factor contributing to burnout which has
made medical students even more vulnerable to
burnout6,7. RESULTS Two hundred and eighty-four medical students
participated in the study. The mean age of the study
participants was 20.56 ± 1.73 with a maximum age
of 25 years and minimum age of 17 years. The
demographic data of study participants is provided
in Table-1 7
been summarized in Table-3. Student
Detail
Screen Time
Overload
Neglect
Lack of
Development
Total Burnout
Score
R
p-
value
R
p-
value
R
p-
value
R
p-
value
All
Student
(N=284)
Screen Time
For Non-
Educational
Purposes
0.06
0.43
0.17
0.02
0.19
0.01
0.19
0.01
Screen Time
For
Educational
Purposes
0.16
0.04
-
0.05
0.49
-
0.07
0.36
0.03
0.73
Student
With
Self-
Percepti
on Of
Good
Health
(N=163)
Screen Time
For Non-
Educational
Purposes
-
0.07
0.46
0.22
0.04
0.22
0.03
0.16
0.14
Screen Time
ForEducatio
nal Purposes
0.22
0.04
-
0.02
0.83
-
0.16
0.13
0.02
0.88
Student
With
Self-
Percepti
on Of
Bad
Health
(N=110)
Screen Time
For Non-
Educational
Purposes
0.10
0.38
0.14
0.24
0.22
0.06
0.24
0.04
Screen Time
For
Educational
Purposes
0.12
0.32
-
0.07
0.52
0.09
0.43
0.04
0.53
Table-2: Correlation of screen time with subtypes of
burnout
Things
Students with self-
perception of good
health
(n=163)
Students with
self-perception
of bad health
(n=110)
p-
value
Total
Burnout
(Mean ± SD)
39.61±9.63
43.91±8.15
<0.001
Overload
13.78±4.58
15.20±5.12
0.017
Neglect
11.69±4.11
13.34±4.18
0.001
Lack Of
Development
14.15±4.47
15.37±4.00
0.021
Table-3: Comparison of burnout scores between
students with self-perception of good health
and self-perception of bad health
DISCUSSION
The increasing prevalence of burnout and its
potentially serious consequences in medical students Student
Detail
Screen Time
Overload
Neglect
Lack of
Development
Total Burnout
Score
R
p-
value
R
p-
value
R
p-
value
R
p-
value
All
Student
(N=284)
Screen Time
For Non-
Educational
Purposes
0.06
0.43
0.17
0.02
0.19
0.01
0.19
0.01
Screen Time
For
Educational
Purposes
0.16
0.04
-
0.05
0.49
-
0.07
0.36
0.03
0.73
Student
With
Self-
Percepti
on Of
Good
Health
(N=163)
Screen Time
For Non-
Educational
Purposes
-
0.07
0.46
0.22
0.04
0.22
0.03
0.16
0.14
Screen Time
ForEducatio
nal Purposes
0.22
0.04
-
0.02
0.83
-
0.16
0.13
0.02
0.88
Student
With
Self-
Percepti
on Of
Bad
Health
(N=110)
Screen Time
For Non-
Educational
Purposes
0.10
0.38
0.14
0.24
0.22
0.06
0.24
0.04
Screen Time
For
Educational
Purposes
0.12
0.32
-
0.07
0.52
0.09
0.43
0.04
0.53 in Table-1. MATERIAL AND METHODS The are various tools to identify and measure
burnout in different situations such as the Maslach
Burnout
Inventory
(MBI)
and
the
Burnout
Assessment Tool (BAT)9. One limitation of the MBI
tool is its reliance on scores of subtypes of burnout
and inability to provide total burnout score. Similarly, the validity and reliability of the
shortened version of the BAT tool have not been
fully explored10. The
Burnout
Clinical
Subtype
Questionnaire
(BSCQ) delineates three distinct domains of burnout
including frenetic subtype, the under-challenged
subtype, and the worn-out subtype. Each subtype
represents a unique manifestation of burnout with its
own set of characteristics and implications. These
subtypes arise from factors such as
work
enthusiasm, motivation levels, lack of appreciation,
and the overall work environment. The BSCQ aims
to capture and assess burnout within these specific
domains11.Multiple
studies
have
reported
an
unprecedently high levels of burnout among medical
students in the UK, Australia, and Pakistan12-14. This
concerning trend continues to escalate despite the
implementation
of
numerous
student-centred
learning approaches. Moreover, emerging factors,
including the pervasive rise in screen time, have
contributed to the increasing prevalence of burnout
among medical students. The increasing prevalence of burnout has a high
probability of leading to physical and mental
ailments such as emotional instability, fatigue and
eating disorders as well as affecting their learning
abilities15. With this background, the present study
aims to explore the correlation of screen time with 16 Relationship Of Burnout with Screen Time In Undergraduate Medical Students The data was stored on the software MS-Excel
software and was later analyzed by SPSS version
23. Categorical variables were presented by
frequencies and percentages whereas mean ± SD
were used to express continuous variables. The
Shapiro-Wilk test was used to check the normality
of data. Student’s t-test was used to compare
different groups for quantitative variables; the
correlation of academic and leisure screen time with
burnout and its subtypes of overload, neglect and
lack of development was analyzed using the Pearson
correlation statistical test. The reference point of
P<0.05 was used to determine significance. The following correlation analysis showed neglect,
lack of development and total burnout score having
a significant correlation with screen time for non-
educational purposes in medical students with a p-
value of 0.022, 0.009 and 0.009 respectively. Screen
time for educational purposes showed a significant
correlation with the overload domain of burnout;
r=0.157 (p=0.039). MATERIAL AND METHODS Students with self-perception of
bad health had a significant correlation of screen
time for non-educational purposes with total burnout
score;
r=0.234(p=0.04). The
results
of
the
correlation analysis are provided in Table-2. The comparison of mean scores of subtypes of
overload, neglect, lack of development and total
burnout in students having self-perception of good
health with students having self-perception of bad
health indicated significant results. Scores of total
burnout and subtypes of overload, neglect and lack
of development were significantly lower in students
with self-perception of good health. The results have
been summarized in Table-3. RESULTS Variables
N (%)
Total Burnout
Scores
P-
Value
Gender
Male
122(43%)
40.96
0.519
Female
162(57%)
41.66
Marital status
Single
277(97.5%)
41.37
0.916
Married
7(2.5%)
41.01
Year of Study
Year-1
74(26.1%)
40.22 ± 8.06
0.435
Year-2
93(32.7%)
41.91 ± 10.64
Year-3
62(21.8%)
42.21 ± 7.27
Year-4
17(6%)
43.41 ± 8.71
Year-5
38(13.4%)
39.95 ± 10.01
Residence
Day Scholar
201(71%)
41.66 ± 9.01
0.335
Hostelites
81(29%)
40.49 ± 9.50
Financial status
Monthly
household
income less
than Rs. 50,000
9(3.4%)
45.67 ± 11.89
0.025*
Monthly
household
income from
Rs. 50,000 -
100,000
43(16%)
44.16 ± 8.04
Monthly
house hold
income of
more than
Rs. 100,000
per month
216(80.6%)
40.67 ± 9.06
Table-1: Demographic data of study participants Table-2: Correlation of screen time with subtypes of
burnout
Things
Students with self-
perception of good
health
(n=163)
Students with
self-perception
of bad health
(n=110)
p-
value
Total
Burnout
(Mean ± SD)
39.61±9.63
43.91±8.15
<0.001
Overload
13.78±4.58
15.20±5.12
0.017
Neglect
11.69±4.11
13.34±4.18
0.001
Lack Of
Development
14.15±4.47
15.37±4.00
0.021
Table-3: Comparison of burnout scores between
students with self-perception of good health
and self-perception of bad health DISCUSSION The increasing prevalence of burnout and its
potentially serious consequences in medical students 17 Relationship Of Burnout with Screen Time In Undergraduate Medical Students necessitate the identification of risk factors and the
development of coping strategies to mitigate the
burnout rate. the academic performance of medical students with
excessive screen time. Despite the intentions and
dedication of students to achieve their academic
outcomes,
excessive
screen
exposure
could
potentially lead to burnout thus adversely affecting
their academic performance. In the present study, we correlated the scores of
subtypes and total burnout scores of medical
students with their screen time and students’ self-
perception of good and bad health. The burnout
scores showed a strong correlation with the screen
time of medical students. The comparison of
burnout scores did not reveal significant resultsin
terms of gender, academic year and between day
scholars and hostilites. However, students with less
monthly household income had significantly higher
burnout scores. These results agree with previous
findings that the financial status of medical students
does contribute to their stress17. Educationists are increasingly emphasizing digital
classrooms and the use of technology, but caution
should
be
exercised
in
implementing
these
approaches. Teachers must be mindful of the
potential drawbacks associated with excessive
online proctoring, synchronous and asynchronous
teaching and learning, as well as the design of
blended and hybrid programs. Maintaining a
delicate balance is crucial, as prolonged educational
screen time and online advertisements can lead to
distractions and a loss of focus for medical students. Therefore, it is imperative to provide teachers with
training on how to develop effective online courses
that mitigate these challenges. According to our results, screen time for non-
educational purposes did show a weak but
significant correlation with the neglect and lack of
development subtypes of burnout and the total
burnout score. To our knowledge, the effect of
screen time on burnout and its subtypes in medical
students has not been extensively explored. The
findings of a review of several research studies
agree with our results showing moderately high
burnout levels in students with high screen time18. DISCUSSION Our results show a significant correlation of
overload, neglect and lack of development with
screen time in students with self-perception of good
health; the results also reveal a significant
association of screen time with total burnout score
in students with self-perception of bad health.The
results of a systematic review provide support for a
positive correlation between screen time and a lower
perceived quality of life in 11 out of 15
studies.19Another study reported a significant
association between recreational screen time and
self-rated health in the adult population after
adjustment by age, socioeconomic status and
physical activity20. These studies support our
findings of a significant correlation of screen time
for non-academic purposes with total burnout score
and hint towards the association of excessive screen
time leading students to engage in online self-
diagnosis,
potentially
fostering
a
negative
perception of their health status The findings of our study highlighted the prevalence
of burnout scores along with its three subtypes
among the students with self-perception of bad
health suggesting negative influence of higher
screen time on the physical and mental health of
medical students. These findings also suggest that
strategies should be developed to explore and
minimize the underlying factors contributing
towards burnout in medical students. The findings of our study were corroborated by the
results of several studies reporting an association of
lack of well-being with burnout in medical students
with high screen time in students.21-23We propose
that medical institutions establish mechanisms to
assess the self-perception of health among medical
students to identify medical students who may have
suboptimal well-being. Potential initiatives to
implement
such
mechanisms
include
the
strengthening of existing mentoring and counselling
programs, as well as the development of new
programs focused on supporting students' stress
management, work-life balance and overall well-
being. Furthermore, the development of new
programs should be tailored to promote the overall
well-being of medical students. These initiatives
could encompass workshops or seminars on stress
management techniques, mindfulness practices,
healthy lifestyle habits, and effective coping
strategies. An important aspect of our results is that a
significant correlation of screen time for education
purposes was only found with the overload subtype
of burnout and was not found to be significantly
correlated with the other two subtypes and the total
burnout score. This finding indicates that students
who take their studies more seriously tend to use
digital tools for educational purposes. Limitations of the Study One limitation of the present study is that the effect
of burnout resulting from increased screen time of
medical students on their academic performance in
summative and formative assessments was not
analyzed. It is proposed that the effect of burnout
and screen time on the academic performance of
medical studentsshould be explored in future
studies. 8. 8. Schaufeli W, Maslach C, Marek T. Professional
burnout: Recent developments in theory and
research. 1st ed. Washington: Taylor and Francis;
1994;207-221 CONCLUSION 9. Deligkaris P, Panagopoulou E, Montgomery AJ,
Masoura E. Job burnout and cognitive functioning: A
systematic review. Work & stress. 2014;28(2):107-
23. We conclude that screen time of medical students
has a significant correlation with burnout and has
the potential to cause physical and mental health
ailments. The findings of the study inform medical
educationists to develop strategies and support
systems to reduce burnout and promote the well-
being of medical students. 10. Schaufeli WB, Desart S, De Witte H. Burnout
Assessment Tool (BAT)—development, validity,
and reliability. International journal of environmental
research and public health. 2020;17(24):9495. 11. Montero-Marín J, García-Campayo J. A newer and
broader definition of burnout: validation of the
“Burnout Clinical Subtype Questionnaire (BCSQ-
36)”. BMC Public Health. 2010; 10:302. doi:
10.1186/1471-2458-10-302 DISCUSSION This finding
necessitates the need for further research to explore In addressing burnout among medical students,
fostering collaboration with external resources like
mental health professionals and support groups is
crucial. Simultaneously, acknowledging the issue,
cultivating a culture of open communication and
destigmatizing mental health concerns within the 18 Relationship Of Burnout with Screen Time In Undergraduate Medical Students International Review of Psychiatry. 2021;33(1-2):37-
42.https://doi.org/10.1080/09540261.2020.1738064 medical community may play a significant role. Given the current trend of increased utilization of
digital learning resources among medical students,
screen time has the potential to become a significant
contributing factor to burnout. 5. Reddy SK, Yennu S, Tanco K, Anderson AE,
Guzman D, Naqvi SM, Sadaf H, Williams J, Liu DD,
Bruera E. Frequency of burnout among palliative
care physicians participating in a continuing medical
education course. Journal of pain and symptom
management. 2020;60(1):80-
6.https://doi.org/10.1016/j.jpainsymman.2020.02.013 Furthermore, the emerging trend of online classes
and
assignments
could
potentially
lead
to
distractions
and
non-educational
web
use,
consequently exacerbating burnout in medical
students. The findings of the present study are
significant for medical educationists and curriculum
planners to formulate appropriate strategies to cope
with academic burnout. 6. Stacey A, D’Eon M, Madojemu G. Medical student
stress and burnout: Before and after COVID-19. Canadian
medical
education
journal. 2020;11(6):e204.https://doi.org/10.36834%2Fcmej.7
0305 7. Fitzpatrick O, Biesma R, Conroy RM, Mc Garvey A. Prevalence and relationship between burnout and
depression in our future doctors: a cross-sectional
study in a cohort of preclinical and clinical medical
students
in
Ireland. BMJ
open. 2019;9(4):e023297.http://dx.doi.org/10.1136/bmjope
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Assistant Professor,
Department of Psychiatry,
Shifa College of Medicine,
Shifa Tameer-e-Millat University,Islamabad. Dr. Arsalan Ahmed,
Final Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Dr. Danish Iltaf Satti,
4th Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Dr. Adil Baloch,
4th Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. REFERENCES Authorship:
KI:
Conceived idea and analyzed Data
FA:
Data Analysis and Manuscript writing
IA:
Designed Study and analyzed data
AS:
Designed Study and analyzed data
NK:
Data Interpretation and Manuscript writing
AA:
Collected Analyzed Data
DIS:
Collected Analyzed Data
AB:
Collected Analyzed Data the “Burnout Clinical Subtypes Questionnaire”
(BCSQ-12). Health Qual Life Outcomes. 2011;
9:74. doi: 10.1186/1477-7525-9-74 17. Morcos G, Awan OA. Burnout in medical school: a
medical student's perspective. Academic Radiology. 2023;30(6):1223-5. doi: 10.1016/j.acra.2022.11.023 Shifa Tameer-e-Millat University,Islamabad. 18. Pandya A, Lodha P. Social connectedness, excessive
screen time during COVID-19 and mental health: a
review of current evidence. Frontiers in Human
Dynamics. 2021; 3:684137. Dr. Arsalan Ahmed,
Final Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Shifa College of Medicine, 19. Stiglic N, Viner RM. Effects of screentime on the
health and well-being of children and adolescents: a
systematic review of reviews. BMJ open. 2019;9(1):
e023191. Dr. Danish Iltaf Satti,
4th Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Dr. Adil Baloch,
4th Year Medical Student,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Authorship:
KI:
Conceived idea and analyzed Data
FA:
Data Analysis and Manuscript writing
IA:
Designed Study and analyzed data
AS:
Designed Study and analyzed data
NK:
Data Interpretation and Manuscript writing
AA:
Collected Analyzed Data
DIS:
Collected Analyzed Data
AB:
Collected Analyzed Data 20. Biaani LG, Palència L, Puig-Ribera A, Bartoll X,
Pérez K. Does adult recreational screen-time
sedentary behavior have an effect on self-perceived
health? Public Health in Practice. 2020; 1:100055. doi: 10.1016/j.puhip.2020.100055 21. Bhugra D, Molodynski A, Ventriglio A. Well-being
and
burnout
in
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students. Industrial
Psychiatry Journal. 2021 Jul;30(2):193. 22. Niedobylski S, Michta K, Wachoá K, Niedziaáek K,
àopuszaĔska U, Samardakiewicz M, Próchnicki M. Academic burnout, self-esteem, coping with stress
and gratitude among Polish medical students–a cross
sectional study. Current Problems of Psychiatry. 2022. 22. Niedobylski S, Michta K, Wachoá K, Niedziaáek K,
àopuszaĔska U, Samardakiewicz M, Próchnicki M. Academic burnout, self-esteem, coping with stress
and gratitude among Polish medical students–a cross
sectional study. Current Problems of Psychiatry. 2022. 22. Niedobylski S, Michta K, Wachoá K, Niedziaáek K,
àopuszaĔska U, Samardakiewicz M, Próchnicki M. Academic burnout, self-esteem, coping with stress
and gratitude among Polish medical students–a cross
sectional study. Current Problems of Psychiatry. 2022. The Authors: Dr. Khurram Irshad, Associate Professor, Department of Physiology, Shifa College of Dentistry, Shifa Tameer-e-Millat University,Islamabad. Dr. Fahad Azam,
Professor,
Department of Pharmacology & Therapeutics,
Shifa College of Dentistry,
Shifa Tameer-e-Millat University, Islamabad. Dr. Fahad Azam,
Professor,
Department of Pharmacology & Therapeutics,
Shifa College of Dentistry,
Shifa Tameer-e-Millat University, Islamabad. Dr. Ifra Ashraf,
Assistant Professor,
Department of Physiology,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Dr. Abida Shaheen,
Professor,
Department of Pharmacology& Therapeutics,
Shifa College of Medicine,
Shifa Tameer-e-Millat University, Islamabad. Department of Pharmacology & Therapeutics, Dr. Ifra Ashraf, Assistant Professor, Department of Physiology, Shifa College of Medicine, Shifa Tameer-e-Millat University, Islamabad. Department of Pharmacology& Therapeutics, 20
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CRLF1 wt Allele
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios CRLF1 wt Allele National Cancer Institute National Cancer Institute Qeios ID: F2SOLD · https://doi.org/10.32388/F2SOLD Source National Cancer Institute. CRLF1 wt Allele. NCI Thesaurus. Code C104951. Human CRLF1 wild-type allele is located in the vicinity of 19p12 and is approximately 14 kb
in length. This allele, which encodes cytokine receptor-like factor 1 protein, may play roles
in nervous system development and immune function. Mutations in this gene are
associated with cold-induced sweating syndrome and Crisponi syndrome. Qeios ID: F2SOLD · https://doi.org/10.32388/F2SOLD 1/1
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Triazoles and Strobilurin Mixture Affects Soil Microbial Community and Incidences of Wheat Diseases
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* Correspondence: avasilchenko@gmail.com Abstract: Pesticides are widely used in agriculture as a pest control strategy. Despite the benefits of pesticides
on crop yields, the persistence of chemical residues in soil have an unintended impact on non-targeted micro-
organisms. In this study, we evaluated the impact of the combined fungicide (difenoconazole, epoxiconazole,
and kresoxim-methyl) on fungal and bacterial communities of Phaeozem. In the fungicide-treated soil, the
Shannon index of both fungal and bacterial communities was decreased, while Chao1 index did not differ
compared to the control soil. Among bacterial taxa, the relative abundance of Athrobacter, Sphingomicrobium,
and Sphingomonas increased in fungicide-treated soil due to their ability to utilize fungicides and other toxic
compounds. Rhizopus and plant-beneficial Chaetomium were the dominant fungal genera, which increased 2-
4 times in the fungicide-treated soil, while the relative abundance of Mortierella and Talaromyces decreased. Fusarium acuminatum was the most abundant phytopathogenic fungus that causes root rot disease of wheat,
but applied fungicide treatment decreased their diversity in the soil 2 times, which is consistent on the observed
plants. Keywords: non-target action; soil microbiome; pesticide contamination; fungicide; soil quality Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 Article Combined Fungicide Treatment Affects Soil Microbial Community and Incidences of Fungal
Diseases on Wheat
Alexey S. Vasilchenko1*, Darya V. Poshvina1, Mikhail V. Semenov1,2, Vyacheslav N. Timofeev3, Artyom A. Stepanov1,
Arina N. Pervushina1 and Anastasia V. Vasilchenko 1 1Laboratory of Antimicrobial Resistance, Institute of Environmental and Agricultural
Biology (X-BIO), Tyumen State University, Tyumen, Russian Federation
2 Dokuchaev Soil Science Institute, Moscow, Russian Federation
3 Scientific Research Institute of Agriculture for Northern Trans-Ural Region - Branch
of Tyumen Scientific Centre SB RAS 1Laboratory of Antimicrobial Resistance, Institute of Environmental and Agricultural
Biology (X-BIO), Tyumen State University, Tyumen, Russian Federation
2 Dokuchaev Soil Science Institute, Moscow, Russian Federation
3 Scientific Research Institute of Agriculture for Northern Trans-Ural Region - Branch
of Tyumen Scientific Centre SB RAS 1Laboratory of Antimicrobial Resistance, Institute of Environmental and Agricultural
Biology (X-BIO), Tyumen State University, Tyumen, Russian Federation
2 Dokuchaev Soil Science Institute, Moscow, Russian Federation
3 Scientific Research Institute of Agriculture for Northern Trans-Ural Region - Branch
of Tyumen Scientific Centre SB RAS * Correspondence: avasilchenko@gmail.com Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 doi:10.20944/preprints202209.0177.v1 Triazoles were introduced into practice in the 70s of last century, since then their
market share has been increasing annually. Triazole fungicides disrupt ergosterol
biosynthesis in fungal cells by inhibiting the enzyme lanosterol 14α-demethylase, and do not
prevent spore germination and early germ-tube growth [7]. In turn, strobilurins were
administered in the German market in 1996 [8]. Strobilurins are the QoI fungicides which bind
to the quinol oxidation (Qo) site of cytochrome b and inhibit mitochondrial respiration [9]. Although triazoles and strobilurines are well known to be effective for major groups
of plant pathogenic fungi, little is known about their effects on prokaryotes. Bacteria do not
have sterols or mitochondria, so the usage of triazoles and QoI fungicides is assumed to have
no effect on them. However, it was found that triadimefon (triazole) had long-term inhibiting
effects on soil bacterial community [10]. Triticonazole and tebuconazole increased the number
of soil bacteria and stimulated the dehydrogenase activity [11], while two other sterol-
targeting fungicides (fenpropimorph and propiconazole) inhibited overall bacterial activity
[12]. Metagenomic analysis showed that some soil microbial genera belonging to
Proteobacteria and Firmicutes decreased significantly, while the abundance of Actinobacteria
increased when strobilurins are present in the soil [13]. Often, pesticides and their
decomposition products serve as carbon sources for soil microorganisms. However, there is
insufficient data on the predominance of pesticide-degrading microorganisms that affect soil
quality. In addition the use of fungicides leads to the problem of the emergence of resistant
pathogens [14]. Agricultural practices based on using combined fungicides will minimize resistant
pathogen clones. Mixing or rotating fungicides with different modes of action is considered
to be an effective approach to slowdown the development of field resistance [15]. Strobilurin
and triazole fungicides have been widely used for plant-protection for several decades. These
fungicides have a wide spectrum of activity and their combination significantly reduces
resistance of phytopathogens. In this study, we used the fungicide mixture which combined strobilurin kresoxim-
methyl and two triazoles (difenoconazole and epoxiconazole). Our primary objective was to
assess the post-treatment effects of the combined fungicide on the fungal and bacterial
communities of Phaeozem. The questions which were answered are included various aspects
of soil health changing under the treatment. Does a fungicide combining strobilurin with
triazoles affect the bacterial community? What is the functional potential of the taxa whose
abundances have been changed? Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 How has the diversity and the abundance of pathogenic
bacterial and fungal taxa changed and is there a relationship with incidences of diseases in
wheat? 2. Results 2.1. The shift of bacterial taxa under fungicide treatment 1. Introduction Every year up to 2.5 million tons of pesticides get into the environment, and then
pesticide residues are found in soils. Only a small dose of pesticides reaches target organisms
[1], while a significant part of them negatively affects beneficial organisms. Exposure of soil
to pesticides could lead to negative effects on soil microbial communities and microbial
processes, consequently reducing the soil quality [2]. The final effect on the soil microbiome
after the application of pesticide may be positive, negative, or neutral depending on soil
properties, pesticides chemical nature, and others. The negative effects of herbicides were showed for Rhizobium, Chlamydomonas,
Azotobacter, Azospirillum, and heterotrophic S-oxidizing and S-reducing bacteria [3,4]. At the
same time, insecticides carbofuran, carbosulfan, thiomethoxam, imidacloprid, chlorpyrifos
did not significantly change the abundance of Rhizobium and phosphorus solubilizing bacteria
[5]. Despite the fact that fungicides contribute to more than 35% of the global pesticide market
[6] their impact on ecosystems has received less attention compared to herbicides and
insecticides. Among fungicides, triazoles and stroiblurins account for the largest part. © 2.1. The shift of bacterial taxa under fungicide treatment Estimation of amount of dominant bacterial taxa in the soils revealed a remarkable
shift in bacterial communities under the fungicide application. The most abundant bacterial
phylum in the control soil sample was Actinobacteriota; Proteobacteria and Acidobacteria
were the second and the third dominant phyla, respectively (Figure 1). The bacterial
community of the fungicide treated soil was represented by the same dominant phyla, but doi:10.20944/preprints202209.0177.v1 their abundances changed. The application of the combined fungicide resulted in an increase
in the number of Actinobacteriota and Proteobacteria, while a decrease in Acidobacteriota and
Verrumicrobiota compared to the control soil. (Figure 1). their abundances changed. The application of the combined fungicide resulted in an increase
in the number of Actinobacteriota and Proteobacteria, while a decrease in Acidobacteriota and
Verrumicrobiota compared to the control soil. (Figure 1). Figure 1. The relative abundances of bacterial phyla (a) and families (b) in the control
and the fungicide-treated soils. Figure 1. The relative abundances of bacterial phyla (a) and families (b) in the control
and the fungicide-treated soils. Figure 1. The relative abundances of bacterial phyla (a) and families (b) in the control
and the fungicide-treated soils. At the family level, the most affected taxa were represented by Micrococcaceae,
Sphingomonadaceae and Nocardioidaceae, which abundances increased in fungicide-treated soil,
while abundances of Gaiellaceae, Xanthomonodaceae and Pyrinomonadaceae decreased (Figure 1) At the family level, the most affected taxa were represented by Micrococcaceae,
Sphingomonadaceae and Nocardioidaceae, which abundances increased in fungicide-treated soil,
while abundances of Gaiellaceae, Xanthomonodaceae and Pyrinomonadaceae decreased (Figure 1)
Five
genera,
including
Arthrobacter,
Sphingomicrobium,
Sphingomonas,
Bradyrhizobium, and a genus of uncultivated bacteria belonging to the Sphingomonadaceae,
have been shown to constitute a major group in the soil bacterial community. All of these taxa
were found to be significantly more abundant in the fungicide-treated soil compared to
untreated one. (Figure 2 a). Five
genera,
including
Arthrobacter,
Sphingomicrobium,
Sphingomonas,
Bradyrhizobium, and a genus of uncultivated bacteria belonging to the Sphingomonadaceae,
have been shown to constitute a major group in the soil bacterial community. All of these taxa
were found to be significantly more abundant in the fungicide-treated soil compared to
untreated one. (Figure 2 a). Moderately presented genera (500–1000 16S rRNA reads) (Figure 2 b) were composed
by 8 genera; they predominated in the soil treated with the fungicide. 2.1. The shift of bacterial taxa under fungicide treatment The minor taxa (< 500
16s rRNA reads) composed by 14 genera, and 5 among them have been less abundant in the
fungicide-treated soil (Figure 2 c). Twelve bacterial genera have been found to be unique to
the each of studied soils (Table S1), however, most of them are unclassified taxa. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 Figure 2. Changes in the number of bacterial taxa in the soil treated with fungicide
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented one (b), and the minor group (c). Figure 2. Changes in the number of bacterial taxa in the soil treated with fungicide
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented one (b), and the minor group (c). 2.2. Alpha diversity of bacterial community 2.3. Functional potential of bacterial community 2.3. Functional potential of bacterial community The reconstruction of metabolic pathways revealed 393 MetaCyc pathways, wherein
31 of them were differentially represented between the control and the fungicides-treated
soils. The bacterial community of the fungicides-treated soil has been characterized by the
prevalence in relative abundance of the predicted biological pathways (Figure 4). Among
them, 12 pathways are associated with biosynthesis of vitamins, carbohydrates, amino acids,
while 17 pathways are involved in degradation and assimilation processes. Figure 4. Pathways that were at differential relative abundances between the control
and the fungicides-treated soils. Corrected p-values were calculated based on Benjamini–
Hochberg FDR multiple test correction. p-value < 0.05 has been considered to be significant. The analysis was based on 16S rRNA sequencing data. Figure 4. Pathways that were at differential relative abundances between the control
and the fungicides-treated soils. Corrected p-values were calculated based on Benjamini–
Hochberg FDR multiple test correction. p-value < 0.05 has been considered to be significant. The analysis was based on 16S rRNA sequencing data. Figure 4. Pathways that were at differential relative abundances between the control
and the fungicides-treated soils. Corrected p-values were calculated based on Benjamini–
Hochberg FDR multiple test correction. p-value < 0.05 has been considered to be significant. The analysis was based on 16S rRNA sequencing data. The relative abundances of 5 pathways were significantly increased in the control soil
comparing to the fungicides-treated one. These pathways were mannan degradation, L-valine
degradation I, L-glutamate degradation VIII, biotin biosynthesis II and peptidoglycan
biosynthesis II (Figure 4). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 2.2. Alpha diversity of bacterial community It has been discovered that the Shannon index in the fungicide-treated soil turned out
to be significantly lower than those of the untreated soil (p-value < 0.05) (Figure 3 a). Chao1
index has shown no statistically significant difference between the fungicide-treated and the
control soils (p-value 0.8273) (Figure 3 b). Figure 3. The Shannon and Chao1 alpha-diversity indices of bacterial communities of
the fungicide-treated and the control soils Figure 3. The Shannon and Chao1 alpha-diversity indices of bacterial communities of
the fungicide-treated and the control soils Figure 3. The Shannon and Chao1 alpha-diversity indices of bacterial communities of
the fungicide-treated and the control soils Figure 3. The Shannon and Chao1 alpha-diversity indices of bacterial communities of
the fungicide-treated and the control soils 2.4. Shift of fungal taxa under the fungicide treatment The fungal community in the untreated soil was represented by dominant phyla
Ascomycota, Mortierellomycota and Basidiomycota (Figure 5). In the fungicide-treated soil,
the dominant phyla were Ascomycota, Mucoromycota, and Mortierellomycota (Figure 5). At
the
family
level,
Rhizopodaceae,
Chaetomiaceae,
and
Aspergillaceae
predominated in the fungicide-treated soil (Figure 5), while the abundance of Mortierellaceae,
Hypocreaceae, Trichocomacea were lower compared to the control soil. Changes in the number of fungi at the genera level were estimated by Log2Fold
values (Figure 6 a). It has been found that Rhizopus sp. was represented moderately in the
untreated soil, while in the fungicides-treated soil its abundance has increased by 8 times
(+4.22 Log2Fold) (Figure 6 b). In a similar way have increased the abundances of Chaetomium doi:10.20944/preprints202209.0177.v1 (+2.06 Log2Fold), Penicillium sp. (+0.64 Log2Fold), and Setophoma (+0.61 Log2Fold) (Figure 6
b). (+2.06 Log2Fold), Penicillium sp. (+0.64 Log2Fold), and Setophoma (+0.61 Log2Fold) (Figure 6
b). b). Fi
5 Th
l ti
b
d
f f
l
h l
( )
d f
ili
(b) i
th
t
l Figure 5. The relative abundance of fungal phyla (a) and families (b) in the control
and the fungicides-treated soils. The most abundant genera in the untreated soil were Mortierella, Talaromyces,
Fusarium, Trichoderma, Trichocladium, and Monocillium. However, in the fungicides-treated soil
the abundances of Talaromyces (-1.43 Log2Fold), Mortierella sp (-1.03 Log2Fold), Trichocladium
(-0.72 Log2Fold), Trichoderma sp. (-0.65 Log2Fold), and Monocillium sp. (-0.62 Log2Fold)
reduced by several times, compared to the intact soil (Figure 6 a). Among the dominant taxa
of the control soil, phytopathogenic Fusarium genus (subsequently was identified as Fusarium
acuminatum) has been found, while in the fungicide-treated soil its abundance was less
for2times (-0.95 Log2Fold). The moderately presented part of fungal soil community (500-1000 ITS reads) in the
control soil was composed by 5 genera . All these genera have increased their abundance in
the fungicide-treated soil (Figure 6 b). Among them, Lachnum was the most abundant genus
(+3.79 Log2Fold), while the abundance of 4 others genera has increased insignificantly (from
+ 0.40 to + 0.15 Log2Fold). The most pronounced shift has occurred with genera that are related to the minor part
(<500 ITS Reads) of the fungal community. It has been found that the abundance of 4 genera
increased in the fungicide treated soil, and 8 genera have decreased (Figure 6 c). 2.4. Shift of fungal taxa under the fungicide treatment The most
pronounced increase in the abundance was undergoing with unidentified genus (related to
Pezizaceae) (+ 4.7 Log2Fold), while genus Hypoxylon was the most reduced in their abundance
(- 5.7 Log2Fold) (Figure 6 c). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 Figure 6. Changes in the number of fungal taxa in the soil treated with fungicides
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented (b), and the minor group (c). Figure 6. Changes in the number of fungal taxa in the soil treated with fungicides
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented (b), and the minor group (c). Figure 6. Changes in the number of fungal taxa in the soil treated with fungicides
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented (b), and the minor group (c). Figure 6. Changes in the number of fungal taxa in the soil treated with fungicides
compared to the control soil. The differences in the abundance of the taxa that comprise the
dominant group (а), the moderately presented (b), and the minor group (c). Within the minor part of fungal community, the following pathogenic fungi have
been found: Septoriella hirta (-3.9 Log2Fold), Aspergillus crustosus (-1.1 Log2Fold), Alternaria
tricina (-0.15 Log2Fold), Alternaria alternata (0.28 Log2Fold) Bipolaris sorokiniana (0.08
Log2Fold). Six fungal genera were discovered to have completely disappeared following
fungicide application, while 2 fungal genera were new for the fungicide-treated soil (Table
S.1). 2.5. Alpha Diversity of fungal community The diversity of fungal community in the control Phaeozem was quite high, while the
fungicide application significantly reduced the Shannon index (p-value < 0.05) (Figure 7 a),
while the Chao 1 diversity indices were found not to different between the compared soils (p-
value 0.8273) (Figure 7 b). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 Figure 7. The Shannon and Chao1 alpha-diversity indices of fungal communities in
the fungicide-treated and the control soils. Figure 7. The Shannon and Chao1 alpha-diversity indices of fungal communities in
the fungicide-treated and the control soils. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 doi:10.20944/preprints202209.0177.v1 Analyzing of wheat at the beginning of the growing season, we have found that up to
8.25 % of growing plants which does not protected by the combined fungicide showed
symptoms of root rot. At the end of growing season, the number of affected plants increased
up to 14.52 ± 0.18 % (Figure 9). At the same time, the applied scheme for fungicide treatment
protected 100% of the plants in the tillering phase, and up to 96.8 ± 0.27 % at the end of
growing season (Figure 9). Figure 9. Manifestation of the wheat diseases in the control and the fungicide-treated groups. Data presents mean ± standard deviation (n=50). * p<0.05 (pair sample t-test). Figure 9. Manifestation of the wheat diseases in the control and the fungicide-treated groups. Data presents mean ± standard deviation (n=50). * p<0.05 (pair sample t-test). The manifestation of leaf spot disease caused by Septoria trici was found to be
insignificant. The percentages of affected leaf surface were 5.11 ± 0.50 % in the control group
and 0.21 ± 0.07 % in the fungicide-treated group. The absence of fungicide in the plant-
protection scheme resulted in the appearance of ear spot disease caused by Stagonospora
nodorum. The manifestation of symptoms in the control group was 3.03 ± 0.10%, and the use
of fungicide reduced the percentage of damaged wheat ears to 0.22 ± 0.05% (Figure 9). 2.6. Functional potential of fungal community Using the ITS sequencing data, 66 MetaCyc pathways were completely restored. Wherein, 51 were differently represented between groups (p-value < 0.02). Only 7 pathways
which are related with ubiquinol and nucleic acids biosynthesis were discovered to be more
abundant in the fungicide-treated soil (Figure 8). Figure 8. Pathways that were at differential relative abundances between control and
fungicides-treated soils. Corrected p-values were calculated based on Benjamini–Hochberg FDR
multiple test correction. p-value < 0.05 has been considered to be significant. The analysis was based on
ITS sequencing data. 2.7. Effect of soil management on fungal disease manifestation Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 doi:10.20944/preprints202209.0177.v1 features of microbial response to this treatment. Firstly, bacterial and fungal diversity has
decreased under the influence of fungicide. Besides, bacterial and fungal communities
differed in their response to the treatment. Finally, when treated with the fungicide, all
dominant taxa remained similar to those in the untreated soil, but their abundances changed. In the fungicide-treated soil, the abundance of Arthrobacter genus increased. This
bacterial genus is known for its ability to decompose strobilurins as the source of carbon [18]. The numbers of Sphingomonas and Sphingomicrobium had also increased. The member of
Sphingomonadaceae are known to have the ability to degrade a variety of aromatic compounds
[19]. Many Sphingomonads have been isolated from environments contaminated with
pesticides, herbicides, and other xenobiotics, which сan be used by bacteria as a sole carbon
source [20]. Recently, two strains of Sphingomonas spp. were isolated from wheat grain and
demonstrated the ability to degrade propiconazole [21]. Thus, at least three bacterial genera
found in the studied soils could be biodegrading strobilurins and triazoles. This may explain
the increase in their abundance in the fungicide-treated soil. In the fungicide-treated soil, there was a slight increase in the relative abundance of
Bradyrhizobium which has a high potential as remediators for fungicide polluted soil [22]. For
example, Bradyrhizobium japonicum bacteria are able to detoxify hexaconazole, colonize plant
tissues and secrete PGP bioactive molecules, even under fungicide pressure [23]. The most pronounced shifts occurred with minor taxa, some of which completely
disappeared in the fungicide-treated soil, while others appeared instead. However, most of
these taxa belong to non-classified uncultivated genera, and their environmental role is
unknown. The fungal community was affected the most by the fungicide combination. The
abundance of Mortierella sp. decreased twice; fungal species belonging to Mortierella are
known to have positive effect on the crop protection, and involved in the reduction of soil
contamination by chemical fertilizers and pesticides [24]. The abundance of Talaromyces has
also been halved, these fungiare known to be effective in preventing the incidence of
Fusarium wilt disease [25]. Rhizopus was the most abundant taxon of fungal community, and its abundance in
the fungicide-treated soil has increased significantly. Rhizopus spp. are known as soil sapro-
trophs, capable to sorption of various toxicants [26]. Since the abundance of this taxon in-
creased by more than 8 times, Rhizopus spp. probably play a more specific ecological role
which are related with fungicide application. 3. Discussion Recent studies have shown that regardless of soil type the overall effect of
difenoconazole/epoxiconazole application led to a decrease in soil microbial biomass and soil
enzymatic activity [1]. Apparently, the effect on microorganisms depends on soil type, since
difenoconazole at concentration of 5 mg/kg did not cause significant changes in
microbiological parameters of clay-loam soil [16], while in loamy-sand soil difenoconazole
concentration of 0.04 mg/kg reduced microbial biomass [17]. At the same time, there are no
available data concerning the effects of kresoxime-methyl on the soil microbial community. We found that the combined fungicide affected both bacterial and fungal
communities. In soil, bacteria and fungi depending on their number were clustered into the
three conditional groups: dominant, moderately and minor presented taxa. There were a few Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 doi:10.20944/preprints202209.0177.v1 Septoriella hirta is considered as an economically important secondary pathogen that increases
the cost of harvesting and declines the quality of the grain [31]. However, the relative
abundances of all of these fungi were insignificant (less than 100 ITS reads). Septoriella hirta is considered as an economically important secondary pathogen that increases
the cost of harvesting and declines the quality of the grain [31]. However, the relative
abundances of all of these fungi were insignificant (less than 100 ITS reads). On the contrary, Fusarium acuminatum was found to be the most abundant
phytopathogen (>1000 ITS reads) which was presented in the soil without fungicide. This
fungal species colonizes the lower stems (crowns) of bread and durum wheat and mostly
related to crown rot [32, 33]. Moreover, F. acuminatum produces toxins bowericin, fusarin C,
moniliformin [34]. In our study, the relative abundance of F. acuminatum has decreased twice
when fungicide was applied, and this correlates with twice reduction in root rot incidents. The description of functional changes in microbialcommunities under the fungicide-
treatment could be based on the assessment of their biosynthetic potential. There was an
increase in the abundance of bacterial metabolic pathways, which are associated mainly with
the conversion of various xenobiotic. The functional change in the fungal community has
shown the opposite trend. The abundance of metabolic pathways was decreased in the
fungicide-treated soil. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 Another fungal taxon which abundance was significantly increased in the fungicide-
treated soil, was Chaetomium genus. This genus is known to be plant-beneficial. For example,
several Chaetomium species are known as biocontrol agents of various phytopathogenic fungi,
such as Fusarium, Helminthosporium, Pythium, Alternaria, and Phytophthora [27-30]. The infection of common root rot of wheat is facilitated by the presence of pathogenes
on seeds and in a soil, and the intensity of damage to the root system can vary under the
influence of weather conditions (precipitation, temperature), agrotechnical measures for soil
preparation. Phytopathogens such as Septoriella hirta, Myrothecium, Alternaria were found in the
fungicide-treated soil in lower shares or were even absent compared to the control one. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 doi:10.20944/preprints202209.0177.v1 was measured according to the international standard ISO 10390. The pHKCl value was
determined potentiometrically in a 1 M KCl solution and the pHH2O value was determined in
an aqueous solution at a soil:solution ratio of 1:5 using a pH-meter Orion Star A 111 (Thermo
Scientific, US) [35]. 4.3. Total soil DNA isolation and sequencing Total soil DNA was isolated from 0.5 g of soil using the FastDNA™ Spin Kit for Soil
DNA Extraction (MP Biomedicals, USA) according to the manufacturer's protocol. DNA was
extracted from three technical replicates per sample to minimize the DNA extraction bias (9
samples of DNA were obtained in total). To assess the yield of total soil DNA , the absorbance
was measured at 230, 260, and 280 nm using NanoPhotometer N120 (Implen, USA). Quality
of total soil DNA was estimated using absorbance ratio as A260 nm/230 nm (DNA/humic acid)
and A260 nm/280 nm (DNA/protein). DNA yield was also quantified fluorometrically with
Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Both NanoPhotometer N120 and Qubit
were used according to manufacturer’s protocols. The extracted DNA was stored in a
freezer (-80 °C) until further analyses. The libraries for sequencing were created using “xGen™ Amplicon Core Kit” with
primers “xGen™ 16S Amplicon Panel v2” for bacteria identification and “xGen™ ITS1
Amplicon Panel” for fungal identification following the manufacturer’s instructions. The
amplicon sequencing was carried out using MiSeq Illumina sequencer (Ilumina Inc., San
Diego, California, USA) and a set of paired ends v2 Illumina (cluster generation and paired
ends sequencing with the power of 2 × 250 bp). 4.2. Phytosanitary control of spring wheat The development of common root rot was determined twice during the growing
season in the tillering phase and before harvesting the crop [36]. The development of aerogenic infections (foliar and blotch disease of wheat) was
monitored from the tillering phase to milky ripeness. Plant material for analysis was taken
from wheat field at three (root rot) and five (aerogenic infections) spatially distant points. The
degree of damage to the leaf surface caused by Septoria trici (foliar disease of wheat) or damage
to the wheat ear caused by Septoria nodorum was expressed in percentage using the universal
scale [37]. 4.1. Site and sampling Soils have been sampled on the territory of the experimental field of the Research
Institute of Agriculture of the Northern Trans-Urals (Coordinates: 57.094, 65.376). Geographical zone: subtaiga subzone of the Tavda province (Turin subprovince). The relief
of the territory is a gently sloping plain with pine-birch and birch grassy forests. The studied
plots were located on arable soil under spring wheat crops (Tríticum durum Desf.). The soil
type under the study was Luvic Phaeozems. Before sowing wheat, the special agrotechnical
process was applied, by first plowing in autumn of 2019 and then plowing in early spring of
2020, followed by 3-fold cultivation during the summer of 2020. Sowing of spring wheat was
done in May 2021. The applied fungicide preparation was “Terapevt-Pro”, (Zemlyakoff, Russia). Terapevt-Pro contains (gram per liter): difenoconazole – 80; kresoxim-methyl – 125;
epoxiconazole – 125. Terapevt-Pro. The fungicide was applied once during the earing phase
of wheat on 7th July 2021 at the rate of 0.7 L per ha. The control (untreated with fungicide)
group of soil was located at a distance of 20 m. Soil sampling was carried out in September 2021 after wheat harvest. Soil samples
were collected using the checkerboard sampling method. Three spatially distant plots (1 m2)
were randomly laid in each studied area. Samples were taken from 0-5 cm of the upper
humus soil layer at four points in the corners and one in the center of the plot. For each plot,
a pooled sample was prepared by mixing incremental samples. The maximum time from the
moment of sampling to their arrival at the laboratory was no more than 2 hours. The soil
samples were sieved with a mesh size of 2 mm and stored at the temperature of minus 80°C. Thus, the three bags of soil were collected at each area. The relative humidity of the soils was 13.37±1.65 %, acidity (pH) – 7.06. The texture of
the studied soils was presented by clay (28 %), silt (26 %) and sand (clay loam) (46 %). Soil pH 4.5. Functional potential analysis Using the 16S rRNA or ITS based ASV tables and the reference sequences generated
by QIIME2, a functional potential of the bacterial community was predicted using the
PICRUSt2 software (version 2.3.0) [44]. The values were converted using the logarithmic
transformation Log2. Data visualization and calculation of statistical indicators were
performed using the Phantasus web application (version 1.11.0). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2022 doi:10.20944/preprints202209.0177.v1 sequences with the reference rRNA sequences from the GTDB database using the IdTaxa
function of the DECIPHER package [40]. The phylogenetic composition of fungal communities was determined by comparing
the ITS2 nucleotide sequences with the reference sequences from the UNITE v 8.3 database
2021-05-10. When necessary, the alignment of nucleotide sequences was carried out using
NCBI nucleotide database. Alpha diversity analysis was carried out using the "estimate_richness" function of the
phyloseq v3.14 package [41], Chao1 and Shannon indices were used for calculation. The
nonparametric Kruskal-Wallis test was used to determine the influence of the factor on the
alpha diversity, The effect was considered significant at p <0.05. Visualization of the alpha
diversity indices was performed using the box-and-whisker chart from the ggplot2 v3.3.5
package [42]. Using the deseq2 package R, the Benjamini-Hochberg multiple comparison was
applied to identify significant differences in the relative diversity of microorganisms between
the compared groups [43]. The difference was considered significant at p <0.05. Dissimilarity
between groups at genus level was expressed in log2FoldChange values. 4.4. Data analysis and statistics The quality of sequenced data was estimated using FastQC v 0.11.9 software [38]. The reads were filtered and trimmed using the Trimmomatic v0.36 program: reads
were removed where average quality throughout the entire length was at least 31 (both for
forward and reverse reads). After filtering, the data was checked using the FastQC program. Further processing of the reads was performed using the package dada2 v4.0.3 [39] of
the R language. After sequence dereplication the main function of the dada2 algorithm was
used - the recovery of the amplicon sequence variant (ASV). The phylogenetic composition of
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by the author contribution or funding sections. This may include administrative and technical sup-
port, or donations in kind (e.g., materials used for experiments). Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In general, the metagenomic analysis of the microbial communities in Phaeozem
showed that fungicide treatment affected alfa diversity of both fungal and bacterial
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Russian Federation within the framework of the Federal Scientific and Technical Program for the
Development of Genetic Technologies for 2019-2027 (agreement №075-15-2021-1345, unique identifier
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GOT2 wt Allele
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Definitions
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios GOT2 wt Allele National Cancer Institute National Cancer Institute Qeios ID: ZCTCFO · https://doi.org/10.32388/ZCTCFO Source National Cancer Institute. GOT2 wt Allele. NCI Thesaurus. Code C51020. Human GOT2 wild-type allele is located within 16q21 and is approximately 27 kb in length. This allele, which encodes aspartate aminotransferase, mitochondrial protein, plays a role
in the transamination between L-aspartate and 2-oxoglutarate to form oxaloacetate and
L-glutamate. Qeios ID: ZCTCFO · https://doi.org/10.32388/ZCTCFO 1/1
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The rise of anti-vaccination legislation in two Midwestern US states: Implications for politics, policy, and society
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Filip Viskupič*, Zachary Liebl, David L. Wiltse
The rise of anti-vaccination legislation in two
Midwestern US states: Implications for politics,
policy, and society First, we mapped the
increase in anti-vaccination bills introduced in the South
Dakota and Minnesota state legislatures since the start of
the COVID-19 pandemic. Second, we discussed the potential
impacts of the growth of these legislative proposals on
politics, public health policy, and society in these two
Midwestern states. The COVID-19 public health emergency ended on May
11, 2023. Even though the immediate threat from COVID-19
diminished, the 3-year experience of the pandemic likely
had long-term consequences on matters of public health,
particularly vaccination. Scholars warned that negative
attitudes toward COVID-19 vaccines people developed during
the pandemic might “spill over” into attitudes toward other
vaccines [1]. They also noted that the experience of the past 3
years might have breathed new life into the anti-vaccine
moment [2]. It is likely that we will see a greater number of
anti-vaccination initiatives compared to the years prior to the
COVID-19 pandemic. Given the high number of anti-vaccina-
tion proposals in recent years, state legislatures are an area
where we could observe the growing opposition to vaccines. Keywords: COVID-19, anti-vaccination legislation, South
Dakota, Minnesota Keywords: COVID-19, anti-vaccination legislation, South
Dakota, Minnesota 1 Introduction This commentary had two goals. First, we mapped the
increase in anti-vaccination bills introduced in the South
Dakota and Minnesota state legislatures since the start of
the COVID-19 pandemic. Second, we discussed the potential
impacts of the growth of these legislative proposals on
politics, public health policy, and society in these two
Midwestern states. The politicization of vaccination was one of the challenges
to the management of the outbreak of COVID-19 in the
United States. Particularly after COVID-19 vaccines became
widely available to the public, vaccination became a con-
tentious issue between the Republican and Democratic
parties. While the communication from Democratic Party This work is licensed under the Creative Commons Attribution 4.0 International License. Open Health 2023; 4: 20230020 or(s), published by De Gruyter.
This work is licensed under the Creative Commons Attribution 4.0 International License. Open Access. © 2023 the author(s), published by De Gruyter.
This work is lice Filip Viskupič*, Zachary Liebl, David L. Wiltse
The rise of anti-vaccination legislation in two
Midwestern US states: Implications for politics,
policy, and society https://doi.org/10.1515/ohe-2023-0020
received September 19, 2023; accepted December 11, 2023 https://doi.org/10.1515/ohe-2023-0020
received September 19, 2023; accepted December 11, 2023 leaders was mostly in line with recommendations from
public health authorities, some Republican Party leaders,
including former President Trump, spread inaccurate infor-
mation about COVID-19 vaccination. The partisan divide
over COVID-19 vaccines and vaccination policy translated
into state-level politics. Republican lawmakers in state
legislatures nationwide proposed bills aimed at banning
COVID-19 vaccination requirements, adding barriers to
vaccinations, and providing vaccination exemptions. leaders was mostly in line with recommendations from
public health authorities, some Republican Party leaders,
including former President Trump, spread inaccurate infor-
mation about COVID-19 vaccination. The partisan divide
over COVID-19 vaccines and vaccination policy translated
into state-level politics. Republican lawmakers in state
legislatures nationwide proposed bills aimed at banning
COVID-19 vaccination requirements, adding barriers to
vaccinations, and providing vaccination exemptions. Abstract: During the COVID-19 pandemic in the United
States, vaccination became a contested issue between poli-
ticians at both national and state levels as well as among
the public, which became increasingly polarized over this
matter. Across the country, a large number of bills were
proposed in state legislatures that had the potential to
reduce vaccination rates. This short commentary mapped
the growth of anti-vaccination legislation in two Midwestern
states: South Dakota and Minnesota. We documented the
increased volume and scope of anti-vaccination bills and
discussed the implications for politics, health, and society. More specifically, it is likely that similar anti-vaccination
bills will be proposed in future legislative sessions, which
will impose further challenges on public health officials
seeking to restore the public’s trust in vaccines. p
g
p
The COVID-19 public health emergency ended on May
11, 2023. Even though the immediate threat from COVID-19
diminished, the 3-year experience of the pandemic likely
had long-term consequences on matters of public health,
particularly vaccination. Scholars warned that negative
attitudes toward COVID-19 vaccines people developed during
the pandemic might “spill over” into attitudes toward other
vaccines [1]. They also noted that the experience of the past 3
years might have breathed new life into the anti-vaccine
moment [2]. It is likely that we will see a greater number of
anti-vaccination initiatives compared to the years prior to the
COVID-19 pandemic. Given the high number of anti-vaccina-
tion proposals in recent years, state legislatures are an area
where we could observe the growing opposition to vaccines. This commentary had two goals. 3 The rise of anti-vaccination
legislation The initial search yielded 248 bills – 22 from South Dakota
and 226 from Minnesota. After excluding bills that appeared
to be unrelated to vaccination policy, we obtained 207 bills –
20 from South Dakota and 187 from Minnesota. In the end,
we classified 151 bills as anti-vaccination bills – 18 from
South Dakota and 133 from Minnesota. Intercoder reliability
was 81.2%, which is in line with similar studies [3]. Table A1
provides a complete list of all anti-vaccination bills from
South Dakota and Minnesota. We uncovered a similar pattern among the anti-vacci-
nation bills proposed in the Minnesota legislature. Between
2013 and 2020, there were 25 anti-vaccination bills pro-
posed. Since 2021, when COVID-19 vaccines became widely
available, 108 bills were proposed, more than a fourfold
increase. The scope of these proposals became increasingly
broad and went beyond COVID-19 vaccines. During the most
recent legislative session, there have been 32 bills proposed,
with only two being COVID-19 specific. One example is
House bill 680, which established a “vaccine recipient bill
of rights” with the right “to determine what is in the indivi-
dual’s own best medical interest” in regard to vaccination. Despite the differences in the political landscapes of
the two states (the South Dakota legislature has half the
members of the Minnesota legislature and South Dakota’s
legislative session is no longer than 40 days), there was a
very similar pattern in the proposed anti-vaccination leg-
islation. First, none of the proposed bills became law. In
South Dakota, where the Republican Party holds a super-
majority in both the House and Senate, 83.3% of the pro-
posed anti-vaccination bills failed to pass the chamber of
origin, and the rest failed to win support in the other
chamber. In Minnesota, where the two parties split the
control of House and Senate during the period we exam-
ined (the Democratic Party currently has a slight majority
in both chambers), 98% of the proposed anti-vaccination
bills were defeated in committee and only 2% came up for
vote in the chamber (pending bills in the current session
excluded). Second, anti-vaccination bills were proposed
overwhelmingly by Republican lawmakers – 94.4% of anti-
vaccination bills in South Dakota and 96.2% of anti-vaccina-
tion bills in Minnesota had a primary sponsor from the
Republican Party. 2 “Animal” OR “AIDS” OR “HIV” OR “Vision” OR “Abortion” OR
“Mammogram” OR “Opioid” OR “Veterinarian” OR “Tribal”). Next, we excluded those bills that did not appear to be related
to vaccination policy. Afterward, the three authors indepen-
dently coded all bills as either anti-vaccination or not anti-
vaccination. Following the extant literature, we defined anti-
vaccination bills as “having the potential to decrease vaccina-
tion rates” [3,4]. Figure A1 visualizes how the anti-vaccination
bills were identified. the spread of the virus. In South Dakota, there were only
three anti-vaccination bills proposed between 2013 and
2020. Since 2021, when COVID-19 vaccinations became a
subject of a heated political debate, there were 15 bills
proposed in total, a fivefold increase. Eight anti-vaccina-
tion bills were proposed in the 2022 legislative session, and
five in the 2023 session. Over time, the proposed anti-vaccination bills in South
Dakota became broader in scope and targeted a wider range
of vaccinations. In 2022, six of the eight proposed bills were
specific to COVID-19 vaccination, generally seeking to pro-
hibit vaccine mandates for COVID-19. However, in 2023, four
of the five proposed anti-vaccination bills targeted vaccina-
tion more broadly, including House Joint Resolution 5003 – a
proposed constitutional amendment that would grant citi-
zens the right to refuse any vaccine or medical treatment. The most sweeping of these proposals was Senate bill 130,
which sought to dilute the religious exemption from pro-
viding evidence of doctrinal teaching of a parent’s religion
to a “sincerely held religious or philosophical belief” held by
an individual parent or guardian. 3 The rise of anti-vaccination
legislation Democratic Party lawmakers rarely served
as co-sponsors – only 11.1% of anti-vaccination bills in South
Dakota and 13.5% of anti-vaccination bills in Minnesota were
co-sponsored by a Democrat. Overall, we found an increase in anti-vaccination bills
introduced since the start of the COVID-19 pandemic. There
was a clear uptick in legislative activity on weakening well-
established vaccination mandates for diseases beyond COVID-
19. While COVID-19 vaccination was likely the catalyst for this
flurry of legislative activity, the reach of the proposed legisla-
tion was far beyond COVID-19 vaccination policy into a policy
sphere where we have traditionally seen bipartisan con-
sensus. The remainder of this commentary discussed the
broader implications of these developments. 2 Methodology * Corresponding author: Filip Viskupič, School of American and Global
Studies, South Dakota State University, School of American & Global
Studies-Box 2212, Brookings, South Dakota, 57006, USA,
e-mail: filip.viskupic@sdstate.edu, tel: +1 605-688-6360
Zachary Liebl: School of American and Global Studies, South Dakota
State University, Brookings, South Dakota, USA
David L. Wiltse: School of American and Global Studies, South Dakota
State University, Brookings, South Dakota, USA We utilized the LegiScan database to search for bills intro-
duced in the South Dakota and Minnesota state legislatures
during the 2013–2023 period. We used the following keyword
combination to search for bills: (“Vaccine” OR “Vaccination”
OR “Immunization”) NOT (“Naloxone” OR “Omnibus” OR 2
Filip Viskupič et al. 6 Implications for society Politics represents one area where we have already seen a
significant impact. During the COVID-19 pandemic, anti-vac-
cination attitudes, which used to occupy the fringes, moved
into mainstream politics. Vaccination policy became one of
the main political cleavages between the Republican and
Democratic parties. Politics represents one area where we have already seen a
significant impact. During the COVID-19 pandemic, anti-vac-
cination attitudes, which used to occupy the fringes, moved
into mainstream politics. Vaccination policy became one of
the main political cleavages between the Republican and
Democratic parties. The increase in anti-vaccination legislative proposals could
negatively affect people’s confidence in vaccines. Given the
fast pace of COVID-19 vaccine development and the per-
ceived novelty of the mRNA technology, an understandable
degree of concern already existed regarding their safety
and effectiveness. The anti-vaccination proposals were
covered extensively by the state and local media in both
states [6,7], which could undermine people’s trust in vac-
cines, science, public health authorities, and healthcare
workers. Scholars already documented a growth in anti-
vaccine discourse on the social network X (formerly known
as Twitter), targeting not only COVID-19 but also influenza
vaccines [8]. Such developments are cause for concern, as
research linked low trust in government and low trust in
medical doctors to vaccine hesitancy [9]. Given the current political climate, politicians particu-
larly at the local and state levels might now have more
incentive to continue proposing laws that restrict access
to vaccination or provide vaccination exemptions. As vac-
cination policy is becoming a major issue, lawmakers have
an incentive to introduce legislative proposals even when
they know that they have little chance of success. Such pro-
posals might increasingly become part of a strategy to draw
attention or raise funds. For example, Dr. Scott Jensen, a
former Minnesota state senator, managed to secure the
Republican Party nomination for the 2022 gubernatorial
election after his anti-vaccination rhetoric brought him to
national prominence [5]. Since the start of the COVID-19 pandemic, diphtheria,
tetanus, pertussis (DTaP), polio, and measles, mumps, and
rubella (MMR) vaccination rates among kindergarten-aged
children have declined in South Dakota in the recent years. In addition, Minnesota recorded 22 cases of measles in 2022,
which was the third-highest annual figure since 2000 [10]. The growing number of increasingly broader anti-vaccina-
tion legislative proposals could likely lead to a further
decline in vaccination rates among vulnerable populations. 6 Implications for society It is likely that similar anti-vaccination bills will be pro-
posed in future legislative sessions beyond South Dakota
and Minnesota. Anti-vaccination legislature might over
time become routinized. Given the overlap between anti-
vaccination and general anti-science attitudes, there is a
possibility that bills targeting other domains of health and
health policy will be proposed in the near future. 4 Implications for politics Although none of the proposed anti-vaccination measures
has been enacted in law, the mere presence of a large
number of such proposals already affected both states. Third, the majority of anti-vaccination bills were pro-
posed after COVID-19 vaccines became widely available to
the public and vaccination became the main policy to stop The rise of anti-vaccination legislation and the implications
3 3 5 Implications for public health
officials and healthcare workers This brief commentary documented the rise of anti-vacci-
nation legislation in South Dakota and Minnesota since the
start of the COVID-19 pandemic. Anti-vaccination senti-
ment has always been present in society; however, it
becomes particularly dangerous when it becomes part of
mainstream politics. COVID-19 vaccination quickly became
a contested issue between Republican and Democratic poli-
ticians, and we saw bills against mandatory COVID-19 vacci-
nation and expanding exemptions. The legislative proposals
became broader in scope and targeted multiple vaccines. We discussed the implications of the rise of anti-vaccination
legislation for society, public health officials, and politics. Given the momentum that anti-vaccination activists and
ideas gained in our study, the “anti-vaccination moment”
is likely to remain in the coming future. The growth of anti-vaccination legislation during COVID-19
added to the challenges facing public health officials and
healthcare providers. Some public health officials and
healthcare providers already faced pressure and were tar-
gets of hate speech and online campaigns due to their
involvement in promoting COVID-19 vaccination. The pro-
position of such anti-vaccination legislation created addi-
tional challenges in communicating to the public the ben-
efits of vaccination. The growth in increasingly broader anti-vaccination
proposals creates challenges for public health officials as
they prepare for future health emergencies. The creation,
implementation, and evaluation of strategies to increase
vaccination confidence will be challenging given the shifting
legislative landscape. Reinforcing trust in the medical com-
munity, which was undermined due to the spread of mis-
information during the COVID-19 pandemic, will be even
more challenging in the context of the growing volume of
anti-vaccination legislation. Funding information: The authors state no funding involved. Author contributions: FV – conceptualization, methodology,
formal analysis, data curation, writing – original draft, writing
– review and editing, supervision. ZL – methodology, formal 4
Filip Viskupič et al. 4 analysis, investigation, data curation, writing – original draft,
writing – review and editing; DW – formal analysis, data
curation, writing – original draft, writing – review and editing. [2]
Larson HJ, Gakidou E, Murray CJL. The vaccine-hesitant moment. N
Engl J Med. 2022;387(1):58–65. [3]
Estep K, Muse A, Sweeney S, Goldstein ND. Partisan polarization of
childhood vaccination policies, 1995-2020. Am J Public Health. 2022;112(10):1471–9. Conflict of interest: The authors state no conflict of
interest. [4]
Goldstein ND, Suder JS, Purtle J. Trends and characteristics of
proposed and enacted state legislation on childhood vaccination
exemption, 2011-2017. Am J Public Health. 2019;109(1):102–7. Ethical approval: The conducted research is not related to
either human or animal use. Ethical approval: The conducted research is not related to
either human or animal use. [5]
News C. Vaccine doubts fuel Dr. Scott Jensen’s Rise In Minnesota
Governor Race. CBS News Minnesota. Vol. 2021; 2021 October. [6]
Haiar J. Anti-vaccine bill fails in South Dakota Senate committee. The Brookings Register; February 16, 2023. [7]
Mercer BSD. House rejects COVID vaccine exemption. KELO-TV;
February 16, 2023. [6]
Haiar J. Anti-vaccine bill fails in South Dakota Senate committee. The Brookings Register; February 16, 2023. Data availability statement: All data generated or ana-
lyzed during this study are included in this published
article and its supplementary information file. [7]
Mercer BSD. House rejects COVID vaccine exemption. KELO-TV;
February 16, 2023. [7]
Mercer BSD. House rejects COVID vaccine exemption. KELO-TV;
February 16, 2023. [8]
Ng QX, Lee DYX, Ng CX, Yau CE, Lim YL, Liew TM. Examining the
negative sentiments related to influenza vaccination from 2017 to
2022: An unsupervised deep learning analysis of 261,613 twitter
posts. Vaccines-Basel. 2023;11(6):1018. [8]
Ng QX, Lee DYX, Ng CX, Yau CE, Lim YL, Liew TM. Examining the
negative sentiments related to influenza vaccination from 2017 to
2022: An unsupervised deep learning analysis of 261,613 twitter
posts. Vaccines-Basel. 2023;11(6):1018. [9]
Viskupic F, Wiltse DL, Meyer BA. Trust in physicians and trust in
government predict COVID-19 vaccine uptake. Soc Sci Quart. 2022;103(3):509–20. [9]
Viskupic F, Wiltse DL, Meyer BA. Trust in physicians and trust in
government predict COVID-19 vaccine uptake. Soc Sci Quart. 2022;103(3):509–20. [10]
Olson J. Minnesota snuffed measles clusters in 2022, but risks
remain. StarTribune; January 13, 2023. [1]
Motta M. Is partisan conflict over COVID-19 vaccination eroding
support for childhood vaccine mandates? NPJ Vaccines. 2023;8(1):5. The rise of anti-vaccination legislation and the implications
5 References [10]
Olson J. Minnesota snuffed measles clusters in 2022, but risks
remain. StarTribune; January 13, 2023. [1]
Motta M. Is partisan conflict over COVID-19 vaccination eroding
support for childhood vaccine mandates? NPJ Vaccines. 2023;8(1):5. [10]
Olson J. Minnesota snuffed measles clusters in 2022, but risks
remain. StarTribune; January 13, 2023. The rise of anti-vaccination legislation and the implications
5 Appendix
All bills identified from
LegiScan (n = 248)
Bills screened
(n = 248)
Bills excluded for not being
related to vaccination policy
(n = 41)
Bills coded
(n = 207)
Bills not coded as anti-
vaccination bills
(n = 56)
Anti-vaccination bills
included in the study
(n = 151)
Figure A1: Flow diagram of the identification of anti-vaccination bills. Appendix Appendix All bills identified from
LegiScan (n = 248) Bills excluded for not being
related to vaccination policy
(n = 41) Bills not coded as anti-
vaccination bills
(n = 56) Anti-vaccination bills
included in the study
(n = 151) Figure A1: Flow diagram of the identification of anti-vaccination bills. 6
Filip Viskupič et al. (Continued) Appendix 6 Table A1: List of anti-vaccination bills, South Dakota and Minnesota, 2013–2023 Table A1: List of anti-vaccination bills, South Dakota and Minnesota, 2013–2023 Minnesota
2023
HF 2720
Employers prohibited from requiring or incentivizing public display of medical information, and civil and criminal penalties
imposed
[Text]
SF 2638
Commissioner of health prohibition from modifying immunization requirements for enrollment in an elementary or secondary
school or child care facility to require immunizations against COVID-19
[Text]
HF 2540
Commissioner of health prohibited from modifying immunization requirements for enrollment in elementary or secondary
school or child care facility to require immunizations against COVID-19
[Text]
SF 923
Government vaccine mandates enforcement prohibition; proof of natural antibodies as an alternative to vaccination allowance
[Text]
HF 1896
Government vaccine mandate enforcement prohibited, and proof of presence of natural antibodies allowed as alternative to
vaccination
[Text]
SF 1106
Consent for vaccination requirement
[Text]
HF 1860
Consent for vaccination required, consent coercion and discrimination prohibited, and criminal penalties imposed
[Text]
SF 2278
Certain health care providers requirement to report adverse vaccination events and disclose certain information
[Text]
SF 1105
Health care providers administering vaccines requirement to disclose certain information to patients
[Text]
HF 1879
Health care providers administering vaccines required to disclose information to patients, reports of adverse reactions to
vaccines required, and content of an informed consent form specified
[Text]
SF 2277
Vaccination consent requirement
[Text]
SF 2279
Vaccination consent addition to the Human Rights Act
[Text]
SF 204
Vaccine requirements establishment prohibition
[Text]
HF 1226
Establishment of vaccine requirements prohibited, and exceptions provided
[Text]
HF 1380
Establishment of vaccine requirements prohibited, and exceptions provided
[Text]
SF 3263
William Shegstad Healthcare Advocates Act establishment
[Text]
HF 3271
William Shegstad Healthcare Advocates Act established, and civil penalties provided
[Text]
SF 488
Vaccine recipient bill of rights establishment
[Text]
HF 680
Vaccine recipient bill of rights established
[Text]
SF 2384
Commissioner of human services rescinding public assistance benefits due to vaccination status prohibition
[Text]
HF 2545
Commissioner of human services prohibited from rescinding public assistance benefits due to vaccination status
[Text]
SF 938
Local enforcement of federal vaccine passports prohibition; civil cause of action establishment
[Text]
HF 1905
Local enforcement of federal vaccine passports prohibited, and civil cause of action created
[Text]
SF 576
Parent or legal guardian requirement to be present for vaccine administration to a minor
[Text]
HF 885
Parent or legal guardian required to be present for vaccine administration to a minor
[Text]
SF 650
State agencies and local units of government prohibition from offering vaccine incentives
[Text]
HF 954
State agencies and local units of government prohibited from offering vaccine incentives
[Text]
SF 811
Higher education immunizations exemption expansion
[Text]
HF 1953
Exceptions from immunizations expanded
[Text]
SF 1076
Limits establishment for contact tracing, digital contact tracing, immunizations, communicable disease testing, and the
required disclosure of certain information
[Text]
HF 704
Contact tracing, digital contract tracing, immunizations, communicable disease testing, and required disclosure of information
limits established; destruction of data required, mandatory digital contact tracing by employers prohibited, and civil penalties
provided
[Text]
SF 937
Employers requiring or incentivizing public safety of medical information prohibition; civil and criminal penalties imposition
[Text]
2021–2022
HF 2530
Vaccine passport, vaccine pass, and vaccine credentials for immunization status requirement prohibited
[Text]
SF 2610
Vaccine credentials for immunization status prohibition
[Text]
HF 4046
Discrimination based on vaccine status or immunity passport prohibited
[Text]
SF 2430
Vaccine passports for COVID-19 immunization status requirement prohibition
[Text]
SF 2474
Vaccine passports for COVID-19 immunization status requirement prohibition
[Text]
SF 2475
Vaccine passports for COVID-19 immunization status requirement prohibition
[Text]
HF 2511
COVID-19; immunization status vaccine passport requirement prohibited
[Text]
SF 3022
Resolution for the State of Minnesota to protect its citizens against unconstitutional and medically irresponsible COVID-19
vaccine mandates
[Text]
HF 2348
COVID-19; a resolution protecting against unconstitutional and medically irresponsible vaccine mandates
[Text]
HF 2899
COVID-19 and influenza vaccine requirements prohibited
[Text]
SF 1172
Disclosure of vaccine ingredients requirement and appropriation
[Text]
HF 1194
Vaccine ingredient disclosure required, and money appropriated
[Text]
(Continued) (Continued) The rise of anti-vaccination legislation and the implications
7 8 Table A1: Continued
Minnesota
2023
SF 3061
Mandated vaccinations expansion
[Text]
SF 1589
Communicable diseases contact tracing, digital contact tracing, immunizations, and required disclosures limits establishment;
data destruction requirement; employer mandatory digital contact tracing prohibition; civil penalties
[Text]
HF 2347
Vaccine recipient bill of rights established
[Text]
SF 3021
Vaccine recipient bill of rights establishment
[Text]
SF 4581
Immunizations exceptions expansion
[Text]
SF 1249
A resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 1330
A resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 667
Employers prohibited from disciplining or discharging employees or applicants for immunization refusal, and civil action
remedies provided
[Text]
SF 2431
Contact tracing, digital contact tracing, immunizations, communicable disease testing, and certain information disclosure
requirements limitation establishment; destruction of certain data requirement; mandatory digital contact tracing by
employers prohibition
[Text]
HF 1583
Contact tracing, digital contact tracing, immunizations, communicable disease testing, and information disclosure limits
established; data destruction required; mandatory employer digital contact tracing prohibited; and civil penalties provided
[Text]
HF 261
COVID-19; vaccine availability provided for school employees
[Text]
HF 2691
Local enforcement of federal vaccine passports prohibited, and civil cause of action created
[Text]
HF 2685
Parent or legal guardian required to be present for vaccine administration to a minor
[Text]
SF 2694
Parent or legal guardian presence requirement for vaccine administration to a minor
[Text]
HF 533
Employers prohibited from disciplining or discharging employees for immunization refusal, and civil action remedies provided
[Text]
SF 1264
Employee immunization refusal employer discipline or discharge prohibition
[Text]
HF 4349
COVID-19; vaccine mandate establishment prohibited
[Text]
SF 2559
COVID-19 public health disaster response establishment; peacetime emergency declared in Executive Order No. 20-01
termination
[Text]
HF 2640
COVID-19; public health disaster response established, and peacetime emergency declared in Executive Order No. Table A1: Continued
Minnesota
2023
SF 2820
Government vaccine mandates prohibition; proof of presence of natural antibodies as an alternative to vaccination
establishment
[Text]
HF 2694
Government vaccine mandate enforcement prohibited, and proof of presence of natural antibodies allowed as an alternative
to vaccination
[Text]
SF 2394
Consent for vaccination requirement; consent coercion and discrimination prohibition; criminal penalties imposition
authorization
[Text]
HF 1245
Vaccination consent required, consent coercion and discrimination prohibited, and criminal penalties imposed
[Text]
HF 2627
Health care providers required to report adverse vaccination events and disclose information
[Text]
HF 2159
Vaccination consent required, consent coercion and discrimination prohibited, and criminal penalties imposed
[Text]
SF 2612
Consent for vaccination requirements
[Text]
SF 2607
Health care provider vaccine disclosure requirements
[Text]
HF 26
Health care providers administering vaccines required to disclose information to patients, adverse reactions to vaccines
reports required, and informed consent form content specified
[Text]
HF 3711
Health care providers administering vaccines required to disclose information to patients, adverse reactions to vaccines
reports required, and content of an informed consent form specified
[Text]
HF 452
Statutory immunization requirement exemption provided for sincerely held religious belief
[Text]
SF 292
Statutory immunization requirements exemption for religious beliefs
[Text]
SF 2613
Consent of vaccination requirement
[Text]
HF 1243
Vaccination consent required
[Text]
HF 1244
Human Rights Act vaccination consent added
[Text]
SF 2611
Human Rights Act vaccination status discrimination addition
[Text]
HF 3214
Vaccine requirement establishment prohibited, and exception provided
[Text]
SF 4489
Establishment of vaccine requirements prohibition
[Text]
HF 3158
COVID-19; vaccine requirement prohibited, and exceptions provided
[Text]
HF 4093
Vaccination of minor consent of a parent or guardian required
[Text]
SF 4332
William Shegstad Healthcare Advocates Act
[Text]
HF 4723
William Shegstad Healthcare Advocates Act established, and civil penalties provided
[Text]
HF 2901
Mandatory immunization damage cause of action established
[Text]
SF 3550
Mandatory immunization damages cause for action establishment
[Text]
HF 4525
Mandatory immunization damages cause of action established
[Text]
HF 3517
COVID-19; state funding prohibited for public and private entities enforcing vaccine mandate or vaccine passport
[Text]
HF 2760
COVID-19; vaccine mandate injuries cause of action provided
[Text]
HF 2759
COVID-19; employer liability cause of action provided for injuries caused by vaccines
[Text]
HF 2843
COVID-19; unemployment insurance benefits ensured for individual terminated for not adhering to vaccine mandate, and
COVID-19 proof of recovery allowed as vaccination substitution
[Text]
SF 2424
Employment discrimination based on vaccination status prohibition
[Text]
SF 2737
Employment discrimination based on vaccination status prohibition
[Text]
HF 3423
Discrimination based on vaccination status prohibited
[Text]
HF 2541
Employment discrimination based on vaccination status prohibited
[Text]
SF 3475
Discrimination based on an individual's vaccination status prohibition
[Text]
HF 3820
Human rights; discrimination based on vaccination status prohibited
[Text]
HF 3841
Natural immunity exemption provided in lieu of employer vaccination or testing requirements
[Text]
SF 3491
Unemployment insurance benefits for an individual terminated for not adhering to the vaccination mandate authorization;
proof of recovery from COVID-19 as a substitution for required vaccination establishment
[Text]
HF 2897
Health freedom sanctuary established, governor's peacetime emergency powers limited, and mandatory vaccinations and
school district mask requirements prohibited
[Text]
SF 2693
Health freedom sanctuary establishment, governor's peacetime emergency powers limitations, and mandatory vaccinations
and school district mask requirements prohibition
[Text]
SF 3035
Health freedom sanctuary establishment, governor's peacetime emergency powers limitations, and mandatory vaccinations
and school district mask requirements prohibition
[Text]
SF 4047
COVID-19; employee injured by vaccination cause of action provided
[Text]
HF 3276
Immunization requirement exemption modified for children in child care facilities and students in school and postsecondary
education institution
[Text] Table A1: Continued Minnesota (Continued) 8
Filip Viskupič et al. 20-01
terminated
[Text]
HF 3736
State agencies and local units of government prohibited from offering vaccine incentives
[Text]
HF 2801
Medical information public display required or incentivized by employers prohibited, and civil and criminal penalties imposed
[Text]
SF 3666
Right establishment for a patient or resident to choose to have support person while receiving care or services
[Text]
2019–2020
SF 2911
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 2862
Vaccines; a resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects
[Text]
SF 3109
Immunization refusal employers discipline or discharge of employee or applicant prohibition
[Text]
HF 2825
Vaccines; a resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that
result in adverse side effects from vaccines
[Text]
SF 3814
Immunization requirements for sincerely held religious beliefs exemption authorization
[Text]
SF 3110
Vaccine minimum safety standards requirement for any vaccine required to enroll or remain enrolled in an elementary or
secondary school
[Text]
HF 4208
Statutory immunization requirement exemption provided for sincerely held religious belief
[Text]
HF 4348
Vaccine minimum safety standards provided for any vaccine required to enroll or remain enrolled in elementary and
secondary school
[Text]
HF 4287
Employers prohibited from discipline or discharge for employee or applicant immunization refusal, and civil action remedies
provided
[Text]
HF 41
Vaccination consent required, consent coercion and discrimination prohibited, and criminal penalties imposed
[Text]
HF 40
Vaccination consent required
[Text]
HF 42
Human Rights Act; vaccination consent added
[Text]
HF 999
Employers prohibited from discipline or discharge for employee or applicant immunization refusal, and civil action remedies
provided
[Text]
SF 1916
Employer discipline or discharge prohibition for employee or applicant refusal to immunize
[Text]
(Continued) Table A1: Continued Minnesota 2023 The rise of anti-vaccination legislation and the implications
9 The rise of anti-vaccination legislation and the implications
9 Table A1: Continued
Minnesota
2023
SF 2781
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
SF 2831
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 3728
Employers prohibited from disciplining or discharging employees for immunization refusal, and civil action remedies provided
[Text]
2017–2018
SF 143
Vaccination and immunization exemption procedures modification
[Text]
HF 2005
Health care providers administering vaccines required to disclose information to parents, reports of adverse reactions to
vaccines required, and informed consent form content specified
[Text]
SF 2660
Mandatory influenza vaccine requirements employee exemptions establishment; employee misconduct definition
modification
[Text]
HF 96
Immunization exemption procedures modified
[Text]
2015–2016
SF 1870
Health care providers vaccination reporting and disclosure requirements
[Text]
HF 1978
Health care providers required to report adverse vaccination events and disclose information
[Text]
2013–2014
HF 2170
Immunization exemption procedures modified
[Text]
South Dakota
2023
SF 2544 Immunization exemption procedures modification
[Text]
HB 1216 Establish and revise requirements related to visitation policies and procedures for certain facilities
[Text]
SB 125
Prohibit the imposition of additional immunization requirements on children
[Text]
HJR5003 Proposing and submitting to the voters at the next general election a new section to Article VI of the Constitution of the State of South
Dakota, relating to the right of a person to reject certain medical procedures
[Text]
HB 1235 Provide a conscience exemption from a COVID-19 vaccination
[Text]
SB 130
Provide for philosophical exceptions to required vaccinations
[Text]
2022
SB 211
Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1262 Prohibit discrimination based on a person's vaccination status or possession of an immunity passport and to declare an emergency[Text]
HB 1258 Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1212 Prohibit certain activities related to a person's COVID-19 vaccination status and provide a penalty therefor
[Text]
HB 1256 Establish employees' exemptions from employer-imposed COVID-19 vaccine requirements
[Text]
HB 1008 Provide a cause of action for certain employees that are required to receive a vaccination as a condition of employment and to declare
an emergency
[Text]
HB 1224 Extend unemployment insurance benefits to individuals who are unemployed because of their refusal to obtain COVID-19 vaccination[Text]
HB 1211 Prohibit the enforcement of contracts limiting competition on certain matters of conscience
[Text]
2021
HB 1097 Provide for philosophical exceptions to required vaccinations
[Text]
HB 1159 Prohibit interference with the right to bodily integrity in contagious disease control
[Text]
2020
HB 1235 Revise provisions regarding immunizations
[Text]
2016
SB 108
Require notice if certain immunizations contain more than trace amounts of mercury
[Text]
2015
HB 1059 Allow authorized entities to access immunization information in certain circumstances
[Text]
2013
SB 98
Revise certain statutory immunization exemption requirements
[Text] Table A1: Continued
Minnesota
2023
SF 2781
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
SF 2831
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 3728
Employers prohibited from disciplining or discharging employees for immunization refusal, and civil action remedies provided
[Text]
2017–2018
SF 143
Vaccination and immunization exemption procedures modification
[Text]
HF 2005
Health care providers administering vaccines required to disclose information to parents, reports of adverse reactions to
vaccines required, and informed consent form content specified
[Text]
SF 2660
Mandatory influenza vaccine requirements employee exemptions establishment; employee misconduct definition
modification
[Text]
HF 96
Immunization exemption procedures modified
[Text]
2015–2016
SF 1870
Health care providers vaccination reporting and disclosure requirements
[Text]
HF 1978
Health care providers required to report adverse vaccination events and disclose information
[Text]
2013–2014
HF 2170
Immunization exemption procedures modified
[Text]
South Dakota
2023
SF 2544 Immunization exemption procedures modification
[Text]
HB 1216 Establish and revise requirements related to visitation policies and procedures for certain facilities
[Text]
SB 125
Prohibit the imposition of additional immunization requirements on children
[Text]
HJR5003 Proposing and submitting to the voters at the next general election a new section to Article VI of the Constitution of the State of South
Dakota, relating to the right of a person to reject certain medical procedures
[Text]
HB 1235 Provide a conscience exemption from a COVID-19 vaccination
[Text]
SB 130
Provide for philosophical exceptions to required vaccinations
[Text]
2022
SB 211
Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1262 Prohibit discrimination based on a person's vaccination status or possession of an immunity passport and to declare an emergency[Text]
HB 1258 Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1212 Prohibit certain activities related to a person's COVID-19 vaccination status and provide a penalty therefor
[Text]
HB 1256 Establish employees' exemptions from employer-imposed COVID-19 vaccine requirements
[Text]
HB 1008 Provide a cause of action for certain employees that are required to receive a vaccination as a condition of employment and to declare
an emergency
[Text]
HB 1224 Extend unemployment insurance benefits to individuals who are unemployed because of their refusal to obtain COVID-19 vaccination[Text]
HB 1211 Prohibit the enforcement of contracts limiting competition on certain matters of conscience
[Text]
2021
HB 1097 Provide for philosophical exceptions to required vaccinations
[Text]
HB 1159 Prohibit interference with the right to bodily integrity in contagious disease control
[Text]
2020
HB 1235 Revise provisions regarding immunizations
[Text]
2016
SB 108
Require notice if certain immunizations contain more than trace amounts of mercury
[Text]
2015
HB 1059 Allow authorized entities to access immunization information in certain circumstances
[Text]
2013
SB 98
Revise certain statutory immunization exemption requirements
[Text] Table A1: Continued
Minnesota
2023
SF 2781
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
SF 2831
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 3728
Employers prohibited from disciplining or discharging employees for immunization refusal, and civil action remedies provided
[Text]
2017–2018
SF 143
Vaccination and immunization exemption procedures modification
[Text]
HF 2005
Health care providers administering vaccines required to disclose information to parents, reports of adverse reactions to
vaccines required, and informed consent form content specified
[Text]
SF 2660
Mandatory influenza vaccine requirements employee exemptions establishment; employee misconduct definition
modification
[Text]
HF 96
Immunization exemption procedures modified
[Text]
2015–2016
SF 1870
Health care providers vaccination reporting and disclosure requirements
[Text]
HF 1978
Health care providers required to report adverse vaccination events and disclose information
[Text]
2013–2014
HF 2170
Immunization exemption procedures modified
[Text]
South Dakota
2023 Table A1: Continued
Minnesota
2023
SF 2781
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
SF 2831
Resolution memorializing the President and Congress to hold vaccine manufacturers liable for design defects that result in
adverse side effects from vaccines
[Text]
HF 3728
Employers prohibited from disciplining or discharging employees for immunization refusal, and civil action remedies provided
[Text]
2017–2018
SF 143
Vaccination and immunization exemption procedures modification
[Text]
HF 2005
Health care providers administering vaccines required to disclose information to parents, reports of adverse reactions to
vaccines required, and informed consent form content specified
[Text]
SF 2660
Mandatory influenza vaccine requirements employee exemptions establishment; employee misconduct definition
modification
[Text]
HF 96
Immunization exemption procedures modified
[Text]
2015–2016
SF 1870
Health care providers vaccination reporting and disclosure requirements
[Text]
HF 1978
Health care providers required to report adverse vaccination events and disclose information
[Text]
2013–2014
HF 2170
Immunization exemption procedures modified
[Text]
South Dakota
2023
SF 2544 Immunization exemption procedures modification
[Text]
HB 1216 Establish and revise requirements related to visitation policies and procedures for certain facilities
[Text]
SB 125
Prohibit the imposition of additional immunization requirements on children
[Text]
HJR5003 Proposing and submitting to the voters at the next general election a new section to Article VI of the Constitution of the State of South
Dakota, relating to the right of a person to reject certain medical procedures
[Text]
HB 1235 Provide a conscience exemption from a COVID-19 vaccination
[Text]
SB 130
Provide for philosophical exceptions to required vaccinations
[Text]
2022
SB 211
Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1262 Prohibit discrimination based on a person's vaccination status or possession of an immunity passport and to declare an emergency[Text]
HB 1258 Protect an individual's conscience from entities requiring the COVID-19 vaccine
[Text]
HB 1212 Prohibit certain activities related to a person's COVID-19 vaccination status and provide a penalty therefor
[Text]
HB 1256 Establish employees' exemptions from employer-imposed COVID-19 vaccine requirements
[Text]
HB 1008 Provide a cause of action for certain employees that are required to receive a vaccination as a condition of employment and to declare
an emergency
[Text]
HB 1224 Extend unemployment insurance benefits to individuals who are unemployed because of their refusal to obtain COVID-19 vaccination[Text]
HB 1211 Prohibit the enforcement of contracts limiting competition on certain matters of conscience
[Text]
2021
HB 1097 Provide for philosophical exceptions to required vaccinations
[Text]
HB 1159 Prohibit interference with the right to bodily integrity in contagious disease control
[Text]
2020
HB 1235 Revise provisions regarding immunizations
[Text]
2016
SB 108
Require notice if certain immunizations contain more than trace amounts of mercury
[Text]
2015
HB 1059 Allow authorized entities to access immunization information in certain circumstances
[Text]
2013
SB 98
Revise certain statutory immunization exemption requirements
[Text] Table A1: Continued Minnesota
|
https://openalex.org/W4288581810
|
https://zenodo.org/records/2564311/files/9617ijcsit08.pdf
|
English
| null |
ON THE IMPLEMENTATION OF GOLDBERG'S MAXIMUM FLOW ALGORITHM IN EXTENDED MIXED NETWORK
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,019
|
cc-by
| 7,211
|
International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 1Department of Computer & Information Technology, King Abdulaziz University KAU
Jeddah, Saudi Arabia.
2Department of Computer Science, King Abdulaziz University KAU, Jeddah,
Saudi Arabia. 1Department of Computer & Information Technology, King Abdulaziz University KAU,
Jeddah, Saudi Arabia. 2Department of Computer Science, King Abdulaziz University KAU, Jeddah,
Saudi Arabia. 2Department of Computer Science, King Abdulaziz University KAU, Jeddah,
Saudi Arabia. 2Department of Computer Science, King Abdulaziz University KAU, Jeddah,
Saudi Arabia Saudi Arabia. KEYWORDS Multipath Routing Protocol, Quality of Service, Wireless Multimedia Sensor Networks, Wireless Sen
Networks ABSTRACT Wireless Sensor Networks (WSNs)have sensor nodes that sense and extract information from surrounding
environment, processing information locally then transmit it to sink wirelessly. Multimedia data is larger in
volume than scalar data, thus transmitting multimedia data via Wireless Multimedia Sensor Networks
(WMSNs) requires stick constraints on quality of services in terms of energy, throughput and end to end
delay.Multipath routing is to discover multipath during route discovery from source to sink. Discover
multipath and sending data via these different paths improve the bandwidth and decrease the end to end
delay. This paper introduces an Energy Location Aware Routing Protocol (ELARP) which is reactive
multipath routing protocol establishing three paths with awareness of node’s residual energy and distance. ELARP has experimented with NS2 simulator. The simulation results show that ELARP enhances QoS for
multimedia data in terms of end to end delay and packet delivery ratio. ENERGY LOCATION AWARE ROUTING PROTOCOL
(ELARP) FOR WIRELESS MULTIMEDIA SENSOR
NETWORKS Ablah AlAmri1 and Manal Abdullah2 1. INTRODUCTION Wireless Multimedia Sensor Networks (WMSNs) are new networks type that have audio or video
sensor nodes integrated with cheap components such as CMOS cameras and microphones. These
sensor nodes have ability to sense the surrounding environment, capture and transmit multimedia
data such as video, audio and even image to the sink[1]. WMSNs have many challenges due to: transmission multimedia data which is large in volume
and required quality of service (QoS) in terms of increasing throughput, and delivery speed of
multimedia data packets to the destination. Because multimedia data packet is very sensitive to
the delay and losses, losing these packets or arriving after deadline leads to distortion in received
multimedia data. Other challenges include: transmit the data wirelessly, and limitations of sensor
nodes capability, limitation on power, memory and capability of the processor[2]. Multipath routing is to discover multipath during route discovery from source node to the sink. Discover multipath and sending data via these different paths improve the bandwidth and
decrease the end to end delay [3]. Multipath routing also provides different advantages such as
balancing the traffic by dividing the traffic across alternative paths in case of use all paths
simultaneously, thus balance the energy between nodes, increase the lifetime of the network and DOI:10.5121/ijcsit.2017.9608 77 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 avoid congestion [4]. Also, multipath routing increases the reliability by protecting from route
failure when uses another path if the main path is fail[5]. avoid congestion [4]. Also, multipath routing increases the reliability by protecting from route
failure when uses another path if the main path is fail[5]. This paper contributes with an Energy Location Aware Routing Protocol (ELARP) which is
reactive multipath routing protocol, establishes three paths from source to the sink with a
consideration of node’s remaining energy and distance to the sink. In WMSNs, considering energy and location of next node during establishing the path is
important to guarantee quality of service QoS for multimedia transmission such as end to end
delay and throughput. During creating the path, next node is selected with best residual energy to
participant in the path and avoid path failure. Considering the location is made by choosing the
node that is much close to the sink. In this research, researchers used the concept of location aware node disjoint paths. 1. INTRODUCTION By node
disjoint paths, no node is sharing between two paths. So that every node joins only one path at a
time. This avoids consuming node energy and causes less congestion which grantee QoS. If the
node share between two paths which is link disjoint path and the node died, two paths are affected
while in node disjoint path only one path is affected. The rest of this paper is organized as follow: literature review is section 2.The proposed ELARP
protocol is described at section 3.Performance metrics that evaluate the protocol is detailed in
section 4.Results and analysis are in section 5.Finally are the conclusion and future works in
section 6. 2.Literature Review The sensor node saves information about hop neighbor such as distance, residual energy
and rate of the link. A multipath routing used metadata to create the paths that meet QoS for multimedia data is
produced by Lan et al[10]. They used routing decision cost function and advanced Dijkstra
algorithm, excluded the neighbor nodes with insufficient delay, residual energy and bandwidth. This protocol described the packet used by metadata. The simulation results show that the
proposed multipath protocol enhances the delay and energy consumption. Guannan et al.[11] proposed a multipath routing and load balancing protocol for WMSNs to
increase reliability, reduce energy, and control the congestion. The proposed protocol is reactive
to reduce the overhead. The protocol creates three disjoint paths (primary, alternate, and backup
paths) from source to sink nodes. The simulation results show an enhancement of network
lifetime and throughput. The protocol shows fast deteriorate of network lifetime at higher
transmission rate. Disjoint path is not suitable for WMSN, the shared node will cause high energy
consumption. Multipath routing protocol proposed by Li et al. [12] is path disjoint based on directed diffusion
(DD) protocol. The protocol modified DD and used cost path which is based on expected
transmission account (ETX) and delay as a metric. After the sink send interest, nodes calculate
the ETX for the last three upstream links and add the mto the packet header. 2.Literature Review Wireless Sensor Networks (WSNs) are used in many applications to abstract information about
certain environment. Many applications such as monitoring and surveillance require sensing the
environment and send multimedia data not only scalar data. Multimedia data may include video,
sound or even image .A WMSN is composed of ordinary nodes of WSNs as well as nodes with
integrated cameras and microphones. WMSNs have sensor nodes that are capable to capture and
communicate streams of multimedia data. There are many researchers produced different routing protocols for WMSNs to better use of
bandwidth, reduce the delay, and improve node’s energy consumptions. This is to show how
WMSNs can be more efficient depending on the constraints and the requirements of QoS on
specific application. WMSNs protocols may be classified based on the routing techniques into
four categories: •
Multichannel: sending the packets via different channels. •
Multichannel: sending the packets via different channels. •
Single path routing: at route discover stage, creating only one path from source to the
sink. Sung-Lee et al. [6] presented routing protocol for MWSNs. It is energy aware that
minimizes the control message overhead. •
Single path routing: at route discover stage, creating only one path from source to the
sink. Sung-Lee et al. [6] presented routing protocol for MWSNs. It is energy aware that
minimizes the control message overhead. •
Multipath routing: discover multipath from source to sink during route discovery. Poojary
and Pai [7] proposed a multipath protocol with power awareness routing. •
Multichannel and Multipath routing: creating a multipath and send the data via
multichannel[8]. edjiah et al [9] introduced a multipath routing protocol that an enhancement of GPSR (Greedy Medjiah et al [9] introduced a multipath routing protocol that an enhancement of GPSR (Greedy
Perimeter Stateless Routing) protocol called GEAMS(Geographic Energy-Aware Multipath
Stream-based) which minimizes the queue size and enhances the network lifetime by adding load Medjiah et al [9] introduced a multipath routing protocol that an enhancement of GPSR (Greedy
Perimeter Stateless Routing) protocol called GEAMS(Geographic Energy-Aware Multipath
Stream-based) which minimizes the queue size and enhances the network lifetime by adding load 78 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 balance. The sensor node saves information about hop neighbor such as distance, residual energy
and rate of the link. balance. 3.2 Application Layer MPEG-4 is a video structure that consists of
4 is a video structure that consists of three types of frames, these frames are: I. Intra – coded frame. I frame is encoded and decoded independently
other types of frames. coded frame. I frame is encoded and decoded independently without reference to any
without reference to any P. Predictive-coded frame. P frame is encoded and decoded dependent
in the sequence of the video. coded frame. P frame is encoded and decoded dependent on previous I or P
in the sequence of the video. previous I or P frames q
B. Bi-directionally predictive coded frame. B frame is encoded and decoded dependent
types of previous and successive
q
directionally predictive coded frame. B frame is encoded and decoded dependent
previous and successive frames in the sequence of the multimedia data. directionally predictive coded frame. B frame is encoded and decoded dependent on other q
B. Bi-directionally predictive coded frame. B frame is encoded and decoded dependent
types of previous and successive
q
directionally predictive coded frame. B frame is encoded and decoded dependent
previous and successive frames in the sequence of the multimedia data. directionally predictive coded frame. B frame is encoded and decoded dependent on other According to this coding relation in MPEG
frames in encoding or decoding, losing one I frame will affect all frames
Picture (GOP).As a result this will affect the quality of
affect itself only. So, I frame is the most important type of frames then P and B frames has
importance. to this coding relation in MPEG-4 structure and the dependent relationship between
frames in encoding or decoding, losing one I frame will affect all frames in same Group of
s a result this will affect the quality of multimedia data. Losing B frame will
affect itself only. So, I frame is the most important type of frames then P and B frames has
4 structure and the dependent relationship between
in same Group of
. Losing B frame will
affect itself only. So, I frame is the most important type of frames then P and B frames has less Decomposing video sequence into
falling between two I frames. GOP is defined by two parameters G (N,M) where N is the distance
between I frames and M is the distance between I and P frames. 3. ENERGY LOCATION AWARE ROUTING PROTOCOL (ELARP) All routing mechanisms that used to discover multipath in WSN and WMSN are based on
different types of discovering the paths, such as start to discover single path each time and repeat
the process until discover all possible paths or specific number of paths. Repeating the process
consumes energy and increases delay. Flooding mechanism is a mechanism to discover maximum
or fixed number of paths which have high energy cost. To avoid this cost, this research suggests
an Energy Location Aware Routing Protocol (ELARP). ELARP mechanism discovers three paths
at the same time with a consideration of node’s remaining energy and distance to the sink. ELARP is used at network layer in layered architecture. Figure 1 shows this architecture. From the figure, application layer MPEG4 distributed code algorithm is used, in transport
layer User Datagram Protocol (UDP) is used to guarantee lower latency because it does not use
hand-shaking mechanism nor end-to-end congestion control . Finally, IEEE 802.11 standard for
scheduling is used in MAC layer. Following subsections discuss the role of each layer in the
WMSN communication process. 79
Figure 1. ELARP Layered Architecture Figure 1. ELARP Layered Architecture 79 International Journal of Computer Science & Information Tec
International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017
hnology (IJCSIT) Vol 9, No 6, December 2017 International Journal of Computer Science & Information Tec
International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017
hnology (IJCSIT) Vol 9, No 6, December 2017 3.1 ELARP Network Model
Network Model The network is composed of N different heterogeneous sensor nodes distributed randomly in
specific flat area, as shown in
capabilities. Sink node is always switched on and it is non
audio and video sensor nodes. All these nodes are mobile nodes with random movement with
same speed in the network area. They have same
neighbour’s information such as energy and location. The network is composed of N different heterogeneous sensor nodes distributed randomly in
specific flat area, as shown in Figure 2. The network has one sink node which has special
capabilities. Sink node is always switched on and it is non-mobile node. The network also has
video sensor nodes. All these nodes are mobile nodes with random movement with
same speed in the network area. They have same transmission range and all nodes has access to
neighbour’s information such as energy and location. The network is composed of N different heterogeneous sensor nodes distributed randomly in
. The network has one sink node which has special
network also has
video sensor nodes. All these nodes are mobile nodes with random movement with
range and all nodes has access to Figure 2. Simulation Network Figure 2. Simulation Network Figure 2. Simulation Network Application layer classified the frames to I,
priority, and GOP size in its header.
classified the frames to I, B, and P types. Then encapsulates frame type, frame
header.
B, and P types. Then encapsulates frame type, frame 3.2 Application Layer video sequence into small units using GOP is referred to the number of frames
between two I frames. GOP is defined by two parameters G (N,M) where N is the distance
between I frames and M is the distance between I and P frames. [13, 14]
to the number of frames
between two I frames. GOP is defined by two parameters G (N,M) where N is the distance Application layer classified the frames to I,
priority, and GOP size in its header. classified the frames to I, B, and P types. Then encapsulates frame type, frame
header. B, and P types. Then encapsulates frame type, frame 80 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 3.3.2 Routing Table Structure ELARP routing table contains five fields. These fields are: destination address, sequence number,
advertised hop count which contains the maximum hop count that available in the rout list, rout
list which is a pair of next hop and hop count and expiration time for this entry. Figure 4 shows
the header of the routing table Destination
address
Rout list
{(nexthopi, hopcounti),
(nexthopj, hopcountj)}
Advertised
hop count
Sequence
number
Expiration
time out
Figure 4. Routing Table Structure Figure 3. Route Request RREQ Packet Format Figure 3. Route Request RREQ Packet Format •
Route Reply (RREP): is a packet format that is used to reply the route request. •
Route Reply Acknowledgment (RREP-ACK): is used to response to route reply. •
Route Reply Acknowledgment (RREP-ACK): is used to response to route reply. •
Route Error (RERR): is a packet format that is used in maintenance phase if failure is
detected. •
Route Error (RERR): is a packet format that is used in maintenance phase if failure is
detected. 3.3 Network Layer ELARP is reactive routing protocol where the path is discovered only when a node has data to
send. Reactive approach is more suitable for WMSN to avoid energy consumption for creating
and maintaining the routing table and also to avoid control messages overhead. 3.3.1 Packet Format ELARP protocol has different types of packet format. These packets are: ELARP protocol has different types of packet format. These packets are: •
Hello Packet: are broadcasted every period of time to update the neighbor list. •
Route Request (RREQ): is a packet format that is used to create the paths and has three
special fields used by ELARP. It contains the fields shown in Figure 3
Source
Addr
Source
Seq#
Broadcast
ID
Dest
Addr
Dest
Seq#
Hop_
Cnt
First_
Hop
Best
Add1
Best
Add2
Best
Add3 •
Hello Packet: are broadcasted every period of time to update the neighbor list. •
Route Request (RREQ): is a packet format that is used to create the paths and has three
special fields used by ELARP. It contains the fields shown in Figure 3
Source
Addr
Source
Seq#
Broadcast
ID
Dest
Addr
Dest
Seq#
Hop_
Cnt
First_
Hop
Best
Add1
Best
Add2
Best
Add3 3.3.3 Next Node Selection Mechanism A reactive approach is work on demand. Whenever an event is detected, it starts route discovery. ELARP protocol has two lists: . Block List: A list contains all nodes that do not have any neighbors. A. Block List: A list contains all nodes that do not have any neighbors. A. Block List: A list contains all nodes that do not have any neighbors. B. Neighbor List: a list contains all neighbor nodes excluding source node, block nodes and
nodes have already joined a path. B. Neighbor List: a list contains all neighbor nodes excluding source node, block nodes and
nodes have already joined a path. 81 81 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 ELARP selects next node in a route based on two criteria: the remaining energy on neighbor node
to avoid hole in the path and distance to the sink. Calculate the weight of the node is based on
equation (1). For each node in the neighbor list Nx of current node x, is given by: ELARP selects next node in a route based on two criteria: the remaining energy on neighbor node
to avoid hole in the path and distance to the sink. Calculate the weight of the node is based on
equation (1). For each node in the neighbor list Nx of current node x, is given by: =
+
(1) (1) Where (, ) is appropriate weight with value between 0 and 1.
is the remaining
energy, and
is the complemented distance to the sink. is the distance between two
nodes and s(the sink node), and is calculated using equation (2). Node that has long distance
should be given less weight, equation (3)is used to calculate this parameter. (2) = ( − )" + (# −#)"
(2) = ( − )" + (# −#)"
(2)
= 1 −
(3)
= 1 −
(3) (3) Neighbor node with high weight has high probability to be a next node in route discovery process
using equation (4). Next Node = +,#(1, 2, … . ,01)
(4) (4) Next Node = +,#(1, 2, … . ,01)
(4) Next Node = +,#(1, 2, … . ,01) 3.3.4
Route Discovery Mechanism When an event occurs and some node has a data to send, the source node checks the neighbor list. If the list is empty, then source node marks itself as block node and updates the block list. Otherwise calculate the weight for each neighbor. Then it selects next node with high weight as
calculated by equation (4). Source node establishes three paths by sending the request to best three neighbors then after the
paths are established, each node select the best neighbors to send the request and this process is
repeated until the paths reaches the sink and generate RREP. A route with minimum hop count is
used to send the data. The other two paths are used as back up. A new route discovery process is
repeated only if all paths are failed. The steps of ELARP mechanism techniques are shown in
Algorithm1. 4. PERFORMANCE METRICS Different performance metrics are used to evaluate the performance of ELARP. These metrics
may be general for WSNs and WMSNs protocols or specific for only multimedia networks: 4.1 General Performance Metrics:
Packet delivery ratio: the ratio between the total numbers of packets received divided by
the total number of packets sent by the source node.
Packet delivery ratio: the ratio between the total numbers of packets received divided by
Packet delivery ratio: the ratio between the total numbe
the total number of packets sent by the source node. the total number of packets sent by the source node.
Network Lifetime: the duration of time from network deployed until the first node dies.
Network Lifetime: the duration of time from network deployed
Drop Packet: the number of packets dropped.
End to end delay: possible delays of a packet being transmitted from source to
destination.
Jitter: the variation delay of receiving packets. 82 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 201 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 4.2 Multimedia Performance Metrics:
Frame loss: the number of frames loss in each type of video frame I, P and B. In this
research, we consider only loss of I frame.
Frame loss: the number of frames loss in each type of video frame I, P and B. In this
research, we consider only loss of I frame.
Peak signal-to-noise ratio (PSNR): is a measure for video quality. PSNR measures the
signal noise by comparing maximum energy signal to corrupting noise. To see the quality
of PSNR to the video, map the PSNR to Mean Opinion Score (MOS) levels[15], where
PSNR>37 is excellent and PSNR<20 is bad. When PSNR is in the range 25-31, it is fair.
Peak signal-to-noise ratio (PSNR): is a measure for video quality. PSNR measures the
signal noise by comparing maximum energy signal to corrupting noise. To see the quality
of PSNR to the video, map the PSNR to Mean Opinion Score (MOS) levels[15], where
PSNR>37 is excellent and PSNR<20 is bad. When PSNR is in the range 25-31, it is fair. Algorithm 1: Route Discovery Algorithm
Require: Route Request Packet (RREQ)
Ensure: Minimum delay path
Output: Route Reply
Initialization: N=3, M=1
If Source Node have data to send
Check Neighbour list
IF (Neighbour list is empty)
Add the node to Block List, Else
foreach node in Neighbour list do
Calculate weight W
End
Sort Neighbour list based on W
Send RREQ to best N node
If node Receive Route Request Packets (RREQ)
If (node address is not in the packet OR RREQ already received)
drop the packet, Else
Set reverse route
If (node is intended receiver)
Send Route Reply Packets (RREP), Else
Check neighbour list
IF (Neighbour list is empty)
Add the node to block List, Else
foreach node in neighbour list do
Calculate weight W
End
Sort neighbour list based on W
Forward RREQ to best M node 5. ELARP RESULT AND ANALYSIS 5. ELARP RESULT AND ANALYSIS ELARP is analysed under two different scenarios. It’s performance is compared with Ad hoc On-
demand Multipath Distance Vector(AOMDV)[16]. To insure correct and no deviated results,
average of ten runs is presented in each case. 5.1 Network Setup The protocol is simulated using network simulation version-2 NS2 [17] with my Evalvid[18] to
test multimedia data transmission. The network consists of 50 heterogeneous sensor nodes and
one sink node. The number of video node is varied from 2 to 10, the number of audio node is
varied from 2 to 10. These nodes are distributed randomly in area 100 x 100 m. All sensor nodes
are mobile nodes with speed equal to 5 m/s. The sink node is centred placed, while the
heterogeneous sensor nodes are distributed randomly in the area. The two test scenarios are:
First, changing number of video and audio nodes and generating non-overlapping two events
during the simulation time. Event’s location are generated randomly. Second, events are
overlapped in three different scenarios. Table 1 summarizes the network parameters. 83 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Table 1: The Network Parameters
Simulator
NS-2.35
Simulation Time
500 s
Simulation Area
100 m* 100 m
Number of Sensor Nodes
50 nodes
Number of Video Nodes
Vary from 2 to 10
Number of Audio Nodes
Vary from 2 to 10
Queue Type
Drop Tail
Propagation Models
Two Ray Ground
Data Rate
1 Mbps
MAC Protocol
IEEE 802.11
Routing Protocol
ELARP, AOMDV
Initial energy of sensor node
5 Joule
Initial energy of video node
30 Joule 5.2 Ratio of Video Node o study the effect of number of video nodes on network performance, researchers increase the
deo and audio nodes from 2 to 16 nodes. Again, the results are taken as average of 10 runs. To study the effect of number of video nodes on network performance, researchers increase the
video and audio nodes from 2 to 16 nodes. Again, the results are taken as average of 10 runs. Packet delivery ratio metric, shown in Figure 5, has ELARP is better than AOMDV on average. It is noticeable that increasing network video nodes would cause decreasing the packet delivered
because of the higher probability of congestion, packet drooped and node death. Figure 5. Packet Delivery Ratio vs. Increased Video Nodes
99.4
99.5
99.6
99.7
99.8
99.9
100
2
4
6
8
10
12
14
16
Packet Delivery Ratio (%)
No of video nodes
Packet Delivery Ratio
AOMDV
ELARP Figure 5. Packet Delivery Ratio vs. Increased Video Nodes ELARP shows minor improvement in network lifetime over AOMDV as shown in Figure 6. Network lifetime decreases from 390s to 350s with increasing video nodes from 2 to 10 nodes. More than 10 video nodes causes dramatic drop in the network lifetime to 220s. ELARP shows stable and linear drop packets over AOMDV as shown in Figure 7.The figure
shows that ELARP packet dropped is between 20 to 60 packets with linear increase with
increasing number of video nodes. While the packet dropped for AOMDV starts at 80 packets
with high fluctuations. 84 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Figure 6. Network Life time vs. Increase Video Nodes
Figure 7. Packet Drop vs. Increase Video Nodes
End to End delay is an important metric especially for WMSNs. Figure 8 shows the
improvement done by ELARP over AODMV. ELARP delay is more linear and stable. Jitter is important metric denoting the stability of the network. ELARP protocol is little low jitter
over AOMDV. Figure 9 shows that more than 10 video nodes among 50 network nodes, jitter
has sharp increase. 5.2 Ratio of Video Node 200
250
300
350
400
2
4
6
8
10
12
14
16
Network Lifetime(Sec.)
No of video nodes
Network Lifetime
AOMDV
ELARP
0
20
40
60
80
100
120
2
4
6
8
10
12
14
16
No of Packets Dropped
No of video nodes
Drop Packet
AOMDV
ELARP 200
250
300
350
400
2
4
6
8
10
12
14
16
Network Lifetime(Sec.)
No of video nodes
Network Lifetime
AOMDV
ELARP Network Lifetime Figure 6. Network Life time vs. Increase Video Nodes Figure 6. Network Life time vs. Increase Video Nodes Figure 7. Packet Drop vs. Increase Video Nodes
0
20
40
60
80
100
120
2
4
6
8
10
12
14
16
No of Packets Dropped
No of video nodes
Drop Packet
AOMDV
ELARP Figure 7. Packet Drop vs. Increase Video Nodes Figure 7. Packet Drop vs. Increase Video Nodes Figure 7. Packet Drop vs. Increase Video Nodes End to End delay is an important metric especially for WMSNs. Figure 8 shows the
improvement done by ELARP over AODMV. ELARP delay is more linear and stable. End to End delay is an important metric especially for WMSNs. Figure 8 shows the
improvement done by ELARP over AODMV. ELARP delay is more linear and stable. Jitter is important metric denoting the stability of the network. ELARP protocol is little low jitter
over AOMDV. Figure 9 shows that more than 10 video nodes among 50 network nodes, jitter
has sharp increase. Frame Loss of types I, P and B is special performance metric for WMSN. The average PSNR for
both ELARP and AOMDV protocols are not affected by increased the number of video nodes. There is no loss of I frame and average PSNR = 27.87, which represents “fair” quality of the
video. 85 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Figure 8: End to End Delay vs. Increase Video Nodes
Figure 9. Jitter vs. 5.2 Ratio of Video Node Increase Video Nodes
0.1
0.11
0.12
0.13
0.14
0.15
0.16
2
4
6
8
10
12
14
16
Delay(S)
No of video nodes
End to End Delay
AOMDV
ELARP
0.0055
0.006
0.0065
0.007
0.0075
0.008
2
4
6
8
10
12
14
16
Jitter (S)
No of video nodes
Jitter
AOMDV
ELARP 0.1
0.11
0.12
0.13
0.14
0.15
0.16
2
4
6
8
10
12
14
16
Delay(S)
No of video nodes
End to End Delay
AOMDV
ELARP End to End Delay Figure 8: End to End Delay vs. Increase Video Nodes 0.0055
0.006
0.0065
0.007
0.0075
0.008
2
4
6
8
10
12
14
16
Jitter (S)
No of video nodes
Jitter
AOMDV
ELARP Figure 9. Jitter vs. Increase Video Nodes 5.3 Events Overlap By event overlap, we mean how much time the events share sending data in the network. This
parameter represents increasing network load. To study the effect of time overlap of reporting
events and sending data, with also increasing he video and audio nodes from two to 16 nodes, the
research increases number of video and audio nodes that reporting events by considering three
cases: Case A: the overlap time is 10 second
Case B: the overlap time is 20 second
Case C: the overlap time is 30 second Case A: the overlap time is 10 second
Case B: the overlap time is 20 second
Case C: the overlap time is 30 second Case A: the overlap time is 10 second Case B: the overlap time is 20 second Case C: the overlap time is 30 second Packet delivery ratio of ELARP is outperforms AOMDV in all the three cases A, B, and C as
shown in Figure 10. ELARP shows also linear distribution regardless of network load. 86 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Figure 10: Packet Delivery Ratiovs. Events Overlap
Although network lifetime is greatly affected by average energy consumption, Figure 11 shows
that both protocols are identical in the three cases. The figure also shows linear drop from 10
video nodes and up to 220 s lifetime
99.26
99.36
99.46
99.56
99.66
99.76
99.86
8
10
12
14
16
Packet Delivery Ratio (%)
No of video nodes
Packet Delivery Ratio
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C 99.26
99.36
99.46
99.56
99.66
99.76
99.86
8
10
12
14
16
Packet Delivery Ratio (%)
No of video nodes
Packet Delivery Ratio
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Packet Delivery Ratio Figure 10: Packet Delivery Ratiovs. Events Overlap Figure 10: Packet Delivery Ratiovs. Events Overlap Although network lifetime is greatly affected by average energy consumption, Figure 11 shows
that both protocols are identical in the three cases. The figure also shows linear drop from 10
video nodes and up to 220 s lifetime. Although network lifetime is greatly affected by average energy consumption, Figure 11 shows
that both protocols are identical in the three cases. 5.3 Events Overlap The figure also shows linear drop from 10
video nodes and up to 220 s lifetime. Figure 11: Network Lifetimevs. Events Overlap
Figure 12 shows packet drop metric where ELARP has minimum drop packets than AOMDV
220
270
320
370
420
8
10
12
14
16
Network Lifetime (S)
No of video nodes
Network Lifetime
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 11: Network Lifetimevs. Events Overlap Figure 11: Network Lifetimevs. Events Overlap Figure 11: Network Lifetimevs. Events Overlap Figure 12 shows packet drop metric where ELARP has minimum drop packets than AOMDV
especially when WMSN is loaded by events overlap. Figure 12 shows packet drop metric where ELARP has minimum drop packets than AOMDV
especially when WMSN is loaded by events overlap. ELARP has achieved good results over AOMDV in end to end delay. Figure 13 shows that while
the number of video nodes increased, the end to end delay decreased for both protocols. The
improvement is better when the network is more loaded (case C). Jitter is nearly constant when WMSN contains less than 10 video nodes and it has linear fast
increase when numbers of video nodes are more than 10 nodes. Figure 14 shows minor
improvement of ELARP over AODMV especially when the network is loaded (case C). 87 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017
Figure 12: Packet Dropvs. Events Overlap
Figure 13. End to End Delayvs. Events Overlap
Figure 14. Jitter vs. 5.3 Events Overlap Events Overlap
28
78
128
178
8
10
12
14
16
No of Packets Dropped
No of video nodes
Drop Packet
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C
0.11
0.12
0.13
0.14
0.15
0.16
0.17
8
10
12
14
16
Delay(S)
No of video nodes
End to End Delay
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C
0.0055
0.006
0.0065
0.007
0.0075
0.008
8
10
12
14
16
Jitter (S)
No of video nodes
Jitter
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 28
78
128
178
8
10
12
14
16
No of Packets Dropped
No of video nodes
Drop Packet
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Drop Packet Figure 12: Packet Dropvs. Events Overlap
28
78
128
8
10
12
14
16
No of Packets
No of video nodes
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C
0.11
0.12
0.13
0.14
0.15
0.16
0.17
8
10
12
14
16
Delay(S)
No of video nodes
End to End Delay
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 12: Packet Dropvs. Events Overlap Figure 12: Packet Dropvs. Events Overlap 0.11
0.12
0.13
0.14
0.15
0.16
0.17
8
10
12
14
16
Delay(S)
No of video nodes
End to End Delay
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 13. End to End Delayvs. Events Overlap Figure 13. End to End Delayvs. Events Overlap 0.0055
0.006
0.0065
0.007
0.0075
0.008
8
10
12
14
16
Jitter (S)
N
f id
d
Jitter
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 14. Jitter vs. Events Overlap 88 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Frame loss of I frame affects the quality of the video data transmitted. ELARP in case B and C
did not loss any I frame. 5.3 Events Overlap AOMDV in same cases show highest loss of I frame as shown in Figure
15. In case A both protocols loss the same number of frames. The PSNR is affected by loss
frames, as shown by Figure 16, more frames lost causes less PSNR. In all cases AOMDV and
ELARP protocol lie in fair MOS level. Figure 15. I Frame Lostvs. Events Overlap
Figure 16. Average PSNR vs. Events Overlap
0
1
2
3
4
5
6
8
10
12
14
16
No of Frame Lost
No of video nodes
I Frame Lost
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C
26.8
27
27.2
27.4
27.6
27.8
28
8
10
12
14
16
PSNR
No of video nodes
PSNR
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 15. I Frame Lostvs. Events Overlap
0
1
2
3
4
5
6
8
10
12
14
16
No of Frame Lost
No of video nodes
I Frame Lost
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 15. I Frame Lostvs. Events Overlap Figure 15. I Frame Lostvs. Events Overlap Figure 16. Average PSNR vs. Events Overlap
26.8
27
27.2
27.4
27.6
27.8
28
8
10
12
14
16
PSNR
No of video nodes
PSNR
AOMDV Case A
ELARP Case A
AOMDV Case B
ELARP Case B
AOMDV Case C
ELARP Case C Figure 16. Average PSNR vs. Events Overlap 6. CONCLUSIONS AND FUTURE WORK QoS is an important issue for WMSNs in term of end to end delay and guarantee data delivery. In
this work, ELARP with awareness of location and energy of the node is developed. A multipath
routing which does aware of remaining energy and location of the node, reduce overhead and
save energy by selecting three paths based on their weight. Only one path is used to send the data
and othersareback up in case of failure. ELARP’s performance is compared against AOMDV
protocol. NS2 simulator is used to simulate ELARP. The results are analysed using two
parameters: increasing number of video nodes in the network and events overlap which reflects
network load. From the results, ELARP enhances the packet drop and delay over AODMV. Increasing number of video nodes more than 10 nodes makes a big difference in protocol 89 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 201 performance. While increasing network load represented by event overlap has also improved
packet delivery ratio and end-to-end delay as well as I frame loss and PSNR. performance. While increasing network load represented by event overlap has also improved
packet delivery ratio and end-to-end delay as well as I frame loss and PSNR. As a future work, ELARP is used in Cross Layer environment where different layers are
collaborated to enhance the performance of ELARP. As a future work, ELARP is used in Cross Layer environment where different layers are
collaborated to enhance the performance of ELARP. REFERENCES [1]
T. Melodia and I. F. Akyildiz, "Research Challenges for Wireless Multimedia Sensor Networks,"
in Distributed video sensor networks, ed: Springer Science & Business Media, 2011, pp. 233-246. [2]
Z. Hamid, F. Bashir, and J. Y. Pyun, "Cross-layer QoS routing protocol for multimedia
communications in sensor networks," in Ubiquitous and future networks (ICUFN), 2012 fourth
international conference on, Puket, Thailand, 2012, pp. 498-502. [3]
J. Ben-Othman and B. Yahya, "Energy efficient and QoS based routing protocol for wireless
sensor networks," Journal of Parallel and Distributed Computing, vol. 70, pp. 849-857, 2010. [4]
D. Ganesan, R. Govindan, S. Shenker, and D. Estrin, "Highly-resilient, energy-efficient multipath
routing in wireless sensor networks," ACM SIGMOBILE Mobile Computing and Communications
Review, vol. 5, pp. 11-25, 2001. [5]
W. Lou, W. Liu, and Y. Zhang, "Performance optimization using multipath routing in mobile ad
hoc and wireless sensor networks," in Combinatorial optimization in communication networks, ed:
Springer, 2006, pp. 117-146. [6] [6]
S.-K. Lee, J.-G. Koh, and C.-R. Jung, "An energy-efficient QoS-aware routing algorithm for
wireless multimedia sensor networks," International Journal of Multimedia and Ubiquitous
Engineering, vol. 9, pp. 245-252, 2014. [7]
D. Kandris, M. Tsagkaropoulos, I. Politis, A. Tzes, and S. Kotsopoulos, "Energy efficient and
perceived QoS aware video routing over wireless multimedia sensor networks," Ad Hoc Networks,
vol. 9, pp. 591-607, 2011. pp
[8]
M. A. Hamid, M. M. Alam, and C. S. Hong, "Design of a QoS-aware routing mechanism for
wireless multimedia sensor networks," in Global Telecommunications Conference, 2008. IEEE
GLOBECOM 2008. IEEE, 2008, pp. 1-6. [9]
S. Medjiah, T. Ahmed, and F. Krief, "GEAMS: a geographic energy-aware multipath stream-
based routing protocol for WMSNs," in Information Infrastructure Symposium, 2009. GIIS'09. Global, 2009, pp. 1-8. [10]
Y. Lan, W. Wenjing, and G. Fuxiang, "A real-time and energy aware QoS routing protocol for
multimedia wireless sensor networks," in Intelligent Control and Automation, 2008. WCICA 2008. 7th World Congress on, 2008, pp. 3321-3326. [11]
G. Sun, J. Qi, Z. Zang, and Q. Xu, "A reliable multipath routing algorithm with related congestion
control scheme in wireless multimedia sensor networks," in Computer Research and Development
(ICCRD), 2011 3rd International Conference on, 2011, pp. 229-233. pp
[12]
S. Li, R. Neelisetti, C. Liu, and A. Lim, "Delay-constrained high throughput protocol for multi-
path transmission over wireless multimedia sensor networks," in World of Wireless, Mobile and
Multimedia Networks, 2008. WoWMoM 2008. 2008 International Symposium on a, 2008, pp. 1-8. REFERENCES [13]
V. Group, "MPEG-4 video verification model version 18.0," Coding of moving pictures and
associated audio MPEG2001/N3908, ISO/IEC JTC1/SC29/WG11, Pisa, Italy,, Jan. 2001. associated audio
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C J C /SC 9/WG
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International Organization for Standardization (1999), Overview of the MPEG-4 standard. [15]
J. Klaue, B. Rathke, and A. Wolisz, "Evalvid-a framework for video transmission and quality
evaluation," Computer Performance Evaluation/Tools, vol. 2794, pp. 255-272, 2003. [16]
M. K. Marina and S. R. Das, "Ad hoc on‐demand multipath distance vector routing," Wireless
communications and mobile computing, vol. 6, pp. 969-988, 2006. [17]
T. Issariyakul and E. Hossain, Introduction to network simulator NS2: Springer Science &
Business Media, 2011. [18]
C.-H. Ke, C.-K. Shieh, W.-S. Hwang, and A. Ziviani, "An evaluation framework for more realistic
simulations of MPEG video transmission," Journal of Information Science & Engineering, vol. 24,
2008. 90 International Journal of Computer Science & Information Technology (IJCSIT) Vol 9, No 6, December 2017 Authors Ablah AlAmri received bachelor degree in computer science in 2012 from King Abdul-Aziz University. Currently, teaching assistant at King Abdul-Aziz University, and master student in Faculty of Computing
and Information Technology, King Abdul-Aziz University. Manal Abdullah:received her PhD in computers and systems engineering, Faculty of Engineering, Ain-
shams University, Egypt, 2002. She has experienced in industrial computer networks with a lot of
reputational vendors of PLC, SCADA, and DCS.Currently she is associate professor in faculty of
Computing and Information Technology, King Abdulaziz University KAU, SA. Dr. Abdullah’s research
interest includes computer networks, Artificial Intelligence, Information Retrieval, Big Data
Analysis,PatternRecognition and Wireless sensor networks. She published more than 90 research papers in
various international journals and conferences. 91
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Magnetic Iron Oxide Nanoparticles for Multimodal Imaging and Therapy of Cancer
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International journal of molecular sciences
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Int. J. Mol. Sci. 2013, 14, 15910-15930; doi:10.3390/ijms140815910 Int. J. Mol. Sci. 2013, 14, 15910-15930; doi:10.3390/ijms140815910 1. Introduction Early detection and treatment of cancer are critical factors for a favorable prognosis. Cancer
diagnosis using nanotechnology is an emerging field. Tremendous efforts in biomedical research have
been devoted to improving the sensitivity and accuracy of diagnosing cancer using early detection
methods and to improving the efficacy of treatment methods. A non-invasive diagnosis of patients can
be achieved using in vivo imaging techniques. These techniques are powerful tools for visualizing the
abnormal state of the body and for monitoring biological situations at the target site. Examples of
non-invasive imaging techniques include the following: computed tomography (CT), magnetic
resonance imaging (MRI), nuclear imaging of positron emission tomography (PET), single-photon
emission computed tomography (SPECT), and optical (or fluorescence) imaging. These imaging
techniques have provided scientists and clinicians with the ability to acquire in vivo images of anatomy
and physiology in both animals and humans [1–6]. MRI uses non-ionizing radiation and is an integral
tool in clinical radiology and experimental settings. MRI is very effective in characterizing soft tissue
and has a high tissue resolution. Additionally, the modality is tomographic with unlimited penetration
into tissues [4,7]. Advances in nanotechnology have permitted new possibilities for theranostics, which are defined as
the combination of therapy and imaging within a single platform [8,9]. Nanotechnology is applied to
molecular imaging in the form of imaging probes capable of enhancing the sensitivity of the image and
the specificity toward the target tissue. Usually, the imaging probe consists of nanoparticles conjugated
with active targeting ligands [10,11]. Other types of tumor targeting are based on passive targeting
mediated by enhanced permeability and retention (EPR) [12,13]. Superparamagnetic iron oxide
nanoparticles (SPION) have a superparamagnetic iron core, which makes them useful as T2 contrast
agents for MRI. SPION can be detected with high sensitivity, and both the iron and polymer
components of SPION are biocompatible and degradable [14]. The size of iron oxide nanoparticles plays a major role in target cell uptake and elimination from the
body. Spleen and liver capture nanoparticles of more than 200 nm in diameter whereas particles having
sizes below 10 nm are selectively filtered by renal systems and eliminated from body [15]. The surface
properties of nanoparticles can be modified to accommodate targeting [16]. With the recent interest in
multimodal imaging, nanotechnology has focused on developing novel probes that combine various
agents to form new all-in-one probes with both multimodal capability and therapeutic functionality [17]. Reju Thomas 1, In-Kyu Park 2 and Yong Yeon Jeong 1,* 1 Department of Radiology, Chonnam National University Hwasun Hospital,
Chonnam National University Medical School, Gwangju 501-746, Korea;
E-Mail: regeth@gmail.com 1 Department of Radiology, Chonnam National University Hwasun Hospital,
Chonnam National University Medical School, Gwangju 501-746, Korea;
E-Mail: regeth@gmail.com 2 Department of Biomedical Science, Research Institute of Medical Sciences,
Chonnam National University Medical School, Gwangju 501-746, Korea;
E-Mail: pik96d@gmail.com 2 Department of Biomedical Science, Research Institute of Medical Sciences,
Chonnam National University Medical School, Gwangju 501-746, Korea;
E-Mail: pik96d@gmail.com * Author to whom correspondence should be addressed; E-Mail: yjeong@jnu.ac.kr;
Tel.: +82-61-379-7102; Fax: +82-91-379-7133. Received: 23 May 2013; in revised form: 16 July 2013 / Accepted: 18 July 2013 /
Published: 31 July 2013 Abstract: Superparamagnetic iron oxide nanoparticles (SPION) have emerged as an MRI
contrast agent for tumor imaging due to their efficacy and safety. Their utility has been
proven in clinical applications with a series of marketed SPION-based contrast agents. Extensive research has been performed to study various strategies that could improve
SPION by tailoring the surface chemistry and by applying additional therapeutic
functionality. Research into the dual-modal contrast uses of SPION has developed because
these applications can save time and effort by reducing the number of imaging sessions. In addition to multimodal strategies, efforts have been made to develop multifunctional
nanoparticles that carry both diagnostic and therapeutic cargos specifically for cancer. This
review provides an overview of recent advances in multimodality imaging agents and
focuses on iron oxide based nanoparticles and their theranostic applications for cancer. Furthermore, we discuss the physiochemical properties and compare different synthesis
methods of SPION for the development of multimodal contrast agents. ywords: SPION; multimodal; multifunctional; theranostics; MRI; CT; SPECT; PET Int. J. Mol. Sci. 2013, 14 15911 2. Synthesis of Iron Oxide Nanoparticles SPION are used as an MRI contrast agent because the T2 relaxivity of a SPION-based agent is
much higher than that of gadolinium agents. The physiochemical properties of SPION, such as charge,
size, and surface chemistry, can influence biodistribution, stability, and metabolism [20]. By giving
proper surface coating that are biocompatible and biodegradable, SPION can avoid immune response
and serum protein adsorption. Surface charge is a major factor in determining the colloidal nature of
nanoparticles, and it can change size of nanoparticles by aggregation. Hence neutral surfaces are more
biocompatible than charged surfaces. Surface charge can also influence plasma protein binding that
directly affects in vivo biodistribution and nanoparticle clearance from the body [21]. A typical SPION is composed of magnetite (Fe3O4) or maghemite (Fe2O3), with appropriate
coatings to maintain aqueous stability. The SPION are synthesized by a wide range of methods,
including co-precipitation, thermal decomposition, and microemulsion (Table 1). There are also recently
discovered methods, such as microwave assisted synthesis, the hydrolysis method, and microfluidic
synthesis [22–24]. Thermal decomposition for the synthesis of SPION is generally more popular as the
nanoparticles obtained this way possess high crystallinity, high magnetization, and a narrow size
distribution. Simultaneously, the nanoparticles are hydrophobic, which is required to provide
appropriate coating for biomedical application [25]. The co-precipitation method is the most commonly
accepted method for synthesizing SPION. It involves the addition of a concentrated base to divalent or
trivalent ferrous salt solutions. The resulting nanoparticles are polydispersed, with a size range from
2–15 nm [15,26]. The particles aggregate and grow, thereby decreasing the surface free energy [27]. The microemulsion technique has the advantage of controlling the size of nanoparticles by acting as a
nano-reactor. This technique involves the formation of SPION either by water-in-oil or oil-in-water
emulsion. The SPION formed have a controlled size distribution between 2 and 12 nm [15]. Superparamagnetism is attributed to the single domain nature of the nanoparticle, which has a net
magnetic dipole. In a magnetic field, the magnetic domains of nanoparticles re-orient themselves in a
manner similar to paramagnetic materials, but the magnetic moment of nanoparticles will be much
higher than that of paramagnetic substances. In the absence of a magnetic field, the dipole randomly
orients with zero magnetic moment. Due to this property, SPION have less chance of aggregation [15]. 1. Introduction Multifunctional nanoparticles enable multimodal imaging with the combination of two or more imaging
modalities or theranostics for simultaneous imaging and therapy. These techniques are expected to
overcome the hurdles of traditional diagnosis and therapy through optimized therapy called
“personalized medicine”[6]. The majority of nanoparticles in development include drug conjugates
and complexes, micelles, dendrimers, vesicles, core–shell particles, microbubbles, and carbon
nanotubes [18]. Among these nanoparticles, magnetic nanoparticles exhibit not only unique material
properties but also an integrated design capacity for cell targeting, imaging, and therapy. This
combination of properties makes nanoparticles ideal platform materials for theranostics. In addition,
magnetic nanoparticles have been proven to serve as workhorses for multimodality preclinical imaging
as they allow MRI, nuclear and optical imaging [15,19]. In this review, we provide an overview of Int. J. Mol. Sci. 2013, 14 15912 recent advances in multimodality imaging, focusing on iron oxide based nanoparticles and on their
theranostic applications for cancer. 2. Synthesis of Iron Oxide Nanoparticles The function of SPION in MRI contrast enhancement is attributed to their ability to change the nuclear
spin relaxation of water protons and cause the region of interest to darken. It is very important to understand the toxic effect and the mechanism by which iron oxide
nanoparticles can cause toxicity before using them for clinical application. Iron oxide nanoparticles are
converted to elemental iron species, and finally merged to our body reserves or utilised for the
formation of haemoglobin. Biosafety of iron oxide nanoparticle (Fe3O4 and MnFe3O4) have been
established up to 200 μg/mL concentration [16]. But when cells are exposed to very high dose of iron
oxide nanoparticles, formation of excess ROS takes place that affects normal functioning of the cell,
leading to apoptosis or cell death [21]. Int. J. Mol. Sci. 2013, 14 15913 Table 1. Comparison of synthesis methods of magnetic nanoparticles. Modified with
permission from [26]. Method
Parameters
Reaction
temperature (°C)
Reaction
time
Solvent
Size
(nm)
Yield
Thermal
decomposition
Nitrogen atmosphere,
reflux condition
100–320
Slow
Water
1.5–8
High
Co-precipitation
Ambient condition
20–90
Fast
Organic 2–15
High
Microemulsion
Ambient condition
20–50
Fast
Organic
2–12
Low 3. Imaging Techniques Imaging techniques used in the clinic generally include MRI, optical imaging, CT, and PET or
SPECT (Table 2). Each imaging technique has its own unique advantages and intrinsic limitations,
such as insufficient sensitivity or spatial resolution, which make it difficult to obtain accurate and
reliable information at the disease site [19]. Table 2. Advantages and disadvantages of imaging modalities. Reprinted with permission
from [28]. Modality
Advantages
Disadvantages
MRI
High spatial resolution
Low sensitivity
Good soft tissue contrast
Relatively long acquisition time
Provides both anatomical and functional
information
Requires expensive equipment
PET
Provides biochemical information
Limited anatomical information
High sensitivity
Requires specialized equipment
Three-dimensional imaging
Requires radio-nucleotide facilities
Can monitor changes in tumour metabolism and
drug biodistribution
Requires expensive equipment
SPECT
Potential to detect multiple probes simultaneously
in contrast to PET
Lower sensitivity than PET
Lower resolution
CT
High-sensitivity anatomical imaging
Limited functional information
Provides three-dimensional image
Poor soft tissue contrast
Requires expensive equipment
Optical
(BLI and
fluorescent)
Wide applicability
Requires genetic manipulation of
investigated cells
Simultaneously monitor several molecular events
Provides limited anatomical
information
Relatively inexpensive
Amenable to smaller research laboratories
Reduced sensitivity with increased
imaging depth 2. Advantages and disadvantages of imaging modalities. Reprinted with permission
28] MRI is a useful problem-solving diagnostic tool in the clinical field because it has higher spatial
resolution and contrast in soft tissue than other imaging modalities. MRI is based on the magnetism
property of protons that align themselves in a very large magnetic field. These protons originate from
water molecules present in our body tissue. A radiofrequency generated at a particular frequency, Int. J. Mol. Sci. 2013, 14 15914 known as the “resonance frequency,” can flip the spin of a proton. When the electromagnetic field is
turned off, the proton flips back to the original state, generating a radiofrequency signal. This process
is called “relaxation.” The receiver coils measure this relaxation, which is turned into an image by a
computer algorithm [29]. MRI contrast agents are used to modify the relaxation rates at time T1 or T2. T1 contrast agents, such as gadolinium chelators, enhance the positive signal on T1-weighted
images, while T2 agents, such as SPION-based contrast agents, decrease the signal intensity on
T2-weighted images. Optical imaging techniques have used different physical parameters of light to interact with tissue,
and a number of different optical imaging approaches have been described. 3. Imaging Techniques These techniques rely on
fluorescence, absorption, reflectance, or bioluminescence as a source of contrast. Optical imaging
consists mostly of NIRF (near-infrared fluorescence), reflectance imaging, and bioluminescence
imaging. Despite its relatively recent emergence, optical imaging is rapidly being implemented in
various clinical and research areas. The advantages of optical imaging are that most of the optical
contrast agents are non-toxic and comparatively inexpensive, versatile, and sensitive [30–32]. Optical
imaging involves an optical contrast agent, either organic or inorganic, that can fluoresce at various
excitation wavelengths. Early optical imaging was used in cancer diagnosis based on the variation in
endogenous fluorescence of neoplastic tissue. However, due to the difficulty in distinguishing the
diagnostic signal component from background fluorescence, exogenous contrast agents were
developed [31]. Nanoparticle based optical contrast agents, such as “quantum dots,” were thus
developed to have superior imaging properties compared to organic counterparts. The major issues
encountered by optical imaging are the inability to quantify the image and the autofluorescence of
normal tissue against the fluorescence of the contrast agent, which can severely impair the image
quality [13]. CT is another imaging technique widely used in the clinic for diagnosis of various diseases. It uses
x-rays to obtain images through slices of the body area. The main advantage of the CT imaging
technique is that it produces images with high spatial resolution. The most popular CT contrast agents
are iodine-based compounds. These agents work by blocking X-rays, thereby providing contrast and
enhancing a part of the body. Iodine-based contrast agents produce side effects, such as vomiting,
itching, and anaphylactic shock. Among non-ionic and ionic contrast agents, ionic agents prove to be
more harmful, especially for patients with renal problems [33–35]. Therefore, recent research has
focused on developing a gold nanoparticle based contrast agent for CT. Gold nanoparticles are
biocompatible and are capable of targeting the tumor by the EPR effect. This material has a very high
X-ray absorption coefficient, which makes it a suitable agent for replacing iodine in CT imaging. Gold
is a metal with a high atomic number and is therefore considered a strong candidate for CT imaging
because it provides better x-ray attenuation and contrast [36]. PET and SPECT fall under the category of nuclear imaging because they are based on the detection
of radioisotopes that emit one or two gamma rays or positrons. 3. Imaging Techniques Both are excellent imaging techniques
due to their specificity, sensitivity, and fast detection time. However, both techniques lack the ability
to display good spatial resolution. PET uses isotopes such as 68Ga, 76Br, 94mTc, 11C, 13N, 15O, 18F, and
64Cu. SPECT uses gamma-emitting heavy radioisotopes such as 123I, 99mTc, and 133Xe [37]. Int. J. Mol. Sci. 2013, 14 15915 4. Multimodal Strategies for Cancer Theranostics Imaging modalities vary in sensitivity, resolution, and quantitative capabilities. The major dilemma
faced in single-modality imaging is the inability to assure the conformance of diagnosis, which is a
crucial factor in determining the treatment. This problem can be solved by multimodal imaging
because each imaging modality offers its own unique benefits. To overcome the intrinsic limitations of
a specific modality, multimodal imaging offers a combination of techniques with complementary
strengths [38,39]. For example, PET images provide high-sensitivity biological and functional
information about cancer. Conversely, CT and MRI can offer high-resolution images to gather
anatomical information. Therefore, a combination of these imaging modalities can provide not only
high sensitivity and resolution simultaneously but also more detailed anatomical or biological
information about cancer. Figure 1 represents a general scheme for combining different modalities into
a single nanoparticle based on an iron oxide core. Multimodal imaging probes that carry more than two
imaging agents have the potential to overcome the limitations of a single imaging modality. They
might also provide detailed information on the target site by targeted delivery. Multimodal imaging for
cancer theranostics is a cutting-edge technology that maximizes the advantages of nanoparticles [7,19]. Iron oxide nanoparticles are an attractive option as a base platform for multimodal agent
development due to their intrinsic material properties that allow for tunable pharmacokinetics and
surface modification. Figure 1. Concept of multimodal contrast agent based on SPION. Figure 1. Concept of multimodal contrast agent based on SPION. Int. J. Mol. Sci. 2013, 14 15916 4.1. MRI/Optical Imaging The development of MRI and NIRF dye conjugated contrast agents is a major research effort in
MRI/optical dual imaging strategies [40,41]. MRI is an excellent anatomical imaging technique, but it
is incapable of measuring molecular events such as protease activity and gene expression. However,
NIRF imaging can produce the functional details of a molecular event. Therefore, these techniques
complement each other to improve overall imaging quality [42]. Among the NIRF dyes, cyanine dye
(Cy5.5) is widely used in SPION. MRI and optical imaging are the most well accepted techniques in
multimodal imaging and are better associated with NIRF-based molecular imaging probes with
emission wavelengths ranging from 650 to 900 nm [43]. Our group fabricated MRI/optical dual-contrast agents specifically for in vivo cancer imaging by
thermally cross-linking SPION using Si-OH containing co-polymer [44]. The optical imaging property
was added by conjugating Cy5.5 dye to SPION after amine modification. The Cy5.5-labeled SPION
was tested in Lewis lung carcinoma-bearing mice and resulted in a tumor specific T2 signal drop of
68% in MRI, with fluorescence lasting up to 4 h in optical imaging. Additionally, the specific
accumulation of Cy5.5-SPION in the tumor was further confirmed by comparing ex vivo images of
harvested tumor and other organs. Figure 2. (A) T2 weighted images. The images are before and after the injection of
CLIO-EPPT in human pancreatic adenocarcinoma. The T2 relaxation rate is 46.5%
(arrow), whereas muscle (arrowhead) is not affected; (B) Color-coded map of NOD/SCID
mice bearing orthotopically implanted human pancreatic adenocarcinoma obtained 24 h
after I.V. injection of CLIO-EPPT that shows high-intensity fluorescence. Reprinted with
permission from [45]. (A)
(B)
In another study performed by Medarova et al., underglycosylated mucin-1 tumor-specific antigen
(uMUC-1) targeted CLIO (dextran-coated crosslinked SPION) was synthesized [45]. CLIO was
modified with NIRF-capable Cy5.5 dye. The EPPT peptides conjugated to CLIO can bind to uMUC-1 (B) (A) (B) (A) (B) In another study performed by Medarova et al., underglycosylated mucin-1 tumor-specific antigen
(uMUC-1) targeted CLIO (dextran-coated crosslinked SPION) was synthesized [45]. CLIO was
modified with NIRF-capable Cy5.5 dye. The EPPT peptides conjugated to CLIO can bind to uMUC-1 Int. J. Mol. Sci. 2013, 14 15917 antigen. EPPT is a synthetic peptide derived from CDR3 Vh region of an adult skeletal muscle
monoclonal antibody, which can specifically bind to uMUC-1 antigen. 4.1. MRI/Optical Imaging This probe could not only
detect orthotopically implanted preclinical models of adenocarcinomas but could also track in vivo
tumor response to chemotherapy in real time. T2 relaxation rate of CLIO-EPPT in the tumor dropped
about 46.5% in orthotopically implanted pancreatic adenocarcinoma (Figure 2A), a change that was
comparable to the 53% signal drop of the subcutaneous tumor model the group has previously studied
as a proof-of-principle [46]. In vivo NIRF optical imaging, using CLIO-EPPT, showed high-intensity
fluorescence (Figure 2B). 4.2. MRI/CT Imaging Anatomical imaging is an important feature requiring the collection of information regarding lesion
occurrence in the body to enable further decisions. Although both MRI and CT are relatively weak in
functional imaging, they are excellent modalities for anatomical imaging. A MRI/CT dual-modal agent
is a combination of two anatomic modalities that share almost the same functional features [35]. The
main difference between MRI and CT is the physics involved in the respective imaging processes. CT
uses X-ray radiation passing through tissue. The image is recorded based on the absorption and
attenuation characteristics of the contrast agent. In contrast, MRI works under the influence of a strong
magnetic field, and the image is analyzed on the basis of the T1 or T2 contrast enhancement induced
by nanoparticles [47]. Gold nanoparticles are most commonly used as CT contrast agents. They are combined with SPION
to form a hybrid known as gold iron oxide nanoparticles (GION), which can be used as a dual-contrast
agent for MRI and CT [48]. Gold acts as the CT contrast moiety, but it provides less spatial resolution
and sensitivity compared to MRI. A dual-contrast agent with an iron oxide core and a gold-layered
shell with PEG coating on the surface was synthesized to provide synergistic effects in both CT and
MRI. The PEG-coated GION was dispersed in water, and its average hydrodynamic size was
approximately 47 nm when measured by TEM. The intensity of the CT images for PEG coated GION
was greater than iodine-based counterparts at the same concentration, but the T2 signal efficiency
decreased compared to oleic acid/oleylamine coated SPION. Our group [49] reported the synthesis of
hybrid nanoparticles by thermal decomposition of Au-oleylamine and Fe-oleate mixtures, followed by
subsequent coating with amphiphilic poly(DMA-r-mPEGMA-r-MA) by nano-emulsion. The resulting
nanoparticles showed acceptable water dispersion and biocompatibility (Figure 3). The in vivo CT
contrast efficiency had improved contrast enhancement (1.6-fold) 1 h post-injection in a murine
hepatoma model. The T2 relaxivity coefficient was greater than that of Resovist®. Additionally, the
in vivo result was noticeably different from hepatoma and normal tissue. The hybrid GION serves as a dual-contrast agent effectively by providing better contrast than
iodine agents. The hybrid gold-SPION also displayed better CT contrast enhancement and relatively
high T2 relaxivity in MRI. These two cases serve as strong examples of multimodal system
development and demonstrate that multimodality is superior to a single-modal system. Int. J. Mol. Sci. 4.2. MRI/CT Imaging 2013, 14 15918 Figure 3. Schematic illustration (a) shows hybrid nanoparticles before and after coating
with amphiphilic poly (DMA-r-mPEGMA-r -MA), also shown in TEM images (b) and (c),
respectively. CT (d) and MRI (e) images in a hepatoma model at various time points. A white arrow indicates the site of the tumor. Reprinted with permission from [49]. A white arrow indicates the site of the tumor. Reprinted with permission from [49]. (d)
(e)
MRI/PET Imaging
MRI/PET imaging evolved from PET/CT and is the preferred modality for anatomic and fu
ging. MRI/PET has a distinct advantage over PET/CT because of the reduced radiation ex
T is a nuclear imaging technique that is similar to SPECT because it is ba
onucleotide-emitting positrons. MRI obtains an image with enhanced spatial resolution,
recting partial-volume effects caused by PET imaging. Multimodal PET/MRI consis
onucleotide conjugated with an MRI agent SPION [50]. (d) ( )
(e) (e) 4.3. MRI/PET Imaging 4.3. MRI/PET Imaging MRI/PET imaging evolved from PET/CT and is the preferred modality for anatomic and functional
imaging. MRI/PET has a distinct advantage over PET/CT because of the reduced radiation exposure. PET is a nuclear imaging technique that is similar to SPECT because it is based on
radionucleotide-emitting positrons. MRI obtains an image with enhanced spatial resolution, thereby
correcting partial-volume effects caused by PET imaging. Multimodal PET/MRI consists of a
radionucleotide conjugated with an MRI agent SPION [50]. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 15920 In another work by Torres Martin de Rosales et al., 64Cu radiolabeling was performed directly on
the inorganic surface rather than the SPION coating using bisphosphonates (BP) [53]. The chelating
agent dithiocarbamate (DTC) was used for conjugating the 64Cu to the BP. The final complex formed
[64Cu (dtcbp) 2] (dithiocarbamate bisphosphonates) was stable in PBS and human serum for 48 h. Then, the [64Cu(dtcbp)2] complex was labeled with clinically available dextran-coated SPION by
virtue of BP’s high affinity to iron oxide. In vivo studies were conducted in a 9.4 T NMR magnet and a
NanoPET–CT scanner. The in vivo model for the study was the lymphatic system, which provided
information regarding the early spread of cancer. A foot pad injection of [64Cu(dtcbp)2]–endorem
resulted in a notable decrease in MR signal in popliteal lymph nodes after 3 h, which provides support
for endorem accumulation. PET imaging of 64Cu-induced signal confirmed the uptake of nanoparticles
in popliteal lymph nodes and iliac lymph nodes (Figure 4). 4.4. MRI/SPECT Imaging There has also been increased interest in combining SPION with SPECT probes for MRI/SPECT
dual-modality imaging (Table 3). One advantage of SPECT is the opportunity to obtain information on
molecular processes using specific radiolabels. SPECT also allows a clinician to determine the
biodistribution of the radiotracer tagged particles in vivo non-invasively in the pico-molar
concentration range. However, a disadvantage of SPECT is that it offers limited anatomical details and
spatial resolution. MRI is used in conjunction with SPECT to obtain quality anatomical images, thus
offering both the structural and functional benefits of dynamic imaging. MRI/SPECT
reduces discomfort associated with multiple sessions and scan times required for separate imaging
procedures [4]. Table 3. Characteristics of nanoparticles for multimodal imaging. Imaging
modality
Types of nanoparticle
Pros
Cons
References
MRI/Optical Cy5.5-SPIONCy5.5-
CLIO-EPPT
Good anatomical detailing,
and functional detailing of
molecular event
Passive targeting
[44,45,54]
MRI/CT
GION, Hybrid Gold
SPION
Good anatomical detailing
Poor functional
imaging capability
No tumor targeting
[48,49]
MRI/PET
64Cu -crude-SPION,
64Cu -PASP-IO
High sensitivity
Different dosage of
PET and MRI agents. [51–53]
MRI/SPECT
111 In -Bam-IO,99mTc-
PEG-IO,99mTc- DPA-
SPION
High sensitivity and
functional information
-
[4,5,55]
Recently, Misri and colleagues have developed an antibody-conjugated MRI/SPECT dual-modality
imaging probe specifically for malignant mesothelioma [4]. Mesothelin targets antigens for
malignant mesothelioma using 111In labeled anti-mesothelin monoclonal antibody (mAbMB) coated on
iron oxide nanoparticles. A cell uptake study showed specific uptake of In-mAbMB-SPION by
mesothelin-positive cells. An in vivo study of a mouse xenograft model of A431K5 tumors showed a Table 3. Characteristics of nanoparticles for multimodal imaging. Imaging
modality
Types of nanoparticle
Pros
Cons
References
MRI/Optical Cy5.5-SPIONCy5.5-
CLIO-EPPT
Good anatomical detailing,
and functional detailing of
molecular event
Passive targeting
[44,45,54]
MRI/CT
GION, Hybrid Gold
SPION
Good anatomical detailing
Poor functional
imaging capability
No tumor targeting
[48,49]
MRI/PET
64Cu -crude-SPION,
64Cu -PASP-IO
High sensitivity
Different dosage of
PET and MRI agents. [51–53]
MRI/SPECT
111 In -Bam-IO,99mTc-
PEG-IO,99mTc- DPA-
SPION
High sensitivity and
functional information
-
[4,5,55]
Recently, Misri and colleagues have developed an antibody-conjugated MRI/SPECT dual-modality
imaging probe specifically for malignant mesothelioma [4]. Mesothelin targets antigens for
malignant mesothelioma using 111In labeled anti-mesothelin monoclonal antibody (mAbMB) coated on
iron oxide nanoparticles. A cell uptake study showed specific uptake of In-mAbMB-SPION by
mesothelin-positive cells. An in vivo study of a mouse xenograft model of A431K5 tumors showed a Table 3. Characteristics of nanoparticles for multimodal imaging. Int. J. Mol. Sci. 2013, 14 15919 Yang et al. developed a water-soluble, SPION-based nanocarrier [51]. A tumor with integrin αvβ3
expression was targeted using PEGylated SPION by cyclic Arg-Gly-Asp-D-Phe-Cys (cRGD) peptides,
and dual modality imaging was attained using PET 64Cu chelators for PET imaging. However,
the MRI relaxivity for cRGD-conjugated SPION was lower than that for clinically available
iron oxide particles (Feridex®). PET imaging better evaluated the capability of cRGD-conjugated
SPION to accumulate in tumors. The same targeting strategy was applied by Lee et al., in which the
αvβ3-integrin expressing tumor was targeted using iron oxide nanoparticles coated with polyaspartic
acid (PASP) [52]. The RGD peptide was used as an integrin αvβ3 targeting agent, and
1,4,7,10-tetraazacyclododecane-N,N',N'',-tetraacetic acid (DOTA) chelators were used after labeling
with 64Cu for PET. DOTA IO-RGD conjugates bound specifically to integrin αvβ3 in vitro. MRI/PET
imaging showed integrinspecific delivery with significant uptake of conjugated RGD-PASP-IO
(Arg-Gly-Asp-D-Phe-Cys-Polyaspartic acid-iron oxide) nanoparticles in the reticuloendothelial system
(RES). The reason stated for the significant uptake was the hydrodynamic size of the nanoparticle,
which is approximately 45 nm. The particle is large enough for RES detection and uptake. Further optimization to control for the size and tumor specificity has been identified as a goal for
future studies. Figure 4. In vivo PET/MRI imaging studies with [64Cu (dtcbp) 2]–Endorem in a mouse. (A) and (B) show signal decreases in popliteal lymph nodes before and after injection
indicated by solid arrows; (C) refers to NanoPET/CT images, showing uptake in popliteal
(represented by solid arrows) and iliac (represented by hollow arrows) lymph nodes;
(D) A whole body NanoPET–CT image of the mouse. Reprinted with permission
from [53]. Int. J. Mol. Sci. 2013, 14 4.4. MRI/SPECT Imaging Imaging
modality
Types of nanoparticle
Pros
Cons
References
MRI/Optical Cy5.5-SPIONCy5.5-
CLIO-EPPT
Good anatomical detailing,
and functional detailing of
molecular event
Passive targeting
[44,45,54]
MRI/CT
GION, Hybrid Gold
SPION
Good anatomical detailing
Poor functional
imaging capability
No tumor targeting
[48,49]
MRI/PET
64Cu -crude-SPION,
64Cu -PASP-IO
High sensitivity
Different dosage of
PET and MRI agents. [51–53]
MRI/SPECT
111 In -Bam-IO,99mTc-
PEG-IO,99mTc- DPA-
SPION
High sensitivity and
functional information
-
[4,5,55] Table 3. Characteristics of nanoparticles for multimodal imaging. Recently, Misri and colleagues have developed an antibody-conjugated MRI/SPECT dual-modality
imaging probe specifically for malignant mesothelioma [4]. Mesothelin targets antigens for
malignant mesothelioma using 111In labeled anti-mesothelin monoclonal antibody (mAbMB) coated on
iron oxide nanoparticles. A cell uptake study showed specific uptake of In-mAbMB-SPION by
mesothelin-positive cells. An in vivo study of a mouse xenograft model of A431K5 tumors showed a Int. J. Mol. Sci. 2013, 14 15921 signal drop post-24-h scan. The biodistribution study using SPECT imaging also showed relatively low
uptake in the other normal organs (lungs, heart, intestine, stomach, muscle, and brain) compared to the
tumor. This result was well correlated with autoradiography images (Figure 5). Figure 5. T2-weighted axial MRI for A431K5 tumors. (A), (B), and (C) show pre-injection
and 24 and 72 h post-injection MR images. The inset represents autoradiographic
images of 20 μM tumor sections; (D) ROI intensity histograms for T2-weighted axial
gradient-echo MR images of A431K5 tumors, which show apparent shifts in a 24-h image
towards low intensity. Reprinted with permission from [4]. In another example, Madru et al. developed 99mTc-labeled PEG coated iron oxide as a multimodal
contrast agent for imaging a sentinel lymph node (SLN) [55]. The SLN is considered the first lymph
node receiving the lymphatic drainage from a malignant tumor, where metastatic cells might anchor
initially. MRI/SPECT both aided in identifying the SLN and provided pre-surgical information
regarding the location and characteristics of the lymph node. SPECT gave a clear visualization of the
lymph node and the highest intensity was found in the popliteal lymph nodes. The clearance of
99mTc-SPION was comparable to the other similar sized nanocolloids. The MR image showed a
non-homogenous uptake of 99mTc-SPION. This approach can be used for breast cancer and malignant
melanoma imaging. In another example, Madru et al. developed 99mTc-labeled PEG coated iron oxide as a multimodal
contrast agent for imaging a sentinel lymph node (SLN) [55]. 4.4. MRI/SPECT Imaging The SLN is considered the first lymph
node receiving the lymphatic drainage from a malignant tumor, where metastatic cells might anchor
initially. MRI/SPECT both aided in identifying the SLN and provided pre-surgical information
regarding the location and characteristics of the lymph node. SPECT gave a clear visualization of the
lymph node and the highest intensity was found in the popliteal lymph nodes. The clearance of
99mTc-SPION was comparable to the other similar sized nanocolloids. The MR image showed a
non-homogenous uptake of 99mTc-SPION. This approach can be used for breast cancer and malignant
melanoma imaging. In another example, Madru et al. developed 99mTc-labeled PEG coated iron oxide as a multimodal
contrast agent for imaging a sentinel lymph node (SLN) [55]. The SLN is considered the first lymph
node receiving the lymphatic drainage from a malignant tumor, where metastatic cells might anchor
initially. MRI/SPECT both aided in identifying the SLN and provided pre-surgical information
regarding the location and characteristics of the lymph node. SPECT gave a clear visualization of the
lymph node and the highest intensity was found in the popliteal lymph nodes. The clearance of
99mTc-SPION was comparable to the other similar sized nanocolloids. The MR image showed a
non-homogenous uptake of 99mTc-SPION. This approach can be used for breast cancer and malignant
melanoma imaging. The dual-modal probe developed by Misri and colleagues displayed better MR relaxivity compared
to previously developed MRI/SPECT agents. The targeting efficiency with mesothelin-positive tumors
was proven, and the SPECT detection motif 111In was used to obtain autoradiography and
biodistribution data. In the work of Madru et al., the radiolabeling efficiency of 99mTc-labeled PEG
coated iron oxide reached 99%. The SLN uptake of nanoparticles was 100% ID/g, suggesting that the
nanoparticle efficiency and SPECT/MRI detection in lymph node should encourage its usage in
other cancers. Int. J. Mol. Sci. 2013, 14 15922 5. Multifunctional Magnetic Nanoparticle for Cancer Theranostics The multi-functionality of nanoparticles further enables the integration of imaging and therapy
(so-called theranostics). Theranostics refer to agents that target molecular biomarkers of a disease and
are expected to contribute to personalized medicine. Among the currently available nano-vehicles,
SPION have received great attention in the development of theranostic nanomedicines because they
are not only used as contrast enhancement agents for MRI but can also deliver therapeutic agents, such
as anticancer drugs and siRNA, to disease sites. In addition, SPION can continuously emit heat
upon exposure to an alternating external magnetic field (AMF) by converting electromagnetic
energy into heat [56–61]. Figure 6 depicts three different approaches in developing a SPION-based
theranostic agent. Figure 6. Different approaches adopted in developing a SPION-based theranostic agen Figure 6. Different approaches adopted in developing a SPION-based theranostic agent. Int. J. Mol. Sci. 2013, 14 15923 The multi-functionality of a SPION-based nanosystem was explored using drug and imaging
modalities. With the development of biocompatible polymer-coated SPION loaded with DOX,
Yu et al. [62] successfully evaluated its tumor-reduction efficacy in lung cancer. Additionally, imaging
from MRI and NIRF provided diagnostic information. Santra [56] developed a theranostic nanoparticle
with similar MRI/optical capabilities and a folate receptor-targeting moiety. The A549 lung cancer cell
line was used to evaluate cell uptake and study toxicity. A high tumor-specific uptake and
doxorubicin-induced cell death were observed. Another unique triple-modal system developed by
Xie et al., was modified in an adjunct study to include a DOX in the HSA (Human Serum Albumin)
matrix to form DOX conjugated HSA–iron oxide nanoparticles [62]. The evaluation of tumor
reduction in 4T1 tumor-bearing mice revealed that the DOX conjugated HSA–iron oxide nanoparticle
was superior to DOX and similar to Doxil. Yu et al. [63] synthesized thermally cross-linked SPION (TCL-SPION) and coated them with a
negatively charged anti-biofouling polymer and Cy5.5 dye. The nanoparticles were then loaded with
positively charged doxorubicin (DOX) using electrostatic interactions. The nanoparticles were
systematically injected into lung cancer bearing mice and resulted in a significant decrease in tumor
size. The nanoparticle uptake in this study was achieved by the EPR effect using a passive targeting
strategy and was evaluated by in vivo MRI and optical imaging (Figure 7). Dox-TCL-SPION were
injected in mice with Lewis lung carcinoma. The data indicate that the tumor reduction was
approximately 63% relative to control and 38% for the DOX group. A T2-weighted image of mice
suggested a 58% drop in signal, indicating significant accumulation in the tumor. An ex vivo
fluorescence study showed intense signal at the tumor area 1 h post-administration and maximum
intensity at 12 h post-injection. Santra et al. developed a theranostic nanoparticle with MRI/optical functionality and targeted
cancer cells expressing folate [56]. A co-encapsulation strategy was adopted to carry a NIR dye and an
anti-cancer drug in the hydrophobic region of polyacrylic acid (PAA), which is coated on SPION by a
modified solvent diffusion technique. Initially, iron oxide coated with PAA formed propargylated
(presence of triple-bond) nanoparticles that served as a precursor for multimodal folate conjugated
nanoparticles by click chemistry. The versatility of the nanosystem was proven by the encapsulation of
taxol and lipophilic fluorescent dye in the hydrophobic region. 5.1. Drug Delivery Chemotherapy is a common therapeutic approach to fighting cancer. However, it is non-selective
and causes significant potential side effects to healthy tissues. To overcome this problem, magnetic
nanoparticles loaded with the drug can serve as potential drug carriers in a new drug delivery strategy
(Table 4). The targeted delivery of therapeutics has the potential to localize therapeutic agents to
specific tissues to enhance treatment efficacy and minimize side effects. Recently, several chemical
drugs, including paclitaxel, doxorubicin (DOX), and methotrexate (MTX), have been combined with
magnetic nanoparticles for cancer therapy [40,56,62]. Table 4. Characteristics of multifunctional magnetic nanoparticles in drug delivery. Nanoparticle property
Modality
Drug
References
cy5.5-SPION
MRI/Optical
Doxorubicin
[63]
PAA-IONPs
MRI/Optical
Taxol
[56]
64Cu-Cy5.5-HSA-SPION
PET/NIRF/MRI
Doxorubicin
[62,64] Table 4. Characteristics of multifunctional magnetic nanoparticles in drug delivery. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 The feasibility of the nanoparticles for
use in in vivo studies was confirmed by encapsulating NIR dialkylcarbocyanine dye and tested for
in vitro uptake in an A549 lung cancer cell line. The fluorescence measurement demonstrated higher
cell uptake for NIR and folate receptor compared with the control group. The cell study showed
targeted uptake of nanoparticles in A549 cells with induction of cell death mediated by the acidic
nature of a tumor cell, which assisted in the release of dye and taxol. Finally, modifying the targeting
ligand can customize the ligand for other cancer types. Int. J. Mol. Sci. 2013, 14 15924 Figure 7. (a) T2-weighted MR images taken at 0 h and 4.5 h after injection of
Dox@TCL-SPION at the LLC tumor on the right back of the mouse; (b) Optical
fluorescence images of major organs and allograft tumors: 1 liver; 2 lung; 3 spleen;
4 tumor; 5 heart; and 6 kidney. Images were taken after intravenous injection of
Dox@TCL-SPION (equivalent to 4 mg of Dox) (above) and free Dox (4 mg) into
tumor-bearing mice (below) (n = 3). The mice were euthanized after 1 h and 12 h. Reprinted with permission from [63]. Reprinted with permission from [63]. (a)
(b)
5.2. Gene Delivery
Gene therapy entails the delivery of a therapeutic gene to the disease tissue to functionally replace a
defective gene and cure the pathological genotypes by expression of a therapeutic gene. Small-interfering
RNA (siRNA) is a class of double-stranded RNA molecules that can inhibit any specific protein
expression at the post-transcriptional level. The process is known as RNA interference (RNAi). Genes
are integrated into SPION, which protect the nucleic acids against enzymatic degradation and facilitate
cellular internalization and endosomal release (Table 5). SPION are believed to be an excellent vehicle
for siRNA delivery because they are biocompatible and target-functionalized [65]. Even hard-to-transfect
cell lines can be delivered with plasmid DNA or siRNA by novel methods such as magnetofection,
where SPION is subjected to oscillating magnetic fields that facilitate caveolae-mediated endocytosis
of SPION and cargo nucleic acid [66,67]. MRI-visible SPION have also been found to carry a
dual-modality function that enhances monitoring of siRNA delivery to the target area [68]. (a) (a)
(b) (a) (b) 5.2. Gene Delivery Gene therapy entails the delivery of a therapeutic gene to the disease tissue to functionally replace a
defective gene and cure the pathological genotypes by expression of a therapeutic gene. Small-interfering
RNA (siRNA) is a class of double-stranded RNA molecules that can inhibit any specific protein
expression at the post-transcriptional level. The process is known as RNA interference (RNAi). Genes
are integrated into SPION, which protect the nucleic acids against enzymatic degradation and facilitate
cellular internalization and endosomal release (Table 5). SPION are believed to be an excellent vehicle
for siRNA delivery because they are biocompatible and target-functionalized [65]. Even hard-to-transfect
cell lines can be delivered with plasmid DNA or siRNA by novel methods such as magnetofection,
where SPION is subjected to oscillating magnetic fields that facilitate caveolae-mediated endocytosis
of SPION and cargo nucleic acid [66,67]. MRI-visible SPION have also been found to carry a
dual-modality function that enhances monitoring of siRNA delivery to the target area [68]. SPION functionalized with PEG grafted polyethylenimine (PEG-g-PEI) was synthesized by
Chen et al. using the CD44v6 single-chain variable fragment as a targeting agent for gastric cancer,
and siRNA was loaded for the therapy [69]. Compared to viral vector delivery, non-viral vector Int. J. Mol. Sci. 2013, 14 15925 PEG-g-PEI had less cytotoxicity and consisted of primary, secondary, and tertiary amines for forming
complexes with siRNA. The strategy used single-chain variable fragment (scFv) targeting CD44
variant 6 (CD44v6) antigens. The transfection efficiency was studied using flow cytometry against
lipofectamine (a control siRNA delivery agent in SGC-7901 gastric cancer cells). The results showed
an efficiency of more than 95% for all three siRNA delivery agents, and fluorescence intensity tests
revealed that the scFvCD44v6-PEG-g-PEI-SPION complex intake was greater than that of the
PEG-g-PEI-SPION complex. To assess cancer-targeting capability, two tumors (SGC-7901 and
A375), one CD44v6 positive and the other negative, were induced in mice, and PEG-g-PEI-SPION
and scFvCD44v6-PEG-g-PEI-SPION contrast agents were injected. As expected, the SGC-7901
tumor area showed a T2 signal drop for the scFvCD44v6-PEG-g-PEI-SPION group and proved
targeting capability. An all-in-one nanoparticle was developed with multimodal imaging and siRNA delivery capability
by Lee et al., in which the fabricated core material was manganese-doped magnetism-engineered iron
oxide (MnMEIO) nanoparticles coated with BSA [61]. This nanoparticle was selected for use in MRI
applications because of its small particle size (15 nm), monodispersed nature and higher magnetic
moment than other iron oxide nanoparticle variants. 5.2. Gene Delivery To conjugate the siRNA and thiolated siRNA
(HS–siGFP–Cy5) targeting agents onto the surface of the nanoparticle, BSA was modified with
pyridyldisulfide groups by N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) treatment. Thus,
activated MnMEIO nanoparticles were treated and functionalized with thiolated poly(ethylene glycol)
(PEG, MW 3400), cyclic Arg-Gly-Asp (RGD) peptide and Cy5-dye labeled thiolated siRNA
(HS–siGFP–Cy5). The effect of MnMEIO–siGFP–Cy5/PEG–RGD nanoparticles was studied in cell
lines expressing GFP (MDA-MB-435-GFP and A549-GFP cells), and the result showed dose-dependent
decreases in GFP expression, which validate the effect of synthesized nanoparticles. Table 5. Characteristics of multifunctional magnetic nanoparticles in gene delivery. Nanoparticle property
Modality
siRNA action
Transfection
efficiency
References
scFvCD44v6,-PEG-g-PEI-SPION
MRI
Not studied
95%
[69]
MnMEIO–siGFP–Cy5/PEG–RGD
MRI/Optical
GFP Suppression
Not studied
[61] Table 5. Characteristics of multifunctional magnetic nanoparticles in gene delivery. 6. Conclusions Multimodal and multifunctional probes are cutting-edge technologies in which the advantages of
nanoparticles are maximized. The use of MRI has prompted the development of a variety of multimodal
probes based on T2 contrast agents. SPION is considered an ideal vehicle for multimodal and
multifunctional applications. The theranostic agents based on SPION are crucial in cancer detection
and the delivery of therapeutic payloads, such as chemotherapeutic drugs and genes. New research is
dedicated to developing multifunctional and multimodal theranostic agents, which form the ultimate
weapon against cancer. However, there are several challenges facing multimodal and multifunctional
techniques, including the standardization of therapy response and the stability of complex nanoparticles
under certain biological conditions. Future research must focus on rectifying the current flaws in
multimodal and multifunctional imaging probes by introducing new biocompatible polymers. Finally,
multimodal and multifunctional techniques may enhance clinical theranostics in the near future. 5.3. Hyperthermia Hyperthermia is a form of treatment in which the temperature of body tissue is raised to 42 °C. This treatment drastically affects the functionality of cellular structures, proteins, cell membrane, nucleic
acid repair enzymes, and induces cell death. Tumor cells can also be killed due to their lower tolerance
of a sudden variation in temperature using magnetic hyperthermia (MHT). SPION possess the inherent
property of generating heat by ferromagnetic resonance. However, the heating process is generated by
Néel and Brownian relaxations during MHT treatment [58]. Therefore, it has been widely studied for
treatment in different types of cancer, such as head and neck cancer, glioma, and cervical cancer [59,60]. The possibility of using SPION in both multimodal and nanoheater applications has been studied by
Hoskins et al. [70]. SPION were synthesized with an average size of 30 nm in diameter and coated
with gold and PEI as the intermediate layer. Finally, PEG was coated on the particles to form Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 15926 Fe3O4-PEI-Au-PEG nanoparticles with improved biocompatibility. To study the hyperthermia effect,
the nanoparticles were suspended in agar to mimic in vivo conditions. Applying laser irradiation at a
532-nm wavelength for 90 s to a nanoparticle concentration of 50 μg/mL induced changes in
temperature of approximately 32 °C. The MRI contrast effect was found to be comparable to that of
Feridex®. PEI acts as a cushion between the core and shell and helps to maintain their physical
properties. The magnetic properties of SPION had also been retained after the gold coating was
applied. This study raises the possibility of using SPION-GOLD hybrid nanoparticles for multimodal
contrast imaging and cancer treatment by hyperthermia. Acknowledgments This work was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (MEST)
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monitor gene therapy with molecular imaging. Methods 2009, 48, 146–160. 69. Chen, Y.; Wang, W.; Lian, G.; Qian, C.; Wang, L.; Zeng, L.; Liao, C.; Liang, B.; Huang, B.;
Huang, K.; et al. Development of an mri-visible nonviral vector for sirna delivery targeting gastric
cancer. Int. J. Nanomed. 2012, 7, 359–368. 70. Hoskins, C.; Min, Y.; Gueorguieva, M.; McDougall, C.; Volovick, A.; Prentice, P.; Wang, Z.;
Melzer, A.; Cuschieri, A.; Wang, L. Hybrid gold-iron oxide nanoparticles as a multifunctional
platform for biomedical application. J. Nanobiotechnol. 2012, 10, 27. © 2013 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 license
(http://creativecommons.org/licenses/by/3.0/). © 2013 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 license
(http://creativecommons.org/licenses/by/3.0/). © 2013 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 license
(http://creativecommons.org/licenses/by/3.0/).
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EDITORIAL
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Editorial A Educação em Revista tem o prazer de publicar o número 19, volume 01
que apresenta ao leitor 10 (dez) artigos na área da Educação com diferentes enfoques: A Educação em Revista tem o prazer de publicar o número 19, volume 01
que apresenta ao leitor 10 (dez) artigos na área da Educação com diferentes enfoques: O primeiro artigo, intitulado “A APROPRIAÇÃO DO RURALISMO
PEDAGÓGICO E A MATERIALIDADE DA ESCOLA RURAL DE SERGIPE
(1947-1951)” usa como fontes de pesquisa relatos orais, mensagens de governadores
do Estado de Sergipe, publicações da Revista Brasileira de Estudos Pedagógicos, a
obra Educação em Sergipe de Nunes Mendonça, publicações de Acrísio Cruz e foto
grafias (dentre outros), a fim de recuperar os moldes do ruralismo pedagógicos no
Estado. Os autores Rony Rei Nascimento Silva e Ilka Miglio Mesquita concluem que
a escola rural – além de ensinar a ler, escrever e contar – pretendia desenvolver na
criança o amor pela terra e pela vida no campo. No artigo “A RACIONALIDADE SUBJACENTE EM PROCESSOS
DE IMPLANTAÇÕES CURRICULARES: UM OLHAR HABERMASIANO
SOBRE RELATOS DE PROFESSORES DE MATEMÁTICA”, Deise Aparecida
Peralta, João Ricardo Neves da Silva e José Augusto Brito Pacheco desenvolvem refle
xões históricas acerca das reestruturações curriculares da disciplina Matemática im
plementadas entre os anos de 1930 e 2013, a partir do relato oral de nove professores
com idade entre noventa e oito e vinte e nove anos. Com base na Teoria da Ação
Comunicativa de J. Habermas, os autores afirmam que a racionalidade técnica – e
não o diálogo com a realidade escolar - fundamentou as ações governamentais. Em “A FILOSOFIA NO ENSINO MÉDIO: O CINEMA COMO
RECURSO DIDÁTICO”, Diego Augusto Doimo e Raimunda Abou Gebran en
tendem o cinema como um recurso didático importante para as aulas de Filosofia
no Ensino Médio, desde que o docente forneça previamente um roteiro de análise, a
partir do qual, o aluno possa acompanhar sua própria aprendizagem, desfazer-se de
concepções previamente estabelecidas e dialogar com a temática colocada. Posta a
problematização a partir do cinema, a leitura do texto filosófico permitiria ao aluno
buscar uma conceituação capaz de instrumentalizar a reflexão acerca dos nossos pro
blemas e do nosso próprio tempo. Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018 Editorial Editorial https://doi.org/10.36311/2236-5192.2018.v19n1.01.p5 Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018
https://doi.org/10.36311/2236-5192.2018.v19n1.01.p5 Editorial No quarto artigo, intitulado “SENTIDOS ATRIBUÍDOS AO MUNDO
DO TRABALHO PELAS CRIANÇAS: A ESCOLA COMO PARTE DESSE
PROCESSO”, as autoras Amanda Antunes Miranda e Raquel Nascimento Coelho
investigam o entendimento que o trabalho adquire para crianças de classe média e
alta com idade entre onze e doze anos, matriculadas em instituições privadas de en 5 5 sino e com bom rendimento escolar. Os entrevistados associam o “bom emprego” ao
preparo intelectual e desqualificam o trabalho voluntário. O autor Juliano Mota Parente identifica os principais elementos que funda
mentam o gerencialismo e a performatividade na gestão educacional brasileira em
seu artigo “GERENCIALISMO E PERFORMATIVIDADE NA GESTÃO DA
EDUCAÇÃO BRASILEIRA”. Segundo o autor, tais diretrizes criam no interior das
escolas um clima de instabilidade; uma cultura de competitividade, de individualis
mo e de disputa de poder. No contexto de performatividade, os processos avaliativos
legitimam o controle do Estado sobre a atividade docente, não levam em considera
ção as especificidades de cada escola e de cada sistema de ensino e impõem a neces
sidade de relatórios, formulários e fichas com informações acerca dos alunos, cuja
confecção demanda um tempo excessivo. No artigo “PROGRAMA ESCOLA DA FAMÍLIA: A ESCOLA A
DESSERVIÇO DA ESCOLA”, Gabriela Guillardi da Silva Maia e Géssica Priscila
Ramos apresentam o programa como assistencialista, cuja proposta – ainda que so
cialmente relevante – fica distanciada daquilo que elas consideram o foco central das
instituições educativas: o desenvolvimento do ensino e da aprendizagem de conheci
mentos efetivamente escolares. A desvinculação entre a “escola dos finais de semana
e a escola dos dias da semana” foi apontada como uma das possíveis razões para o
insucesso da proposta. As autoras Gysely Josiara Ferreira Moura, Lucia Israel da Conceição Silva,
Tatiene Germano Reis Nunes e Rosely Cardoso Maia apresentam os resultados de
uma pesquisa da qual participaram seiscentos e cinquenta e oito jovens com idade
entre treze e vinte e quatro anos, de ambos os sexos, matriculados em escolas públicas
de Educação Básica na cidade de Belém. A pesquisa apresentada no artigo intitulado
“RELAÇÕES ENTRE PARES E VIOLÊNCIA NA ESCOLA: PERCEPÇÕES DE
ESTUDANTES PARAENSES” evidencia a forte exposição dos jovens e adolescentes
à violência no contexto escolar e questiona as relações estabelecidas no interior das
instituições de ensino. Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018 Editorial Em “O MEDIADOR ESCOLAR NO SEGUNDO SEGMENTO
DO ENSINO FUNDAMENTAL: INTERLOCUÇÕES ENTRE ESPAÇOS,
DASAFIOS E POSSIBILIDADES DA INCLUSÃO ESCOLAR”, os autores
William Scheidegger Moreira e Mara Monteiro da Cruz apresentam o relato de uma
experiência desenvolvida em um Colégio de Aplicação Universitário. O mediador es
colar – função exercida por um bolsista da Universidade – acompanha o desempenho
de um aluno diagnosticado com deficiência intelectual leve, transtorno de ansiedade,
atraso cognitivo e motor. O artigo destaca a importância de adaptações curriculares
que favoreçam o processo de aprendizagem dos alunos. No artigo “PRÁTICAS DE LEITURA NA EJA: CONTRIBUIÇÕES PARA
A FORMAÇÃO POLÍTICA DE LEITORES NO ENSINO MÉDIO”, João Paulo 6 6 Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018 Editorial da Conceição Alves e Ana Claudia Figueiredo Martins Penha apresentam os resul
tados de uma pesquisa realizada junto a professores da EJA/Ensino Médio em uma
escola pública localizada na cidade de Macapá. Segundo os autores, ainda que frágeis,
as práticas de leitura realizadas na escola são de grande importância no sentido da
formação política. Finalizando este número, os autores Octávio José Zimbico e José Inocêncio
Narciso Cossa, no artigo intitulado “INFLUÊNCIA DO APOIO DOS PAIS E
ENCARREGADOS DE EDUCAÇÃO NO DESEMPENHO ESCOLAR DOS
EDUCANDOS EM MOÇAMBIQUE” discutem o papel da família no aprovei
tamento de estudos a partir dos resultados de uma pesquisa desenvolvida junto a
uma escola comunitária localizada no sul de Moçambique. Foi desenvolvida uma
pesquisa qualitativa baseada em documentos escritos, questionários e entrevistas com
professores, membros da direção da escola, pais e alunos. Os autores concluem que
a escola reproduz as desigualdades sociais, legitimando e naturalizando o capital cul
tural dominante. Desejamos a todos (as) uma boa leitura! Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018 Cláudia da Mota Darós Parente
Emery Marques Gusmão
Editoras Cláudia da Mota Darós Parente
Emery Marques Gusmão
Editoras Cláudia da Mota Darós Parente
Emery Marques Gusmão
Editoras 7 8 Educação em Revista, Marília, v.19, n.1, p. 5-8, Jan.-Jun., 2018
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A droplet-based microfluidic platform enables high-throughput combinatorial optimization of cyanobacterial cultivation
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OPEN
A droplet‑based microfluidic
platform enables high‑throughput
combinatorial optimization
of cyanobacterial cultivation
Jialan Cao1*, David A. Russo2, Ting Xie1, G. Alexander Groß1 & Julie A. Z. Zedler3*
Cyanobacteria are fast-growing, genetically accessible, photoautotrophs. Therefore, they have
attracted interest as sustainable production platforms. However, the lack of techniques to
systematically optimize cultivation parameters in a high-throughput manner is holding back progress
towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidic
platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial
cultivation. We successfully grew three different unicellular, biotechnologically relevant,
cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus
sp. UTEX 3154. This was followed by a highly-resolved 1D screening of nitrate, phosphate, carbonate,
and salt concentrations. The 1D screening results suggested that nitrate and/or phosphate may be
limiting nutrients in standard cultivation media. Finally, we use 2D screening to determine the optimal
N:P ratio of BG-11. Application of the improved medium composition in a high-density cultivation
setup led to an increase in biomass yield of up to 15.7%. This study demonstrates that droplet-based
microfluidics can decrease the volume required for cyanobacterial cultivation and screening up to a
thousand times while significantly increasing the multiplexing capacity. Going forward, microfluidics
have the potential to play a significant role in the industrial exploitation of cyanobacteria.
Cyanobacteria have attracted interest for sustainable biotechnology due to their fast photoautotrophic growth,
minimal nutritional requirements, and metabolic capacity for heterologous product formation. Transfer of
existing systems to an industrial scale is, however, still rare and yet to come of age. Despite this, the market for
cyanobacterial products is rapidly growing and cyanobacteria already account for half of the European algae
production1. The existing products, however, are largely limited to “Spirulina” (Arthrospira spp.) which can be
grown in high pH and high salt conditions. To efficiently exploit cyanobacterial diversity, and increase their
biotechnological relevance, several challenges need to be solved. One key aspect is strain-specific optimization
of growth conditions. To achieve this, high-throughput screening methods that are suitable for studying cyanobacterial chassis are needed. Standard high throughput methods for heterotrophic systems, such as micro-well
cultivation and live-cell monitoring systems, are often incompatible with cyanobacteria due to the requirement
for controlled light exposure, extended cultivation periods and autofluorescence interference with optical measurements. In addition, increasing of throughput by reducing volume raises new challenges in terms of oxygen
supply, evaporation control and homogeneity. Thus, it is of great importance to establish alternative, cost-efficient,
technologies that allow high-throughput optimization of cyanobacterial cultivation. Here, microfluidics has the
potential to tackle this challenge.
In microfluidics, liquids or gases are handled in technical channels with dimensions in the micrometer to
lower millimeter range. The central idea is the miniaturization of large-scale technical laboratory processes
to improve handling, material requirements and to enable process automation. The beginning of the twentieth century brought a tremendous boom in microfluidics research, leading to the development of numerous new microfluidic platforms like droplet microfluidics, paper analytical devices, organ-on-a-chip and open
1
Department of Physical Chemistry and Microreaction Technology, Institute for Chemistry and Biotechnique,
Ilmenau University of Technology, Weimarerstr. 32, 98693 Ilmenau, Germany. 2Bioorganic Analytics,
Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstr. 8, 07743 Jena,
Germany. 3Synthetic Biology of Photosynthetic Organisms, Matthias Schleiden Institute for Genetics,
Bioinformatics and Molecular Botany, Friedrich Schiller University Jena, Dornburgerstr. 159, 07743 Jena,
Germany. *email: jialan.cao@tu-ilmenau.de; julie.zedler@uni-jena.de
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microfluidics2. Most of the above-mentioned platforms can be assigned to one of three main categories: continuous-flow, microarray, and droplet-based3. Continuous flow microfluidic systems are featured by the continuous manipulation and flow of a single-phase fluid through closed microchannels or conduits. Many reports are
showing that continuous-flow systems have been successfully used in drug discovery applications4. However,
this system is difficult to scale for high throughput screening. Furthermore, this format is limited by the fact that
parabolic flow profiles are inherent. Due to this, analyte molecules are in close contact with the channel walls
resulting in precipitation, fouling and contamination. In microarray microfluidic systems, the liquid phase is
single-phase, but discrete volumes may be separated from the stream to undergo further processing. The advantages of this technique are ease of screening large number of different samples and easy coupling with current
industry high-throughput screening technologies (e.g. microtiter plates). The major disadvantage of microarray
systems is that robotics is required for high-throughput, and precise, handling of small volumes. In comparison
to the methods previously described, droplet-based microfluidic systems present several advantages. First, they
operate with discrete, physically separated, fluids in microchannels and exhibit the highest multiplex capability. Here, the sample or reagent phase is interrupted by a water-immiscible carrier phase, forcing it to split into
discrete droplets. Since the droplets are separated from each other and the channel walls by the immiscible
carrier phase, such droplets do not have the disadvantages that occur with continuous flows, such as fouling or
contamination. The volume of droplets ranges from pL to nL, making the reaction volume roughly a thousand
to a million times smaller than conventional cultivation conditions. Furthermore, various unit operations for
droplet manipulation such as mixing, dilution, splitting, merging and incubation are readily a vailable5,6.
From an application point of view, droplet microfluidics is an established tool in chemical analytics and micro
reaction technology and has played a pivotal role in the development of high-throughput analysis of biological systems. It was first recognized as a promising tool for biological applications more than two decades ago.
Starting with micro capillary e lectrophoresis7 and micro flow-through P
CR8,9, miniaturized fluidic techniques
became of interest for fast and efficient bioanalytical procedures. The ability to miniaturize volumes and multiplex
sample handling and analysis then also attracted interest in molecular biology as well as in microbiology. For
cultivation, droplet-based microfluidics by micro segmented-flow was first introduced to separate soil microorganisms from complex soil microbial c ommunities10. In small droplets, the principle of stochastic confinement applies. In other words, rare individuals and their released molecules can accumulate to high densities,
thus, enabling single cell e xperiments11–13. The segmented-flow principle enables an easy screening of sample
series with concentration gradients and allows the measurement of organismic responses to stress factors such
as antibiotics, nanoparticles or heavy m
etals14–16 with high resolution. In addition, this approach allows the
screening of two and three-dimensional concentration spaces with a small amount of consumables in a single
experimental run. Thus, opening the possibility of conveniently detecting combinatorial effects of, for example,
toxins, pharmaceutical drugs and n
utrients17. Furthermore, droplet-based microfluidics is in use for detection of
antibiotic resistances and for the development of new antibiotics18. Besides bacteria, the micro segmented-flow
technique has also proven to be suitable for eukaryotic microorganisms such as Chlorella19 or even embryos
of multicellular organisms20 and for the characterization of the concentration-dependent response of complex
unknown environmental bacterial communities on toxic s ubstances21,22. As applied to cyanobacterial systems,
microfluidics is an emerging technology with only a handful of applications. For instance, a microfluidic tool
for harvesting cyanobacterial biomass was developed for Synechocystis sp. PCC 6 80323 (hereafter PCC 6803).
In addition, microfluidic technology has been used for lactate productivity screening in a PCC 6803 CRISPRi
library24 as well as to screen for ethanol p
roduction25.
Here, we established a droplet-based microfluidic setup that allows for high-throughput medium composition optimization for biotechnologically relevant cyanobacteria. We tested the compatibility of our system with
three different cyanobacterial strains using biomass accumulation as the screening parameter. Our microfluidic
setup allowed for continuous cultivation and analysis of the cells where individual medium parameters such
as macronutrient content as well as salt and bicarbonate concentrations were varied in small steps allowing for
highly parallel, high-throughput screening. In addition, we demonstrated that our platform is able to screen two
parameters simultaneously to explore the two-dimensional effect space of combined variables. In conclusion,
we demonstrate that microfluidics can be used to improve medium composition and cultivation conditions in a
cost- and time-effective manner to unlock the exploitation of biotechnologically relevant cyanobacteria.
Materials and methods
Cyanobacterial strains and growth conditions. Three cyanobacterial wild-type strains were used: a
non-motile, glucose-tolerant substrain of Synechocystis sp. PCC 6803 (originally obtained from Patrik Jones,
Imperial College London), Synechococcus elongatus UTEX 2973 (originally obtained from Himadri Pakrasi,
University of Washington) (hereafter UTEX 2973) and Synechococcus sp. UTEX 3154 (a spontaneous mutant of
Synechococcus sp. PCC 1 190126 which does not require vitamin B12 for growth, obtained from the UTEX culture
collection) (hereafter UTEX 3154). PCC 6803 and UTEX 2973 were grown in BG-11 m
edium27 supplemented
with 10 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES) buffer (pH 8.0) (BG-11 TES
medium). Medium A
28 with D7 m
icronutrients29 (AD7 medium) (pH 7.5) was used for cultivation of UTEX
3154. All cultures were maintained at 30 °C with 20–50 µmol photons m
−2 s−1 white light [Lumilux cool white
L 15 W/840 fluorescent lamps (Osram, Germany)] on BG-11 or AD7 medium plates supplemented with 1.5%
bacto-agar. Liquid pre-cultures for inoculation of the microfluidic coils were grown in glass tubes bubbled with
3% CO2-supplemented air at 30 °C with approximately 60 µmol photons m−2 s−1 white light for 3 to 4 days (midto-late exponential growth phase) (culture volume: 20 mL). These cultures were then counted and diluted with
growth medium to obtain 1 07 cells m
L−1 and used for droplet inoculation. The initial cell density of each droplet
−1
was 10 cells nL with a total droplet volume of 500 nL.
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Control and data analysis
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e ector 2
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Waste
Tube coil
for storage
Figure 1. Experimental droplet-based microfluidic setup for 1D and 2D screening of cultivation parameters
used in this study. The illustration on the left shows a 5-channel syringe pump and the droplet generator which
generates the droplets. The individual aqueous cell medium droplets are separated by the immiscible carrier
medium. Droplet size/volume, spacing and composition can be adjusted via the controlled flow rate program of
the syringe pump. The generated droplets are measured by a combined photo-fluorimetric micro flow-through
detector unit. The droplet sequences are then collected and incubated in PTFE tube coils in an internally
illuminated incubator.
To test improved nitrogen:phosphorus (N:P) ratios based on 2D screening experiments, standard BG-11
medium and BG-11 medium with adjusted NaNO3 and K2HPO4 concentrations were prepared. Liquid cultures
were inoculated for PCC 6803 and UTEX 2973 in a high-density cultivation setup (HDC 6.10 starter kit CellDEG, Germany)30 using 25 mL culture vessels with a culture volume of 10 mL. The cultures were supplemented
through a membrane with CO2 at a partial pressure of approximately 32 mbar (reference T = 20 °C) by a carbonate
buffer (3 M K
HCO3 and 3 M K
2CO3, ratio 4:1)30. The growth setup was placed on a Unimax 1010 orbital shaker
(Heidolph Instruments, Germany) and incubated with shaking at 280 rpm at 30 °C with 50 µmol photons m
–2 s–1
white light [Lumilux cool white L 15 W/840 fluorescent lamps (Osram, Germany)]. Main cultures for the growth
experiment were inoculated in triplicates from a pre-culture grown in the same medium and conditions to a
starting optical density (OD) at 750 nm of 0.3 and monitored for 7 days by measuring OD at 750 nm every 24 h
using a GENESYS 10S UV–Vis Spectrophotometer (Thermo Scientific, Germany). For PCC 6803, the optimized
BG-11 medium contained 0.4 mM K
2HPO4 and 27 mM N
aNO3 and for UTEX 2973 0.45 mM K
2HPO4 and
30 mM NaNO3. All other elements of the medium were kept the same as in the standard BG-11 TES medium.
Microfluidic cultivation
Microfluidic arrangement. Details on the fluidic devices, the optical detection unit and the applied meth-
ods for realizing concentration-graded droplet sequences were reported e arlier15. Here, a similar experimental
setup (Fig. 1) was used for one- and two-dimensional screening of medium parameters. Briefly, the system is
based on a syringe pump with six dosing units (Cetoni GmbH, Germany). The microfluidic droplets were generated by a self-developed droplet generator comprising of a 6-port m
anifold31. Droplets with varying compositions are generated by controlled dosing of effectors, culture medium and cell suspension into a flow of carrier
liquid [perfluoromethyldecalin (PP9)] The droplet generator was connected via fluoroethylenepropylene (FEP)
tubing (inner diameter 1.0 mm and outer diameter 1.6 mm) to the computer-operated syringe pump, utilizing
syringes with volumes of 500 µL (effector solution and cell suspension), 1000 µL (culture medium) and 5000 µL
(carrier liquid). Generated segments are transported at a constant flow rate of 200 µL min–1 through an optical
detector unit for the simultaneous measurement of absorbance and fluorescence data of the droplets. Data were
recorded directly through the visually transparent FEP tubing (Fig. 1). Absorbance was measured with four light
emitting diodes (Agilent, United States) with peak wavelengths of 470, 505, 615 and 750 nm. Measurement of
fluorescence was carried out with a laser diode with a peak wavelength of 405 nm (Changchun New Industries
Optoelectronics, China) with a longpass filter (425 nm) (Laser Components, Germany) on the detection side.
The emitted photons were recorded by a photomultiplier module (Hamamatsu, Japan). To store and incubate
the generated droplet sequences, polytetrafluoroethylene (PTFE) tube coils (inner diameter 0.5 mm and outer
diameter 1.6 mm) with a length of four meters for 1D and seven meters for 2D screening were used.
Experimental parameters. The syringe pump flow rates of the different fluids were controlled using a
LabVIEW program (National Instruments, USA). To investigate the dose–response relationships for single substances (1D screening), a syringe pump control program with continuous change of the desired effector against
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b
a
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e ector 2
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120
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Figure 2. Syringe pump control program for the generation of 1D and 2D gradients. (a) Highly resolved
dose response screening for single substances (1D screening). The base cultivation medium was continuously
substituted over time by the effector-spiked medium and (b) two-dimensional concentration spaces in 11
combinatorial steps, resulting in 121 concentration combinations (2D screening).
a diluting medium was used (Fig. 2a). For two effectors (2D screening), a stepwise increase of the concentrations
with a resolution of 10% was applied. Hence, for two effectors, 11 different concentration steps (0, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100%) were combined resulting in 121 different combinations (Fig. 2b). The flow rates of
the carrier liquid and the cell suspension were set to 136 µL m
in–1 and 32 µL m
in–1 respectively. The flow rates
of the effector solutions and cultivation medium were varied within a total flow rate of 32 µL m
in–1. Therefore,
–1
the overall flow rate of the segment generation process was kept constant at 200 µL min . An initial cell density
of 5000 cells per 500 nL segment (107 cells m
L−1) was applied. The generation of the highly resolved 1D and 2D
screening sequences required approximately four and nine minutes. After droplet formation and initial photofluorimetric analysis (t = 0), the droplets were gathered in the subsequent collection tube coils for incubation.
The tube coils were incubated for 7 days at 30 ± 2 °C, 3% CO2 and under 20 ± 5 µmol photons m−2 s−1 illumination. The different droplet sequences were analyzed daily by passing through the photo-fluorimetric detection
unit to monitor cell density.
Effector screening concentration range. For effector screening, only the concentrations of our test
variables were altered, while the remaining medium components were kept at the same concentrations. Stock
solutions with two-fold concentrations for 1D screening and four-fold concentrations for 2D screening of the
highest final concentration were prepared. These stocks were then used to generate highly resolved concentration gradients using the syringe pump control programs as shown in Figs. 1 and 2. In all experiments, PP9 (F2
Chemicals Ltd, Lancashire, UK) was used as an immiscible carrier liquid.
Data processing.
The photo-fluorometric data were recorded with 250 Hz sampling rate. Droplet sequences
were measured immediately after formation and after seven days of incubation in the coils (for the generation
of growth curves, daily measurements were taken). The droplet data were analyzed offline using a custom LabVIEW program which elucidates droplet-specific data from the spectral sensor raw data: number, size, distance
between two neighboring droplets as well as extinction and fluorescence measurements. Individual droplets
were detected if the absorbance value exceeded the background and achieved a set threshold value. The droplet size correlates with the droplet passage time through the sensor and is determined by the time interval the
absorbance exceeds a set threshold value. Due to the inhomogeneous cell distribution in the droplets, growth
behavior was analyzed by calculating the integral of the absorbance and fluorescence signal with respect to the
droplet size. The recorded autofluorescence intensity of the droplets I(t) was normalized to the initial measurement intensity I(t0) using Eq. (1). Whereas the background was taken on the FEP-tube filled with carrier liquid.
Data are given as normalized autofluorescence units (NFU).
405/425
NFUt
=
It − It0
It0
(1)
Statistics and reproducibility.
In total, the growth kinetic and 1D screening experiments were repeated
three times and the 2D screening experiments two times. An example of a typical data distribution can be
found in Supplementary Fig. 1 where each circle represents the intensity for an individual droplet. The droplets
were then grouped into 32 concentration ranges from 0 to 100% of effector concentration (corresponding to a
3.1% concentration resolution). The mean and standard deviation were calculated for these 32 concentrations
ranges and the resulting curve was plotted as a dose–response curve (Supplementary Fig. 1 blue triangles). For
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the growth kinetic experiment, each data point represents the average and standard deviation of 50 ± 5 droplets
across two independent coils. For 1D and 2D screening experiments, a total of 440 ± 21 and 814 ± 26 droplets,
respectively, were generated per coil. These droplets were then distributed across the chemical gradients and
approximately 10 (1D screening) and 8 (2D screening) droplets were analyzed per tested concentration. In sum,
each data point represents the average and standard deviation of 20 ± 6 (1D screening) and 16 ± 6 (2D screening)
droplets. This redundancy was applied in order to validate measurement reliability and to control for stochastic
effects that can derive from small reaction volumes and cell numbers. Comparison of final biomass values in the
high-density cultivation setup were done with an independent sample Student’s t-test with a significance value
of α = 0.05.
Results and discussion
In this study we introduce a microfluidic method that allows us to rapidly screen one- and two-dimensional (1D
and 2D) culture medium parameters to optimize the growth conditions of industrially relevant cyanobacteria.
Our results show that our droplet-based microfluidic approach is well suited to screen culture media conditions
in a fast and efficient way, with low material input and a reduced amount of incubator space. In addition, we
observed that commonly used medium formulations are not optimized for maximum growth and small changes
can result in significantly higher biomass outputs. This method should be widely applicable to a variety of freshwater and saltwater strains and has the potential to facilitate high-throughput strain optimization.
Both freshwater and marine unicellular cyanobacteria can be successfully cultivated in micro‑
droplets. To test the applicability of microdroplet cultivation to cyanobacteria we chose three biotechnologi-
cally relevant species. PCC 6803 is a freshwater model cyanobacterium that has been extensively characterized
and for which many molecular tools exist. UTEX 2973 is a freshwater fast-growing relative of Synechococcus
elongatus PCC 7942. Due to their genetic proximity, an average nucleotide identity (ANI) > 99.8%32, the tools
developed for Synechococcus elongatus PCC 7942 are largely transferable to UTEX 2973. UTEX 2973 is of particular interest for biotechnological applications because of its fast growth phenotype with doubling times
reported as fast as 1.5 h33. As a third strain, we chose a saltwater strain. A recently isolated strain, Synechococcus
sp. PCC 11901 (hereafter PCC 11901), is genetically tractable and has clear biotechnological potential due to its
reported doubling time of approximately 2 h and its ability to grow at high light intensities and a range of salinities. Under optimized conditions, PCC 11901 can accumulate up to 33 g of dry cell weight per liter. Curiously, it
has an ANI of 96.76% when compared to the commonly used Synechococcus sp. PCC 7002 s train26. Therefore, it
may be possible to utilize tools previously developed for Synechococcus sp. PCC 7002 in PCC 11901. However,
this strain is a vitamin B
12 auxotroph. Hence, we decided to use a closely related, spontaneous mutant of this
strain, UTEX 3154, that can grow without an external supply of vitamin B12.
First, we proceeded to test whether these three cyanobacterial strains could grow in the droplet-based microfluidic setup. For the initial screening, sensors detecting autofluorescence (excitation: 405 nm, emission: 425 nm)
and optical density (OD) at 470, 505, 615 and 750 nm were tested over a period of 7 days. In our microfluidic
setup, OD reflects the reduction of the intensity of transmitted light by use of a microflow-through photometer.
OD typically correlates well with the final cell number. However it does not allow to distinguish between alive
and dead cells. Growth can also be monitored by measuring the endogenous cellular autofluorescence with a
micro flow-through fluorimeter. The fluorescence can be used to evaluate the approximate number of physiological active cells and, typically, is a more sensitive parameter than OD. The observed increase of the signals
in all sensor channels clearly demonstrates cell growth (Supplementary Fig. 2, Supplementary Data Table S1).
However, the highest intensities were observed in the O
D470 (Fig. 3a) and the autofluorescence (Fig. 3b) channels.
Therefore, in further experiments, we decided to use the autofluorescence channel to evaluate biomass accumulation. In some cases, biomass accumulation in the droplet storage coils was visible by the naked eye already after
4 days (Fig. 3c). The high cell density was confirmed by the observation of selected individual droplets by light
microscopy (Fig. 3d). The typically reported doubling times for PCC 6803 are in the range of 10 to 12 h34,35 and
approximately 2 h for UTEX 2 97332 and UTEX 315426. In line with this, our data show that PCC 6803 grows
significantly slower than UTEX 2973 and UTEX 3154 (Fig. 3a,b). Between UTEX 2973 and UTEX 3154 we
observed similar exponential growth rates and final biomass values despite the longer lag phase of UTEX 2973.
Overall, the individual growth profiles fit with published literature26,32,36 and show that microdroplet cultivation
is suitable for unicellular freshwater and saltwater strains.
Microdroplet technology facilitates high‑throughput, high‑resolution dose response screen‑
ing. Following the successful droplet-based cultivation of PCC 6803, UTEX 2973 and UTEX 3154, we pro-
ceeded to apply the methodology to investigate the response of the cyanobacteria to a variety of medium parameters. Therefore, we designed 1D screening experiments for media optimization. We used the standard growth
medium for the respective strains (BG-11 for PCC 6803 and UTEX 2973, AD7 for UTEX 3154) and varied one
parameter at a time by microfluidic means. The varied parameters included the nitrogen (N) source, phosphorus (P) source as well as medium salinity and sodium bicarbonate concentrations (Table 1). Furthermore, we
proceeded to look at combinatorial effects using 2D screening where both the N and P concentrations were
varied simultaneously. An overview of the concentration range of all tested effectors for 1D and 2D screening
experiments is shown in Table 1. A summary of the optimum values for the medium parameters tested in the
1D screening is shown in Table 2.
We first started 1D testing of different concentrations of N
aNO3 ranging from 2 to 50 mM. The data show
that the freshwater PCC 6803 and UTEX 2973 achieved maximum biomass values at approximately 10 and
20 mM NaNO3, respectively (Fig. 4a, Supplementary Data Table S2). For the saltwater UTEX 3154, the maximum
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a
b
0.3
UTEX 3154
1.5
UTEX 3154
UTEX 2973
UTEX 2973
0.2
1.0
PCC 6803
PCC 6803
NFU
OD470 nm
c
0.1
0.5
0.0
0.0
0
d
1
2
3
4
5
6
7
0
1
2
3
Time (d)
4
5
6
7
Time (d)
PCC 6803
UTEX 2973
UTEX 3154
Figure 3. Growth of cyanobacteria in microdroplet setup. Growth kinetics of cyanobacterial strains (UTEX
3154, UTEX 2973 and PCC 6803) in the microfluidic coils measured with multi-channel photo-fluorimeter
device showing (a) absorbance (optical density OD) at 470 nm and (b) normalized autofluorescence in the
microfluidic setup over a 7 day period. Data points are averages of 50 ± 5 droplets. Error bars represent standard
deviation. Normalized autofluorescence is shown as NFU. (c) Image of a microfluidic coil carrying UTEX 2973
droplets. (d) Light microscopy images of cyanobacteria PCC 6803, UTEX 2973 and UTEX 3154 in droplets after
4 days of growth (scale bar: 100 µm).
Medium component
1D screening range
2D screening range
BG-11 TES medium
NaNO3 (mM)
2–50
0–30
17.6
AD7 medium
12
K2HPO4 (mM)
0.05–5
0–0.5
0.175
–
KH2PO4 (mM)
0.05–5
0–0.5
–
0.37
NaHCO3 (mM)
0–100
–
–
–
NaCl (mM)
0–1300
–
–
308
Table 1. Range of concentrations for 1D and 2D screening experiments of selected medium components used
in this study. Reference values of concentrations in standard BG-11 TES and AD7 medium are given.
Medium component
PCC 6803
UTEX 2973
UTEX 3154
NaNO3 (mM)
10
20
30
K2HPO4 (mM)
2.5–3.5
0–0.5
–
KH2PO4 (mM)
–
–
0.75
NaHCO3 (mM)
50
NS
NS
NaCl (mM)
100–650
0–300
0–1100
Table 2. Summary of the concentration ranges where the highest growth was observed in the 1D screening.
biomass value was achieved at approximately 30 mM N
aNO3 (Fig. 4a). The typical concentration of N
aNO3
present in the freshwater cyanobacterial growth medium BG-11 is 17.6 mM. Comparing this value with the
limiting NaNO3 concentrations for PCC 6803 and UTEX 2973 (10 and 20 mM, respectively), we can conclude
that N is not typically the limiting nutrient in BG-11 medium. Accordingly, an earlier study showed that during
batch cultivation of PCC 6803 in BG-11 medium one of the major medium limitations may be sulfate ions37.
Regarding the saltwater medium AD7, the concentration of NaNO3 is 12 mM. Considering that the biomass
accumulation of UTEX 3154 only peaked at around 30 mM NaNO3, our data suggest that N may be a limiting
nutrient in AD7. This is supported by the original PCC 11901 strain publication where the authors determined
the ideal NaNO3 concentration to be between 24 and 48 mM26. Overall, these data show that, given enough N,
all three cyanobacteria are rapidly limited by other nutrients. Therefore, we proceeded to apply our microfluidic
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a
b
4
3
2
NFU
NFU
3
1
1
0
0
0
c
2
10
20
30
40
50
0
2
3
4
5
K2HPO4 / KH2PO4 [mM]
NaNO3 [mM]
3
1
UTEX 3154
2
NFU
UTEX 2973
PCC 6803
1
0
0
20
40
60
80
100
NaHCO3 [mM]
Figure 4. Highly resolved dose–response screenings of key macronutrients in UTEX 3154, UTEX 2973 and
PCC 6803. (a) Normalized autofluorescence after 7 days in response to varying concentrations of N
aNO3.
(b) Normalized autofluorescence after 7 days in response to varying concentrations of K
2HPO4 (UTEX 2973,
PCC 6803) or KH2PO4 (UTEX 3154). (c) Normalized autofluorescence after 7 days in response to varying
concentrations of bicarbonate (NaHCO3). Data points are averages of 10 ± 3 droplets. Error bars represent
standard deviation. Normalized autofluorescence is shown as NFU.
approach to test the effect of varying concentrations of phosphate ( K2HPO4 for UTEX 2973 and PCC 6803 and
KH2PO4 for UTEX 3154) up to a maximum concentration of 5 mM. The data show that the maximum biomass
values were achieved between 2.5 and 3.5 mM phosphate for PCC 6803 and UTEX 2973 and 0.75 mM phosphate
for UTEX 3154 (Fig. 4b, Supplementary Data Table S3). Looking at the formulations of the base medium, phosphate is present at a concentration of 0.175 mM K
2HPO4 in BG-11 and 0.37 mM K
H2PO4 in AD7. Therefore, our
microfluidic growth data suggest that both media are P deficient. This is particularly the case for BG-11 which has
a K2HPO4 concentration 5 to 6 times lower than the levels at which we observed the highest biomass accumulation. It has been suggested that media designed for the growth of algae and cyanobacteria are often P limited
due to a lack of compatibility with the Redfield r atio38. This ratio describes the amount of carbon (C), N and P
typically present in both phytoplankton biomass and in dissolved nutrient pools and has been determined to be
106 C:16 N: 1 P. Based on the media formulation used in the study, AD7 presents a N:P ratio of 32:1 and BG-11
100:1. Thus, supporting our previous hypothesis that both media, and BG-11 in particular, may be P limited.
The autotrophic growth of cyanobacteria requires a source of inorganic C which is typically provided as CO2.
CO2 can be supplied by air (0.04% CO2), CO2 enriched air (typically 1–5%) or in the form of bicarbonate salts.
Installing a gas supply to cyanobacterial cultures can be costly and presents a logistical challenge for parallel
experimentation. Therefore, sodium bicarbonate is a popular low-cost inorganic C source for cyanobacterial
medium. Here, we tested the addition of NaHCO3 to BG-11 and AD7 in the range of 0 to 100 mM (Fig. 4c, Supplementary Data Table S4). The addition of bicarbonate improved the growth of PCC 6803 from 35 mM and
achieved a maximum biomass accumulation at approximately 50 mM. For UTEX 2973 and UTEX 3154 there
was no significant difference in biomass accumulation in the tested range. In our previous experiments we have
shown that BG-11 is P limited (Fig. 4b). This could explain the lack of response of UTEX 2973 to the varying
bicarbonate concentrations. In regard to UTEX 3154, a previous study in Synechococcus sp. PCC 7002 showed
that significant biomass accumulation was only visible with bicarbonate concentrations higher than 500 m
M39.
This suggests that the range tested here may not comprise the ideal bicarbonate values for this cyanobacterium.
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3
UTEX 3154
UTEX 2973
2
NFU
PCC 6803
1
0
0.0
0.5
1.0
1.5
NaCl [M]
Figure 5. 1D screening of NaCl tolerance in cyanobacteria. Normalized autofluorescence after 7 days in
response to varying concentrations of NaCl shown for three different cyanobacterial strains, UTEX 3154,
UTEX 2973 and PCC 6803. Data points are averages of 10 ± 3 droplets. Error bars represent standard deviation.
Normalized autofluorescence is shown as NFU.
However, Synechococcus sp. PCC 7002 and UTEX 3154 present significant genetic differences therefore it is difficult to make definite conclusions. Finally, another factor could be light limitation. In our experiments we used
20 ± 5 µmol photons m–2 s–1, while the maximum growth rates of UTEX 2973 were reported at 1500 µmol photons
m–2 s–1, 42 °C and 5% CO233. However, this is not directly comparable as the light path in our microfluidic setup is
1.0 mm as opposed to the 27 mm used to determine the maximum growth rates of UTEX 2973 in the literature.
Further tests would be needed to establish a robust comparison.
Salinity of the growth medium is a critical factor as high salinities can induce a variety of stresses and consequently pose a challenge to cell s urvival40. In addition, future large-scale cultivation of cyanobacteria should be
done in seawater due to the limited freshwater resources present on Earth. Therefore, there has been an increased
interest in prospecting for and developing salt-tolerant cyanobacterial chassis. Here, we used our microfluidic
platform to determine the salt tolerance of PCC 6803, UTEX 2973 and UTEX 3154 (Fig. 5, Supplementary Data
Table S5). As expected, the two freshwater strains exhibit lower salt tolerances than UTEX 3154. UTEX 2973
exhibits the lowest salt tolerance with a decline in biomass accumulation starting at 0.3 M NaCl. PCC 6803
maintains similar levels of biomass accumulation until 0.65 M with a sharp decline observed thereafter. Total
inhibition was observed of both PCC 6803 and UTEX 2973 at 0.7 M NaCl. UTEX 3154 accumulates similar
biomass levels until approximately 0.8 M, whereafter a sharp decline is also observed. It is worth noting here that
the base AD7 used in this study contains 308 mM NaCl and 8 mM KCl therefore the data show that UTEX 3154
can tolerate salt concentrations up to 1.1 M NaCl. These values are in accordance with the published literature
on salt tolerance in c yanobacteria41. Our data show that this microfluidic setup can be used for high-resolution
screening for optimum salinity cultivation conditions of single-celled cyanobacteria and could serve as an effective high-throughput method to screen for strains with increased salt tolerance in future studies.
Simultaneous screening of phosphorus and nitrogen is essential for medium optimiza‑
tion. The above results demonstrate that our microfluidic platform can rapidly generate droplet screening
sequences with a wide range of varying parameters (i.e. 1D screening). However, it is often of interest to screen
a combination of parameters. Therefore, we took advantage of the capabilities of the droplet-based technique
to simultaneously vary two independent parameters (2D screening). The results of a 2D medium optimization experiment are 2D response diagrams where we can easily estimate the effect of the two variables on one
readout parameter (e.g. biomass accumulation). For the 2D proof of concept experiment we decided to screen
N (NaNO3) and P (K2HPO4 or KH2PO4) sources in conjunction (Fig. 6, Supplementary Data Table S6, S7, S8).
These two parameters were chosen because the N:P ratio is one of the key parameters that influences algal
growth and rapidly optimizing N and P concentrations is fundamental for cost-efficient scale-up of microalgal
cultures42. Our data show that final biomass values in PCC 6803 and UTEX 2973 peaked at the maximum N
values tested (0.45 mM K
2HPO4/30 mM NaNO3 for PCC 6803 and 0.4 mM K
2HPO4/30 mM NaNO3 for UTEX
2973) (Fig. 6a,b). Interestingly, an increase in N at low P values or an increase of P at low N values was not sufficient to obtain high biomass values. Only when both parameters were increased simultaneously was a significant
increase in final biomass values observed. This contrasts with the conclusions from the 1D screening data where
P seemed to be limiting the culture. This suggests that it is more efficient to find a good balance between N and
P instead of just increasing one parameter. Therefore, 2D screening is fundamental to understanding nutrient
dynamics in cyanobacterial cultures. Regarding the N:P ratio, both strains achieved their maximum biomass
values at a ratio of approximately 100:1. This is the same ratio as BG-11 which suggests that the classic cyanobacterial medium has a good N:P ratio but would benefit from higher concentrations of both N and P. For UTEX
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3
15
2
12
9
18
15
2
12
21
1
18
15
12
6
6
1
3
3
0
0
0
0.
0
05
0.
1
0.
15
0.
2
0.
25
0.
3
0.
35
0.
4
0.
45
0.
5
0.
0
05
0.
1
0.
15
0.
2
0.
25
0.
3
0.
35
0.
4
0.
45
0.
5
0.
0
05
3
0
0.5
9
9
6
1.0
24
3
21
NaNO3 [mM]
18
27
K2HPO4 [mM]
K2HPO4 [mM]
5
24
0.
24
30
0.
4
0.
45
27
21
NaNO3 [mM]
30
27
Synechococcus sp. UTEX 3154
0.
3
0.
35
30
4
0.
2
0.
25
4
c
Synechococcus elongatus UTEX 2973
0.
1
0.
15
b
Synechocystis sp. PCC 6803
NaNO3 [mM]
a
KH2PO4 [mM]
Figure 6. 2D screening of nitrogen and phosphorus source in cyanobacterial cultivation using standard growth
media. (a) PCC6803 normalized autofluorescence after 7 days in response to varying concentrations of N
aNO3
and K2HPO4 in BG-11 medium. (b) UTEX 2973 normalized autofluorescence after 7 days in response to varying
concentrations of N
aNO3 and K2HPO4 in BG-11 medium. (c) UTEX 3154 normalized autofluorescence after
7 days in response to varying concentrations of N
aNO3 and KH2PO4 in AD7 medium. Data points are averages
of approximately 5 ± 3 droplets.
a
b
12
PCC 6803 BG-11
10
UTEX 2973 BG-11
10
PCC 6803 opt. N/P
UTEX 2973 BG-11 opt. N/P
8
OD750
8
OD750
12
6
6
4
4
2
2
0
0
0
50
100
150
0
50
100
150
Time (h)
Time (h)
Figure 7. High-density cultivation in BG-11 medium containing N:P ratios optimized through 2D microfluidic
screening. Growth of (a) PCC 6803 and (b) UTEX 2973 in standard vs. optimized N/P ratio observed by
measuring optical density (OD) at 750 nm. n = 3 and error bars represent standard deviation.
3154 no clear trend was observed within the tested N:P range (Fig. 6c). Together with the 1D screening data, this
suggests that N may be the limiting nutrient in the AD7. However, the possibility that a nutrient other than N or
P limits the culture remains open.
Improved N:P ratios significantly increase biomass accumulation in high density cultiva‑
tion. To assess whether the optimal N:P concentrations suggested by the microfluidic 2D experiments can
be translated to a larger scale cultivation setup, we carried out growth assays in a laboratory setting. To this
end, PCC 6803 and UTEX 2973 were grown in 25 mL high-density cultivators where gaseous C
O2 is supplied
via integrated semi-permeable membranes. With this setup, growth was compared in standard BG-11 medium
(0.175 mM K2HPO4, 17.6 mM N
aNO3) and BG-11 with the optimized N:P concentrations obtained from the
2D N:P microfluidic screening experiment. More specifically, 0.45 mM K
2HPO4 with 30 mM N
aNO3 for PCC
6803 and 0.4 mM K
2HPO4 with 30 mM N
aNO3 for UTEX 2973. For both PCC 6803 and UTEX 2973 significant
increases (p < 0.05) of final biomass values of 7.4% (Fig. 7a) and 15.7% (Fig. 7b), respectively, could be observed.
Overall, this confirmed that the findings from the microfluidic experiments are transferable to biotechnologically relevant high-density cultivation setups.
Droplet microfluidics significantly increases the multiplexing capacity of cyanobacterial cul‑
tivation. The cyanobacterial cultivation method that offers the highest multiplex capability is currently the
microtiter plate (MTP). Assuming a 96-well MTP with 200 µL in each well, and that each microdroplet is the
functional equivalent of a well on an MTP, we can establish a comparison between a MTP and the microfluidic
approach established in this work. For example, for a 1% concentration resolution of a single substance (1D
screening), 100 concentration steps are required. If 10 droplets are screened per concentration step, this is a total
of 1000 droplets. With a volume of 0.2 µL/droplet, a total of 200 µL of medium is required in the microfluidic
reactor. On the other hand, for the same experiment in an MTP, this would require 200 mL of medium across
10 MTPs. For the same 1% resolution in the case of a binary mixture (2D screening), 10,000 concentration steps
are required. Therefore, a total of 2 mL of medium is required in the microfluidic reactor. To screen the same
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amount of effector combinations in a MTP, 2L of medium across 100 MTPs would be required. The entire microfluidic preparation process, including cell counting and dilution, medium and effector preparation, sequence
generation and measurement requires approximately 4 h. For 1D screening, the preparation of 10 MTPs with
100 concentration steps could feasibly be achieved in 4 h, albeit with a much higher risk of error. However,
when moving to 2D screening, the preparation and cultivation of 100 MTPs becomes unfeasible. With droplet
microfluidics, the move from 1 to 2D screening requires very little extra preparation time and the experimental
footprint remains the same.
Conclusions
To achieve cost-effective scale-up of cyanobacteria it is crucial to screen for the ideal cultivation conditions. In
particular, medium optimization can significantly increase biomass and product yields. However, the cost and
time investment required to test different levels of key nutrients, and the possible interactions amongst them, is
prohibitive with standard cultivation methods. Recently, miniaturized multiplex screening (e.g. microfluidics)
has been suggested as a route to tackle the large parameter field of medium optimization. In this study we used
a droplet-based microfluidic technique to improve the cultivation of three different biotechnologically relevant
cyanobacterial strains. Our proof of concept demonstrated that the strains could be successfully cultivated with a
medium requirement one thousand times lower than MTPs. Furthermore, growth data could be collected online
with non-invasive, in-situ, measurements. The 1D screening data confirmed that our microfluidic platform is
well-suited for the investigation of cyanobacterial response towards single nutrients. In addition, the 2D screening
allowed us to explore the two-dimensional space of nutrient interactions with only a small increase in the time
and reagents required. In conclusion, this study shows that microfluidics can play a valuable role in improving the
cost-effectiveness of cyanobacterial cultivation. We also expect that this microfluidic approach can be generalized to other applications such as expression level optimization of engineered cyanobacteria and bioprospecting.
Data availability
All cyanobacterial strains used in this study are available from the Pasteur Culture Collection, Paris (France) or
the UTEX Culture Collection of Algae, University of Texas, Austin (USA). The raw data of all experiments are
provided in the Supplementary Data files.
Received: 9 July 2022; Accepted: 5 September 2022
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Acknowledgements
We thank Prof. Michael Köhler for the fruitful discussion. We thank Frances Möller and Steffen Schneider for lab
assistance. JC gratefully acknowledges financial support from the project “Screen | in drop lines” (2016FE9016)
by Thüringer Aufbaubank and a habilitation scholarship from the TU Ilmenau, DAR was supported by the
Alexander von Humboldt Foundation and by the I MPULSEproject (IP-2020-03, Friedrich Schiller University Jena).
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under
Germany’s Excellence Strategy—EXC 2051—Project-ID 390713860 (JAZZ) and the by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—CRC 1127/2—239748522 (JAZZ).
Author contributions
Conceptualization and study design: J.C., D.A.R. and J.A.Z.Z. Experimental work and data analysis: J.C., D.A.R.,
T.X. and J.A.Z.Z. Microfluidic device and system development: G.A.G., J.C. Writing—original draft: J.C., D.A.R.
and J.A.Z.Z. Writing—review & editing: J.C., D.A.R., G.A.G. and J.A.Z.Z. Funding acquisition and project administration: J.C., D.A.R., G.A.G. and J.A.Z.Z. All authors have read and approved the final manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-022-19773-6.
Correspondence and requests for materials should be addressed to J.C. or J.A.Z.Z.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
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SINTESIS TEKS KE GAMBAR: TINJAUAN ATAS DATASET
|
Electric, Electronic, Instrumentation, Control, Telecommunication
| 2,024
|
cc-by
| 6,837
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SINTESIS TEKS KE GAMBAR: TINJAUAN ATAS DATASET Suci Ramadhani Arifin Kata kunci : Sintesis, Teks Ke Gambar,
Dataset Kata kunci : Sintesis, Teks Ke Gambar,
Dataset Kata kunci : Sintesis, Teks Ke Gambar,
Dataset Salah
satu
tantangan
dalam
pengembangan dataset adalah memperoleh
pasangan teks dan gambar dalam jumlah
besar yang berkualitas tinggi dan relevan
[4]. Beberapa teknik yang digunakan antara Abstrak - Penelitian ini bertujuan untuk
melakukan analisis mendalam terhadap
berbagai dataset yang digunakan dalam
riset sintesis teks ke gambar. Fokus utama
penelitian ini adalah pada pemahaman
karakteristik
masing-masing
dataset,
pengaruh pemilihan dataset terhadap hasil
penelitian, serta keunggulan dan kelemahan
setiap dataset. Beberapa dataset yang
diteliti meliputi MS COCO, CUB-200-
2011, dan Oxford 102 Flower, bersama
dengan dataset-domain khusus lainnya
yang relevan. Metode penelitian mencakup
analisis deskriptif terhadap jumlah gambar,
karakteristik visual, dan deskripsi teks yang
melibatkan setiap dataset. Data yang
diperoleh dianalisis secara kualitatif untuk
mendapatkan wawasan mendalam tentang
setiap dataset. Hasil analisis diharapkan
dapat memberikan panduan bagi peneliti
dalam memilih dataset yang sesuai dengan
tujuan penelitian mereka dalam sintesis teks
ke gambar. Penelitian ini diakhiri dengan
rekomendasi
dan
kesimpulan
yang
merangkum
temuan
utama
dan
relevansinya dalam konteks riset ini. relevan dari input teks menggunakan
pemodelan oleh mesin [1]. Banyak aplikasi
menarik
dapat
diwujudkan
dengan
teknologi ini, seperti pembangkit ilustrasi
otomatis, asisten desain grafis, hingga
penerjemah bahasa isyarat. Kunci keberhasilan sintesis teks ke
gambar
adalah
ketersediaan
dataset
pelatihan yang berkualitas tinggi [2]. Dataset
yang
besar
dan
bervariasi
diperlukan agar mesin dapat memahami
pemetaan kompleks dari bahasa alami ke
representasi visual [3]. Oleh karena itu,
studi mendalam terhadap dataset menjadi
penting dalam kemajuan riset sintesis teks
ke gambar. Berbagai
jenis
dataset
telah
dikembangkan untuk riset sintesis teks ke
gambar, seperti dataset generik dan spesifik
domain, dalam bahasa Indonesia maupun
Inggris. Dataset
generik mengandung
gambar dan teks deskripsi dalam berbagai
domain, sementara dataset spesifik domain
fokus pada satu tema tertentu seperti burung
atau bunga. EEICT EEICT
https://ojs.uniska-bjm.ac.id/index.php/eeict e-ISSN: 2615-2169
p-ISSN: 2654-4296 e-ISSN: 2615-2169
p-ISSN: 2654-4296 EEICT https://ojs.uniska-bjm.ac.id/index.php/eeict bermanfaat. Oleh karena itu, riset dataset
masih terbuka lebar untuk eksplorasi lebih
lanjut. lain crowdsourcing dengan melibatkan
kontributor manusia, atau metode otomatis
seperti scraping dan crawling data online
[5]. Ukuran dataset yang besar diperlukan
agar model machine learning dapat belajar
pola kompleks dari teks ke gambar [6]. Dataset skala besar umumnya berisi ratusan
ribu hingga jutaan pasang data latih. Namun
dataset kecil dengan puluhan ribu pasang
data juga masih berguna untuk riset awal. EEICT
htt
// EEICT
https://ojs.uniska-bjm.ac.id/index.php/eeict I.
PENDAHULUAN Sintesis teks ke gambar merupakan
bidang penelitian kecerdasan buatan yang
bertujuan untuk menghasilkan gambar Vol. 7 No. 1 Tahun 2024 Page | 10 e-ISSN: 2615-2169
p-ISSN: 2654-4296 III. HASIL DAN PEMBAHASAN Dalam penelitian ini, peneliti meninjau
beberapa dataset utama yang digunakan
dalam penelitian sintesis teks ke gambar. Dataset-dataset tersebut mencakup MS
COCO, Flickr30k, CUB-200-2011, dan
beberapa dataset-domain khusus lainnya. Data
tambahan
seperti
panoptic
segmentation [30], depth estimation [9],
dan keypoint detection [31] memperkaya
MS COCO untuk riset computer vision
lainnya. https://ojs.uniska-bjm.ac.id/index.php/eeict model sintesis teks ke gambar. model sintesis teks ke gambar. EfficientDet
[13]
yang
menunjukkan
performa state-of-the-art. Hasil analisis kemudian digunakan
untuk
membahas
keunggulan
dan
kelemahan
masing-masing
dataset. Penelitian ini juga menyoroti bagaimana
karakteristik dataset dapat mempengaruhi
hasil penelitian dalam bidang sintesis teks
ke gambar. Optimasi model deteksi objek real-time
juga banyak memanfaatkan MS COCO,
misalnya dengan model pruning [14],
weight quantization [15], dan neural
architecture search [16]. Di bidang image segmentation, MS
COCO menjadi dataset acuan untuk model
seperti PSPNet [17], DeepLabv3+ [18],
CCNet [19] , Context Embedding [20],
hingga Segmenter [21]. Temuan penelitian akan menghasilkan
rekomendasi terkait dengan pemilihan
dataset yang sesuai untuk berbagai jenis
penelitian
sintesis
teks
ke
gambar. Rekomendasi
ini
akan
memberikan
panduan bagi peneliti yang ingin memilih
dataset
yang
sesuai
dengan
tujuan
penelitian mereka. Beberapa
penelitian
terbaru
menerapkan self-supervised representation
learning untuk segmentasi gambar di MS
COCO, seperti MoCo-v2 [22] dan SwAV
[23]. Penelitian ini akan diakhiri dengan
kesimpulan yang merangkum temuan
utama
dan
relevansi
dalam
konteks
penelitian sintesis teks ke gambar dan
membahas potensi arah penelitian masa
depan yang muncul dari peninjauan dataset
ini. Dalam captioning gambar, MS COCO
sangat populer untuk pengembangan dan
evaluasi algoritma terbaru seperti CNN-
RNN [24], attribute-based [25], hingga
cross-modality transfer learning [26]. Metode attention based juga banyak
diteliti untuk captioning di MS COCO,
seperti Meshed-Memory Transformer [27],
adaptive attention [28], dan dual attention
[29]. EEICT
htt
// https://ojs.uniska-bjm.ac.id/index.php/eeict II. METODE Dalam penelitian ini, fokus utama
adalah
pada
analisis
dataset
dan
memberikan wawasan yang mendalam
tentang
pengaruh
pemilihan
dataset
terhadap penelitian sintesis teks ke gambar. Analisis tersebut akan didasarkan pada data
yang
ditemukan
dan
informasi
terdokumentasi yang ada. Selain ukuran, keragaman data juga
penting agar model tidak overfitting pada
konteks terbatas [7]. Data harus mencakup
berbagai objek, latar belakang, sudut
pandang, kondisi pencahayaan, dan variasi
lainnya baik pada teks maupun gambar. Langkah pertama dalam penelitian ini
adalah mengumpulkan sumber daya yang
diperlukan untuk melakukan review dataset
dalam konteks sintesis teks ke gambar. Sumber daya ini termasuk dataset itu
sendiri, artikel penelitian terkait, serta
informasi dokumentasi resmi dataset. Jenis anotasi pada dataset meliputi
label objek, atribut, relasi antar objek,
caption/deskripsi teks, terjemahan bahasa,
hingga informasi region of interest dalam
gambar. Semakin kaya anotasi data,
semakin memudahkan mesin memahami
hubungan teks dan gambar [8]. Seleksi dataset yang akan dibahas
dalam
penelitian
merupakan
langkah
penting. Dalam tinjauan ini, peneliti
memilih sejumlah dataset yang secara luas
digunakan dalam penelitian sintesis teks ke
gambar, seperti MS COCO, Flickr30k,
CUB-200-2011, dan dataset-domain khusus
lainnya yang relevan. Tinjauan ini bertujuan memberikan
gambaran komprehensif mengenai berbagai
dataset untuk sintesis teks ke gambar yang
telah dikembangkan. Dengan demikian, review dataset yang
komprehensif
ini
diharapkan
dapat
membantu peneliti dalam memilih dan
memanfaatkan dataset untuk riset sintesis
teks ke gambar. Setelah dataset terpilih, berikutnya
dilakukan
analisis
deskriptif
terhadap
masing-masing dataset yang mencakup
jumlah
total
gambar
dalam
dataset,
karakteristik visual dari gambar, jumlah
deskripsi teks yang ada untuk setiap
gambar, keragaman kata kunci dan gaya
deskripsi teks serta informasi terkait lisensi
dan hak cipta dataset. Selain
itu,
tinjauan
ini
dapat
mengidentifikasi peluang penelitian dataset
di masa depan guna mendukung kemajuan
riset dan aplikasi sintesis teks ke gambar. Secara keseluruhan, kajian mendalam
terhadap dataset merupakan landasan
penting untuk pengembangan teknologi
sintesis teks ke gambar yang handal dan Data yang dikumpulkan akan dianalisis
secara kualitatif. Analisis akan mencakup
pemahaman mendalam tentang kelebihan
dan kekurangan masing-masing dataset,
serta dampaknya pada pengembangan Page | 11 Vol. 7 No. 1 Tahun 2024 Page | 11 e-ISSN: 2615-2169
p-ISSN: 2654-4296 e-ISSN: 2615-2169
p-ISSN: 2654-4296 https://ojs.uniska-bjm.ac.id/index.php/eeict dataset yang relatif kecil untuk melatih
model deep learning skala besar [38]. Jumlah gambar COCO sekitar 200 ribu
dinilai terbatas dibandingkan kebutuhan
model sekarang, beberapa gambar dengan
konten serupa memiliki anotasi objek yang
berbeda
di
COCO
[39]
dan
ketidakseimbangan jumlah gambar tiap
kategori objek [40]. Sehingga dataset lain
mulai dikembangkan. EEICT
htt
// EEICT
https://ojs.uniska-bjm.ac.id/index.php/eeict transformer [48]. Teknik multimodal fusion informasi
tekstual dan visual menjadi tren mutakhir
pada CUB-200-2011, seperti dual modality
graph
network
[49]
dan
text-image
embedding [50]. Beberapa tantangan pada CUB-200-
2011 antara lain variasi pose burung, warna
bulu yang serupa, dan latar belakang
kompleks [51]. Data augmentasi dapat
membantu mengatasi ini. Namun total anotasi MS COCO
mencapai 2.5 juta gambar, jauh lebih besar
dari dataset lainnya. Ini menjadikannya
sumber data paling kaya untuk riset
computer vision. Meskipun ada dataset serupa yang
lebih baru, CUB-200-2011 masih relevan
sebagai benchmark untuk algoritma fine-
grained image classification terkini [52]. CUB-200-2011
diharapkan
terus
diperbaharui dengan versi yang lebih besar
dan variatif di masa mendatang. 3.3. Oxford 102 Flower MS COCO terus diperbaharui dengan
tambahan anotasi dan task oleh komunitas
peneliti computer vision global. Hal ini
menjadikan MS COCO akan tetap relevan
di masa mendatang sebagai benchmark
dataset untuk pengembangan algoritma
computer vision mutakhir. Oxford
102
Flower
merupakan
dataset gambar bunga yang dirilis tahun
2008. Dataset ini berisi 8.189 gambar dari
102 spesies bunga yang diambil dari
beragam sudut pandang dan latar belakang
[53]. 3.1. MS COCO MS
COCO
(Microsoft
Common
Objects in Context) pertama kali dirilis pada
tahun 2014 dan menjadi salah satu
benchmark utama untuk object detection,
segmentation, dan captioning [9]. Dataset
ini berisi lebih dari 200 ribu gambar yang
dianotasi secara detail. Contoh pemanfaatan data depth di MS
COCO antara lain untuk depth completion
[32], depth estimation [33], hingga 3D
reconstruction [34]. Selain itu, MS COCO juga populer
untuk riset domain lain seperti VQA [35],
action recognition [36], hingga image
retrieval [37]. Dalam bidang object detection, MS
COCO
banyak
digunakan
untuk
mengevaluasi model seperti Mask R-CNN
[10], YOLOv3 [11], RetinaNet [12], hingga Meski demikian, beberapa kekurangan
MS COCO diantaranya adalah ukuran Vol. 7 No. 1 Tahun 2024 Page | 12 e-ISSN: 2615-2169
p-ISSN: 2654-4296 EEICT
htt
// 3.6. Fashion-Gen Fashion-Gen adalah dataset fashion
generative modeling yang dirilis tahun
2021. Dataset ini berisi 365.126 pasangan
gambar fashion item dan teks deskripsinya
yang di-generate dengan teknik generative
modeling [74]. Teknik yang digunakan antara lain
generative adversarial networks, variational
autoencoders, dan transformer-based model
untuk
mensintesis
atau
memodifikasi
gambar wajah dari input tekstual [64]. Keunggulan Multi-Modal CelebA-HQ
adalah resolusi gambar tinggi dan pasangan
teks-gambar
yang
berkualitas. Tetapi
cakupan domainnya terbatas pada wajah
selebriti [65]. Fashion-Gen
dapat
dimanfaatkan
untuk riset generative multimodal teks-
gambar. Beberapa
penelitian
memanfaatkannya
untuk
text-to-image
generation [75], image editing [76], dan text
attribute manipulation [77]. Multi-Modal CelebA-HQ bermanfaat
untuk riset personalisasi gambar berbasis
teks seperti editing wajah, avatar generasi,
dan penerapan augmented reality [66]. Teknik yang digunakan antara lain
generative adversarial networks, variational
autoencoders, transformer-based model,
dan deep generative models lainnya untuk
mensintesis dan memanipulasi gambar
fashion dari teks [78]. https://ojs.uniska-bjm.ac.id/index.php/eeict yang lebih besar, Oxford 102 Flowers
masih relevan sebagai benchmark algortima
fine-grained classification [59]. representation
learning,
retrieval,
dan
domain adaptation [69]. representation
learning,
retrieval,
dan
domain adaptation [69]. Beberapa
tantangan
LAION-5B
adalah data tidak berlabel,
beragam
sumbernya, dan berisiko mengandung
konten berbahaya [70]. Diperlukan kurasi
dan filtering yang hati-hati [71] (Srinivasan
et al., 2022). Beberapa
tantangan
LAION-5B
adalah data tidak berlabel,
beragam
sumbernya, dan berisiko mengandung
konten berbahaya [70]. Diperlukan kurasi
dan filtering yang hati-hati [71] (Srinivasan
et al., 2022). Pengelompokan gambar per spesies
perlu
diperhatikan
agar
latihan
dan
pengujian menggunakan gambar berbeda
untuk menghindari data leakage [60]. 3.4. Multi-Modal CelebA-HQ LAION-5B telah dikurasi menjadi
LAION-400M dan LAION-2B yang lebih
kecil namun berkualitas dan aman [72]. Namun skala data asli tetap penting untuk
domain adaptasi [67]. Multi-Modal
CelebA-HQ
adalah
dataset wajah manusia yang dikembangkan
dari CelebA-HQ dengan penambahan data
tekstual. Dataset ini berisi 30.000 gambar
wajah resolusi tinggi beserta caption dan
metadata terkait [61]. Pengelolaan dataset besar seperti
LAION-5B
memerlukan
infrastruktur
komputasi yang memadai dan kerja sama
multidisiplin [73]. Multi-Modal CelebA-HQ cocok untuk
riset multimodal generatif teks-gambar. Beberapa
penelitian
memanfaatkannya
untuk text-guided image synthesis [62] dan
text-based image editing [63]. EEICT
htt
// EEICT
https://ojs.uniska-bjm.ac.id/index.php/eeict 3.2. CUB-200-2011 CUB-200-2011
adalah
dataset
gambar burung yang dirilis tahun 2011. Dataset ini berisi 11.788 gambar dari 200
spesies burung yang disertai anotasi
bounding box, part locations, dan atribut
[41]. Oxford 102 Flower sering digunakan
sebagai benchmark untuk riset fine-grained
image classification. Beberapa penelitian
terbaru menerapkan deep metric learning
[54], graph neural networks [55], dan
capsule networks [56]. CUB-200-2011
sering
dijadikan
benchmark untuk riset fine-grained image
classification. Beberapa penelitian terbaru
menggunakan
graph
convolutional
networks [42] dan co-attention model [43]. Data
augmentation
dan
transfer
learning dengan model CNN seperti ResNet
dan DenseNet populer dilakukan untuk
meningkatkan akurasi klasifikasi pada
dataset ini [57]. Beberapa penelitian menerapkan few-
shot learning [44] dan zero-shot learning
[45] pada CUB-200-2011 untuk mengatasi
minimnya data latih pada beberapa kelas. Beberapa tantangan pada Oxford 102
Flowers antara lain perbedaan sudut
pandang, variasi ukuran, bentuk dan warna
bunga yang mirip pada beberapa spesies
[58]. Metode
attention-based
neural
networks juga banyak diteliti dengan
inovasi cross-attention [46] dan dynamic
attention [47]. Demikian pula penggunaan Meskipun sudah ada dataset serupa Vol. 7 No. 1 Tahun 2024 Page | 13 e-ISSN: 2615-2169
p-ISSN: 2654-4296 e-ISSN: 2615-2169
p-ISSN: 2654-4296 3.7. ImageNet Conceptual
Images
12M
adalah
kumpulan gambar konsep yang dikurasi
dari berbagai sumber dan dirilis tahun 2020. Dataset ini berisi 12 juta gambar dengan
1.625 konsep/kelas [95]. ImageNet adalah dataset gambar untuk
penelitian computer vision yang pertama
kali dirilis tahun 2009. ImageNet berisi 14
juta gambar yang dikategorikan ke dalam
puluhan ribu kelas berdasarkan WordNet
hierarchy [81]. Conceptual Images 12M cocok untuk
riset pembelajaran mesin konsep visual
seperti
zero-shot
learning,
few-shot
learning,
transfer
learning,
dan
pengklasifikasian konsep [95]. ImageNet telah menjadi benchmark
standar untuk penelitian deep learning pada
task image classification, object detection,
segmentation dan retrieval [82]. Beragam
arsitektur
deep
neural
network seperti AlexNet [83], VGG [84],
ResNet
[85],
dan
EfficientNet
[86]
berkembang
berkat
evaluasi
yang
komprehensif di ImageNet. Beberapa penelitian memanfaatkan
dataset ini untuk zero-shot classification
dengan CLIP model [96], metric learning
[97], dan contrastive learning [98]. Keunggulan Conceptual Images 12M
adalah cakupan konsep yang luas dan
beragam. Tantangannya
adalah
tidak
adanya pemisahan data latih-uji secara
eksplisit [97]. Beberapa tantangan ImageNet antara
lain adanya bias dan kesalahan label kelas
pada sebagian data [87]. Pembersihan
dataset perlu dilakukan agar tetap relevan. Meski demikian, ImageNet masih
menjadi
salah
satu
dataset
paling
berpengaruh dalam kemajuan deep learning
untuk pengolahan gambar [88]. 3 8 Google’s Open Images Skala Conceptual Images 12M yang
besar menantang keterbatasan sumber daya
komputasi dan mendorong inovasi model
efisiensi tinggi [96]. 3.9. Conceptual Images 12M 3.9. Conceptual Images 12M EEICT
htt
// https://ojs.uniska-bjm.ac.id/index.php/eeict 3.5. LAION-5B LAION-5B adalah kumpulan gambar
berukuran besar yang dirilis tahun 2022,
berisi 5 miliar pasangan teks dan gambar
yang diambil dari internet [67]. Keunggulan Fashion-Gen adalah data
latih yang besar dan beragam atributnya. Tetapi data bersifat sintetis dan terbatas
pada domain fashion item tertentu [79]. LAION-5B
dapat
dimanfaatkan
untuk melatih model AI skala besar,
khususnya multimodal [68]. Data ini cocok
untuk
riset
generative
modeling, Fashion-Gen cocok untuk penelitian
generatif multimodal dan penerapannya Vol. 7 No. 1 Tahun 2024 Page | 14 e-ISSN: 2615-2169
p-ISSN: 2654-4296 3.10. Flickr30k 3.8. Google’s Open Images https://ojs.uniska-bjm.ac.id/index.php/eeict seperti virtual try-on, fashion image
retrieval, dan sistem rekomendasi fashion
generatif [80]. Open Images menjadi salah satu dataset
skala besar paling beragam untuk riset
computer vision saat ini [89]. https://ojs.uniska-bjm.ac.id/index.php/eeict answering [101], image retrieval [102], dan
visual grounding [103]. IV. KESIMPULAN Penelitian ini memberikan tinjauan
komprehensif terhadap berbagai dataset
yang digunakan dalam riset sintesis teks ke
gambar. Sintesis teks ke gambar merupakan
bidang penelitian kecerdasan buatan yang
menjanjikan,
dengan
potensi
aplikasi
seperti
pembangkit ilustrasi
otomatis,
asisten desain grafis, dan penerjemah
bahasa isyarat. Salah satu tantangan pada Flickr30k
adalah caption yang dibuat manusia
memiliki banyak variasi sehingga evalusi
automatik kurang mencerminkan kualitas
caption
[104]. Beberapa
penelitian
mengusulkan evaluasi manusia [105]. Captioning gambar out-of-domain
merupakan tantangan Flickr30k lainnya
karena data latih berasal dari domain
gambar tertentu Flickr [106]. Transfer
learning dinilai dapat mengatasi domain
gap ini. Kunci keberhasilan dalam riset ini
adalah ketersediaan dataset pelatihan yang
berkualitas tinggi. Dataset yang besar dan
bervariasi diperlukan agar model dapat
memahami pemetaan kompleks dari bahasa
alami ke representasi visual. Oleh karena
itu, pemilihan dan analisis mendalam
terhadap dataset menjadi kritis dalam
kemajuan riset ini. Flickr30k
diharapkan
terus
berkembang dengan versi yang lebih besar
dan beragam untuk mendukung riset
captioning automatic, visual grounding,
hingga visualsemantic embeddings di masa
mendatang. Beberapa dataset utama yang dibahas
meliputi MS COCO, CUB-200-2011,
Oxford 102 Flower, Multi-Modal CelebA-
HQ, LAION-5B, Fashion-Gen, ImageNet,
Google's Open Images, Conceptual Images
12M, Flickr30k, dan Filtered YFCC100M. Masing-masing
dataset
memiliki
karakteristik, keunggulan, dan tantangan
tersendiri. 3.11. Filtered YFCC100M Vol. 7 No. 1 Tahun 2024 EEICT
h
// EEICT
https://ojs.uniska-bjm.ac.id/index.php/eeict 3.8. Google’s Open Images Flickr30k merupakan dataset gambar
yang dirilis tahun 2014 untuk riset
captioning gambar. Dataset ini berisi
31.000 gambar yang diambil dari Flickr dan
masing-masing
dilengkapi
5
caption
deskriptif oleh manusia [99]. Google's
Open
Images
adalah
kumpulan gambar skala besar yang dirilis
tahun 2018. Dataset ini berisi 9 juta gambar
yang dianotasi untuk riset object detection,
classification, dan visual relationship [89]. Open Images banyak dimanfaatkan
untuk pengembangan dan evaluasi model
object detection seperti YOLOv4 [90],
EfficientDet [13] dan DetectoRS [91]. Flickr30k menjadi dataset standar
untuk pengembangan dan evaluasi model
captioning gambar. Beberapa penelitian
memanfaatkan Flickr30k untuk model
seq2seq [100], CNN-RNN [28], hingga
attention-based captioning [28]. Data Open Images juga digunakan
pada riset domain lain seperti human action
recognition [92], image retrieval [93],
hingga visual question answering [94]. Data Flickr30k juga sering digunakan
pada riset terkait seperti visual question Dengan lebih dari 500 kelas objek, Vol. 7 No. 1 Tahun 2024 Page | 15 e-ISSN: 2615-2169
p-ISSN: 2654-4296 e-ISSN: 2615-2169
p-ISSN: 2654-4296 e-ISSN: 2615-2169
p-ISSN: 2654-4296 EEICT
htt
// Saran [5] [5]
E. S. Jo and T. Gebru, “Lessons from
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termasuk memperoleh pasangan teks dan
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Short Paper – MIDL 2021 submission Short Paper – MIDL 2021 submission Medical Imaging with Deep Learning – Under Review 2021 Abstract In this work, we propose the use of Recurrent Inference Machines (RIMs) to perform T1
mapping. The RIM is a neural network framework that learns an iterative inference process
using a model of the signal, similar to conventional statistical methods for quantitative MRI
(QMRI), such as the Maximum Likelihood Estimator (MLE). Previously, RIMs were used
to solve linear inverse reconstruction problems. Here, we show that they can also be used
to optimize non-linear problems. The developed RIM framework is evaluated in terms
of accuracy and precision and compared to an MLE method and an implementation of
the ResNet. The results show that, compared to the other techniques in Monte Carlo
experiments with simulated data, the RIM improves the precision of estimates without
compromising in accuracy. Keywords: Quantitative MRI, Deep learning, Recurrent inference machines. Editors: Under Review for MIDL 2021 Editors: Under Review for MIDL 2021 Editors: Under Review for MIDL 2021 2021 E.R. Sabidussi et al. Recurrent Inference Machines as Inverse Problem Solvers for
MR Relaxometry e.ribeirosabidussi@erasmusmc.nl 2. Materials and Methods Recurrent Inference Machines
The RIM is a recurrent neural network framework that
learns an efficient iterative inference method and a prior that uses the neighborhood con-
text. The framework uses the gradients of a likelihood function to plan efficient parameter
updates. At a given optimization step j 2 {0, ..., J −1}, the RIM receives as input the
current estimate of parameters, ˆj, the gradient of a log-likelihood function L with respect
to , r, and a vector of memory states hj to keep track of optimization progress and per-
form more efficient updates. The network outputs an update to the current estimate and
the memory state to be used in the next iteration. The update equations for this method
are given by {∆ˆj+1, hj+1} = gγ(ˆj, r, hj) and ˆj+1 = ˆj +∆ˆj+1, where ∆ˆj+1 is the
incremental parameter update at step j + 1 and gγ represents the neural network portion
of the framework, parameterized by γ. Predictions are compared to a known ground-truth
and training losses are accumulated at each step. The optimal network model is learned
via ˆγ = arg minγ(1/J) PJ−1
j=0 k−ˆj+1k2
2. Signal model and likelihood function
The joint log-likelihood of the N acquired im-
ages is given by L(, σ|S) =
1
σ2
PN
n=1kf n(ˆ) −Snk2
2, where ˆdenotes the parameter
estimates, and σ the noise standard deviation (STD). In this work, we assume σ to be
known. As signal model for the CINE sequence (Atkinson and Edelman, 1991) we use
fn() =
"""A
⇣
1 −B exp(−⌧n
T1 )
⌘""", in which A is proportional to the proton density and re-
ceiver gain, B captures the efficiency of the inversion pulse, and T1 is the longitudinal
relaxation time. Training dataset
The RIM is trained with simulated ground truth tissue parameters
and simulated weighted images S. To generate training samples with a spatial distribution
that resembles the human brain, ten 3D virtual brain models from BrainWeb (Aubert-
Broche et al., 2006) were selected. We randomly extract 2D patches (40 ⇥40 pixels) from
the brain images, with patch centers drawn uniformly from the model’s brain mask. T1
values were simulated in the range of [0.3, 3.5] s and A values in between [0.65, 1] a.u. B
values were simulated as 2 −Γ, where Γ is the half-normal distribution, with STD of 0.2. 1. Introduction T1 relaxation time is a promising biomarker for a range of diseases. Conventionally, in
MR relaxometry, T1 maps are estimated by fitting a known signal model to every voxel
of a series of weighted images with varying contrast settings. This approach is used by
widely employed methods, such as the Maximum Likelihood Estimator (MLE). However,
without explicit definition of regularization priors, estimates have reduced precision if the
data is noisy. In this paper, we propose a new framework for MR relaxometry based on
the Recurrent Inference Machines (RIMs) (Putzky and Welling, 2017). Here, we show in
Monte Carlo experiments with simulated data that the RIM estimates T1 maps with higher
precision than MLE and ResNet implementations. 2. Materials and Methods S
was generated with additive zero mean Gaussian noise with STD drawn from a log-uniform
distribution in the range [0.0065, 0.255] (SNR from 100 to 3). We used 31 inversion times
(⌧) varying linearly from 0.139 to 0.937 seconds. Evaluation
The RIM was compared to the MLE estimate, obtained by maximizing the
L(, σ|S), and an implementation of the ResNet (He et al., 2015), trained with the same
training data. The prediction accuracy was evaluated in terms of the Relative Bias between
the reference parameter values and the estimated parameters ˆc 2 {ˆ1, ..., ˆC} for each
repeated experiment c, defined as Relative Bias [%] =
1
C
PC
c=1 [(ˆc −) ↵] ⇥100%,
where ↵denotes the element-wise division. The Coefficient of Variation (CV) was used to
measure the precision of the predictions, defined as CV [%] =
⇣
STDC (ˆc) ↵1
C
PC
c=1 ˆc⌘
⇥
100%, where STDC is the standard deviation over C estimates ˆ. To assess each method’s robustness to noise and mapping quality, simulated T1 weighted
images were generated as the training dataset, using a 2D slice of a virtual brain model not
included in the training. For the same ground-truth T1, A and B maps, C = 100 realisations 2 Recurrent Inference Machines for MR Relaxometry Figure 1: Results of the Monte Carlo experiment as a function of SNR levels. Figure 1: Results of the Monte Carlo experiment as a function of SNR levels. of acquisition noise were simulated per SNR 2 [3, 5, 10, 30, 60, 100]. The Relative Bias and
CV were computed per pixel and their distribution over all pixels within a brain mask is
presented. of acquisition noise were simulated per SNR 2 [3, 5, 10, 30, 60, 100]. The Relative Bias and
CV were computed per pixel and their distribution over all pixels within a brain mask is
presented. of acquisition noise were simulated per SNR 2 [3, 5, 10, 30, 60, 100]. The Relative Bias and
CV were computed per pixel and their distribution over all pixels within a brain mask is
presented. 3. Results and Conclusion Figures 1(a)-(c) show the Relative Bias measured for A, B and T1 maps. For most cases
where SNR > 3, all methods produced quantitative maps with comparable median Relative
Bias, but both neural networks displayed a larger range of values than the MLE. The CV
for all SNR levels is shown in Figs. 1(d)-(f) for the same data. The RIM presented lower
CV than the other methods for all SNRs, while, comparatively, the MLE showed higher
CV, accentuated in low SNR. We proposed a new method for T1 mapping based on the RIM framework. Experiments
with simulated data show that the proposed RIM produces T1 estimates with similar accu-
racy and higher precision than the MLE and ResNet methods. These results suggest that
the RIM is a promising technique for quantitative MRI. Acknowledgment
This project was funded by the European Union’s Horizon 2020 pro-
gramme, under the grant agreement No 764513. P. Putzky and M. Welling.
Recurrent inference machines for solving inverse problems.
arXiv, 1706.04008, 2017. References D. J. Atkinson and R. R. Edelman. Cineangiography of the heart in a single breath hold
with a segmented turboflash sequence. Radiology, 178(2):357–360, 1991. B. Aubert-Broche, M. Griffin, G. B. Pike, A. C. Evans, and D. L. Collins. Twenty new
digital brain phantoms for creation of validation image data bases. IEEE Transactions
on Medical Imaging, 25(11):1410–1416, 2006. K. He, X. Zhang, S. Ren, and J. Sun. Deep residual learning for image recognition. arXiv,
1512.03385, 2015. P. Putzky and M. Welling. Recurrent inference machines for solving inverse problems. arXiv, 1706.04008, 2017. 3 3
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https://openalex.org/W3216512770
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https://authors.library.caltech.edu/113285/1/aa42327-21.pdf
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English
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OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star
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Astronomy & astrophysics
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cc-by
| 11,109
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ABSTRACT Aims. With the aim of interpreting anomalous lensing events with no suggested models, we conducted a project of reinvestigating
microlensing data collected in and before the 2019 season. In this work, we report a multi-planet system, OGLE-2019-BLG-0468L,
that was found as a result of this project. Methods. The light curve of the lensing event OGLE-2019-BLG-0468, which consists of three distinctive anomaly features, could
not be explained by the usual binary-lens or binary-source interpretations. We find a solution that explains all anomaly features with a
triple-lens interpretation, in which the lens is composed of two planets and their host, making the lens the fourth multi-planet system
securely found by microlensing. y
y
g
Results. The two planets have masses of ∼3.4 MJ and ∼10.2 MJ, and they are orbiting around a G-type star with a mass of ∼0.9 M⊙
and a distance of ∼4.4 kpc. The host of the planets is most likely responsible for the light of the baseline object, although the possibility
of the host being a companion to the baseline object cannot be ruled out. Key words. gravitational lensing: micro – planets and satellites: detection OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets
around a G-type star OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets
around a G-type star
Cheongho Han1, Andrzej Udalski2, Chung-Uk Lee3, Doeon Kim1, Wei Zhu4,
Michael D. Albrow5, Sun-Ju Chung3,6, Andrew Gould7,8, Kyu-Ha Hwang3, Youn Kil Jung3,
Hyoun-Woo Kim3, Yoon-Hyun Ryu3, In-Gu Shin3, Yossi Shvartzvald9, Jennifer C. Yee10, Weicheng Zang4,
Sang-Mok Cha3,11, Dong-Jin Kim3, Seung-Lee Kim3,6, Dong-Joo Lee3, Yongseok Lee3,11,
Byeong-Gon Park3,6, Richard W. Pogge8, Chun-Hwey Kim12, Woong-Tae Kim13
(The KMTNet Collaboration),
Przemek Mróz2,14, Michał K. Szyma´nski2, Jan Skowron2, Radosław Poleski2, Igor Soszy´nski2,
Paweł Pietrukowicz2, Szymon Kozłowski2, Krzysztof A. Rybicki2, Patryk Iwanek2,
Krzysztof Ulaczyk15, Marcin Wrona2, Mariusz Gromadzki2
(The OGLE Collaboration),
David Buckley16, Subo Dong17, and Ali Luo18
(Affiliations can be found after the references)
Received 29 September 2021 / Accepted 5 November 2021 Cheongho Han1, Andrzej Udalski2, Chung-Uk Lee3, Doeon Kim1, Wei Zhu4,
Michael D. Albrow5, Sun-Ju Chung3,6, Andrew Gould7,8, Kyu-Ha Hwang3, Youn Kil Jung3,
Hyoun-Woo Kim3, Yoon-Hyun Ryu3, In-Gu Shin3, Yossi Shvartzvald9, Jennifer C. Yee10, Weicheng Zang4,
Sang-Mok Cha3,11, Dong-Jin Kim3, Seung-Lee Kim3,6, Dong-Joo Lee3, Yongseok Lee3,11,
Byeong-Gon Park3,6, Richard W. Pogge8, Chun-Hwey Kim12, Woong-Tae Kim13
(The KMTNet Collaboration), (
),
Przemek Mróz2,14, Michał K. Szyma´nski2, Jan Skowron2, Radosław Poleski2, Igor Soszy´nski2
Paweł Pietrukowicz2, Szymon Kozłowski2, Krzysztof A. Rybicki2, Patryk Iwanek2,
Krzysztof Ulaczyk15, Marcin Wrona2, Mariusz Gromadzki2
(The OGLE Collaboration),
David Buckley16, Subo Dong17, and Ali Luo18
(Affili i
b f
d f
h
f
) (The OGLE Collaboration), David Buckley16, Subo Dong17, and Ali Luo18 1. Introduction multi-planet systems, microlensing has so far detected three reli-
able two-planet systems, OGLE-2006-BLG-109L (Gaudi et al. 2008; Bennett et al. 2010), OGLE-2012-BLG-0026L (Han et al. 2013; Beaulieu et al. 2016), and OGLE-2018-BLG-1011L (Han
et al. 2019), in addition to two candidate two-planet systems,
OGLE-2014-BLG-1722L (Suzuki et al. 2018) and KMT-2019-
BLG-1953L (Han et al. 2020a). Compared to the total number of
over 100 planetary systems found with microlensing, the number
of multi-planet systems is small. However, given the relatively
low efficiency of detecting multi-planet systems with microlens-
ing (Zhu et al. 2014), these numbers already somewhat indicate
that a perhaps substantial fraction of microlensing planets have
additional planetary-mass companions (Madsen & Zhu 2019). Studies based on radial velocity (RV) observations have shown
that beyond ∼1 AU about 10% of stars have giant planets
(Cumming et al. 2008; Fulton et al. 2021). One question to ask is
how common it is for such cold giant planets to have massive
planetary-mass companions. On one hand, cold giant planets
have eccentric orbits that are generally attributed to dynamical
interactions with additional massive companions, suggesting that
perhaps the majority of them have such companions, at least at
birth (Juri´c & Tremaine 2008; Chatterjee et al. 2008). On the
other hand, after over two decades of searches, RV surveys have
only been able to identify the presence of massive companions
to ∼20–30% of known cold giants (Wright et al. 2009; Rosenthal
et al. 2021). This discrepancy can potentially be reduced with
more discoveries of giant planet systems, but unfortunately RV
becomes extremely inefficient in detecting planets with relatively
low masses and/or long orbital periods. Given the potential of microlensing in studying the multi-
plicity distribution of cold planets and thus the architecture of
planetary systems in the cold region, it is important to detect
more, secure multi-planet microlensing systems. This requires
high-cadence observations over a large number of microlensing
events as well as detailed light curve modelings of all anomalous
events. The signal produced by multiple planets differs from that
produced by a single planet because the individual planets induce
their own caustics and these caustics often result in a complex Being most sensitive to cold planets located around or
beyond the water snow line, gravitational microlensing can
play an important role in completing the demographic census
of exoplanets (Gaudi 2012; Zhu & Dong 2021). Astronomy
&
Astrophysics Astronomy
&
Astrophysics A&A 658, A93 (2022)
https://doi.org/10.1051/0004-6361/202142327
© ESO 2022 Astronomy
&
Astrophysics Article published by EDP Sciences 1. Introduction 2021c),
KMT-2019-BLG-0797 was a binary-lensing event occurring on a
binary stellar system, a 2L2S event (Han et al. 2021b), and KMT-
2019-BLG-1953 was a strong candidate planetary event with a
lens composed of two planets and the host (Han et al. 2020a). The events in this group have the common characteristic that
interpreting the lensing light curves of the events requires one
to add extra source or lens components into the modeling. to the Optical Gravitational Lensing Experiment IV (OGLE-IV)
photometry system. As we show in Sect. 4, the source is much
fainter than the baseline magnitude, and the baseline flux comes
mostly from a blend. y
Figure 1 shows the light curve of OGLE-2019-BLG-0468. The rising of the source flux induced by lensing was first found
by the OGLE-IV (Udalski et al. 2015) survey in the early part of
the 2019 season, on April 13, 2019 (HJD′ ≡HJD −2 450 000 ∼
8586). The OGLE team utilizes the 1.3 m telescope at the
Las Campanas Observatory in Chile, which is equipped with
a camera that yields a 1.4 deg2 field of view. The source flux
increased until t1 ∼8586.3 and then declined during 8586 ≲
HJD′ ≲8590, producing a weak bump at around t1. The flux
suddenly increased at t2 ∼8592.9, suggesting that the source
crossed a caustic induced by the multiplicity of the lens. The
detailed structure of the light curve for the three nights during
the period 8596 ≲HJD′ ≲8598 could not be delineated because
the OGLE observation of the event was not conducted for that
time. When the event was observed by the OGLE survey again
on HJD′ ∼8599, the source flux continued to decline until it
reached the baseline. In this work we present the results found from the reanaly-
sis of the lensing event OGLE-2019-BLG-0468. The light curve
of the event was previously investigated with 2L1S and 1L2S
interpretations, but no plausible solution was suggested. From
the reanalysis of the event based on more sophisticated models,
we find that the event was produced by a triple-lens (3L1S) sys-
tem, in which the lens is composed of two giant planets and their
host star. We present the analysis of the event according to the fol-
lowing organization. In Sect. 2 we describe observations of
the lensing event and the characteristics of the observed light
curve. In Sect. 1. Introduction In terms of Article published by EDP Sciences A93, page 1 of 10 A&A 658, A93 (2022) Fig. 1. Light curve of the microlensing event OGLE-2019-BLG-0468. The arrows labeled t1 (8586.3), t2 (8592.9), and t3 (8595.7) indicate the
three epochs of major anomalies. The curves drawn over the data points
represent the 1L1S (dotted curve) and 3L1S (close-close model, solid
curve) models. The zoomed-in view of the anomaly region around t2
and t3 is shown in Fig. 2. pattern due to the interference between the caustics (Danˇek &
Heyrovský 2015a,b, 2019). As a result, these signals, in most
cases, cannot be described by the usual lensing models based on
the binary-lens (2L1S) or binary-source (1L2S) interpretations. This implies that some anomalous events with signals produced
by multiple planets are probably left unanalyzed. The amount of microlensing data was dramatically decreased
during the COVID-19 pandemic because many of the major
survey telescopes were shut down. In order to make the best
use of this downtime, we conducted a project in which pre-
vious microlensing data collected by the Korea Microlens-
ing Telescope Network (KMTNet) survey in and before the
2019 season were systematically reinvestigated. The aim of
the project is to find events of scientific importance among
those with no presented analyses. One group of events for
this investigation are those with weak anomalies. Investigating
such events led to the discoveries of 16 microlensing planets:
KMT-2018-BLG-1025Lb (Han et al. 2021e), KMT-2016-BLG-
2364Lb, KMT-2016-BLG-2397Lb, OGLE-2017-BLG-0604Lb,
OGLE-2017-BLG-1375Lb (Han et al. 2020e), KMT-2018-BLG-
0748Lb (Han et al. 2020d), KMT-2019-BLG-1339Lb (Han et al. 2020b), KMT-2018-BLG-1976, KMT-2018-BLG-1996, OGLE-
2019-BLG-0954 (Han et al. 2021d), OGLE-2018-BLG-0977Lb,
OGLE-2018-BLG-0506Lb, OGLE-2018-BLG-0516Lb, OGLE-
2019-BLG-1492Lb, KMT-2019-BLG-0253 (Hwang et al. 2022),
and OGLE-2019-BLG-1053 (Zang et al. 2021). Fig. 1. Light curve of the microlensing event OGLE-2019-BLG-0468. The arrows labeled t1 (8586.3), t2 (8592.9), and t3 (8595.7) indicate the
three epochs of major anomalies. The curves drawn over the data points
represent the 1L1S (dotted curve) and 3L1S (close-close model, solid
curve) models. The zoomed-in view of the anomaly region around t2
and t3 is shown in Fig. 2. Another group of targeted events are those with known
anomalies but for which interpretations of the anomalies have
not been presented. From the investigation of such events, it was
found that KMT-2019-BLG-1715 was a planetary event involved
with three lens masses and two source stars (Han et al. 1. Introduction 3 we depict various models tested to explain the
observed light curve, including 2L1S, 1L2S, 2L2S, and 3L1S
models. In Sect. 4 we characterize the source star and estimate
the angular Einstein radius. In Sect. 5 we estimate the physical
lens parameters using the available observables of the event. In
Sect. 6 we discuss the possibility that the baseline object is the
lens. We then summarize our results and conclude in Sect. 7. The event was also located in the field covered by the KMT-
Net survey (Kim et al. 2016). The KMTNet group utilizes three
identical telescopes, each of which has a 1.6 m aperture and is
mounted with a camera that yields a 4 deg2 field of view. For the
continuous coverage of lensing events, the telescopes are dis-
tributed over the three continents of the Southern Hemisphere,
at the Siding Spring Observatory in Australia (KMTA), the
Cerro Tololo Inter-American Observatory in Chile (KMTC), and
the South African Astronomical Observatory in South Africa
(KMTS). For both the OGLE and KMTNet surveys, observa-
tions of the event were done mainly in the I band, and a fraction
of V-band images were acquired for the measurement of the
source color. The event was identified by the KMTNet survey
from the post-season inspection of the 2019 season data, and it
was labeled as KMT-2019-BLG-2696. Hereafter we use only the 2. Observations and data The source of the lensing event OGLE-2019-BLG-0468 lies
in the Galactic bulge field at the equatorial coordinates
(RA, Dec)J2000 = (17:45:37.44, −24:26:50.2), which correspond
to the Galactic coordinates (l, b) = (3◦.834, 2◦.336). The apparent
baseline I-band magnitude of the source is Ibase = 17.8 according A93, page 2 of 10 C. Han et al.: OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star Fig. 2. Zoomed-in view of the anomaly region around the epochs t2 and
t3. Notations are the same as in Fig. 1. separation at t0, and the event timescale, respectively. Adding an
extra lens or source component into the modeling requires one to
include extra parameters. For a 2L1S event, these extra param-
eters are (s, q, α), which denote the binary separation, the mass
ratio between the lens components, and the angle between the
direction of the source motion and the binary axis (source trajec-
tory angle), respectively. For a 1L2S event, the extra parameters
are (t0,2, u0,2, qF), the first two of which are the closest approach
time and separation of the source companion, and the last param-
eter indicates the flux ratio between the companion (S 2) and
primary (S 1) source stars. In all cases of the tested models,
we included an additional parameter, ρ, which denotes the ratio
of the angular source radius, θ∗, to the angular Einstein radius,
θE, that is, ρ = θ∗/θE (normalized source radius), to account for
possible finite-source effects in the lensing light curve. To dis-
tinguish parameters related to S 1 and S 2, we use the notations
(t0,1, u0,1, ρ1) and (t0,2, u0,2, ρ2), respectively. ,
,
,
,
p
y
From the modeling of the observed light curve with the 2L1S
and 1L2S interpretations, it was found that the data cannot be
explained by these models. In Fig. 3 we plot the model curves
and residuals of the 2L1S (dashed curve) and 1L2S (dot-dashed
curve) models. The lensing parameters of these models are listed
in Table 1 together with the χ2 values of the fits. Fig. 2. Zoomed-in view of the anomaly region around the epochs t2 and
t3. Notations are the same as in Fig. 1. We made an additional check with a 2L2S model, in which
both the lens and source are binaries; such a system is exempli-
fied by MOA-2010-BLG-117 (Bennett et al. 2018), OGLE-2016-
BLG-1003 (Jung et al. 3.2. 3L1S model p
p
p
(
)
In addition to the photometric data, we also obtained two
spectra, with a 1000 s exposure for each, of the baseline object on
the night of June 3, 2021 (HJD′ = 9398), which is ∼2.2 yr after
the event, using the Robert Stobie Spectrograph (Burgh et al. 2003) mounted on the South African Large Telescope (SALT;
Buckley et al. 2006). The spectroscopic data were reduced using
a custom pipeline based on the PySALT package (Crawford et al. 2010), which accounts for basic charge-coupled device charac-
teristics, removal of cosmic rays, wavelength calibration, and
relative flux calibration. To estimate the stellar parameters (Teff,
log g, and [Fe/H]), we interpolated the observed spectra in an
empirical grid (Du et al. 2019), which was constructed with mas-
sive spectra collected by the Large Sky Area Multi-Object Fibre
Spectroscopic Telescope (LAMOST). Unfortunately, it was dif-
ficult to securely estimate the stellar parameters due to the low
signal-to-noise ratios of the spectra. We then tested a model in which the lens is a triple system (3L1S
model). In the first step of this modeling, we checked whether
a 2L1S model can describe part of the anomalies, although it
turned out that the model could not explain all anomaly features. We did this check because lensing light curves with three lens
components (M1, M2, and M3) can, in many cases, be approxi-
mated by the superposition of two 2L1S light curves produced
by M1–M2 and M1–M3 lens pairs (Bozza 1999; Han et al. 2001). This is exemplified by the light curve of OGLE-2012-BLG-0026
(Han et al. 2013), which is generated by a lens composed of two
planets and a host star, and that of OGLE-2018-BLG-1700 (Han
et al. 2020c), which is produced by a lens composed of a planet
and binary stars. From the modeling conducted with the partial
data, excluding those after t2, it is found that the light curve
is well approximated by a 2L1S model with binary parameters
(s, q, α) ∼(0.86, 3 × 10−3, 154◦). This suggests the possibility
that the event may be produced by a triple-lens (3L) system Modeling with the inclusion of a third lens component
requires one to add three extra parameters to those of the 2L1S
model. 3.2. 3L1S model These parameters are (s3, q3, ψ), which represent the M1–
M3 separation, the M3/M1 mass ratio, and the orientation angle
of M3 as measured from the M1–M2 axis with the center at the
position of M1, respectively. In the first round of the 3L1S mod-
eling, we searched for the lensing parameters related to M3, that
is, s3, q3, and ψ, with a grid approach by fixing the parameters
related to M2, that is, s2, q2, and α, as the ones obtained from
the preliminary 2L1S modeling conducted with the use of the 2. Observations and data 2017), KMT-2019-BLG-0797 (Han et al. 2021b), and KMT-2018-BLG-1743 (Han et al. 2021a). The model
curve and residual from the 2L2S solution are presented in Fig. 3,
and the lensing parameters of the solution are listed in Table 1. This model provides a better fit than the 2L1S and 1L2S models
by ∆χ2 = 295.2 and 2069.7, respectively. However, the model still
leaves substantial residuals, indicating that a new interpretation
of the light curve is needed. OGLE event number, according to the order of discovery, to des-
ignate the event. The light curve constructed with the additional
KMTNet data revealed that there existed an additional peak at
t3 ∼8595.7 that was not covered by the OGLE data. Figure 2
shows the zoomed-in view of the light curve around the epochs
t2 and t3. The light curve of the event was constructed by conducting
photometry of the source using the pipelines of the individual
survey groups: Wo´zniak (2000) for OGLE and Albrow et al. (2009) for KMTNet. In order to consider the scatter of the data
and to make χ2 per degree of freedom for each data set unity, the
error bars estimated by the pipelines were readjusted according
to the prescription depicted in Yee et al. (2012). 3. Light curve interpretation 3.1. 2L1S, 1L2S, and 2L2S models 3.1. 2L1S, 1L2S, and 2L2S models We first modeled the observed light curve under the assumption
of lens (2L1S model) or source (1L2S model) binarity, which is
the most common cause of lensing light curve anomalies. The
lensing light curve of a single-lens single-source (1L1S) event
is characterized by the three parameters (t0, u0, tE), which repre-
sent the time of the closest lens-source approach, the lens-source A93, page 3 of 10 A&A 658, A93 (2022) A&A 658, A93 (2022)
Fig. 3. Comparison of the four tested models, 1L2S,
2L1S, 2S2L, and 3L1S (close-close solution). The lower
panels show the residuals from the individual models. Table 1. Lensing parameters of the 1L2S, 2L1S, and 2L2S models. Parameter
2L1S
1L2S
2L2S
χ2
2244.3
4018.8
1949.1
t0,1 (HJD′)
8594.721 ± 0.024 8595.614 ± 0.013 8585.635 ± 0.124
u0,1
0.060 ± 0.003
0.0020 ± 0.0001
0.0084 ± 0.0014
t0,2 (HJD′)
–
8585.745 ± 0.149 8595.361 ± 0.022
u0,2
–
0.0070 ± 0.0009
0.0053 ± 0.0003
tE (days)
29.10 ± 1.68
297.36 ± 1.69
181.05 ± 9.90
s
0.623 ± 0.008
–
0.110 ± 0.004
q
0.067 ± 0.002
–
1.252 ± 0.307
α (rad)
2.257 ± 0.010
–
1.191 ± 0.014
ρ1 (10−3)
–
–
–
ρ2 (10−3)
–
–
–
qF
–
0.22 ± 0.02
7.15 ± 0.44
Notes. HJD′ ≡HJD −2 450 000. partial data. Figure 4 shows the ∆χ2 distribution in the log s3–
log q3 parameter plane obtained from this first-round modeling. We note that there exist two locals in the ∆χ2 map, indicating that
there are degenerate solutions: at (log s3, log q3) ∼(+0.15, −2.0)
and ∼(−0.15, −2.0). We discuss this degeneracy more below. In
the second round, we refined the local solutions found from the
first-round modeling using a downhill approach based on the
Markov chain Monte Carlo (MCMC) method. In this process,
we released all parameters as free parameters. We find that the anomaly features of the observed light curve
are well explained by a 3L1S model. The model curve is plotted
over the data points in Figs. 1 and 2, and the residual from the
model is compared to those of the other models (1L2S, 2L1S,
and 2L2S models) in Fig. 3. We find two solutions, in which
s2 < 1.0 and s3 < 1.0 for one solution and s2 < 1.0 and s3 >
Fig. 4. 3.1. 2L1S, 1L2S, and 2L2S models Distribution of ∆χ2 in the log s3–log q3 parameter plane
obtained from the preliminary 3L1S grid searches for the parameters
related to the third lens component. Red, yellow, green, cyan, and blue
are used to denote points with ∆χ2 < n(12), < n(22), < n(32), < n(42),
and < n(52), respectively, where n = 10. 1.0 for the other solution. We refer to the individual solutions as
“close-close” and “close-wide” solutions, respectively. The lensing parameters of the two 3L1S solutions are listed
in Table 2. Also listed in the table are the χ2 values of the fits
and the flux parameters of the source, fs, and the blend, fb, as
measured on the OGLE flux scale. We note that the estimated
source flux, fs ∼0.03, is much smaller than the blend flux, Fig. 3. Comparison of the four tested models, 1L2S,
2L1S, 2S2L, and 3L1S (close-close solution). The lower
panels show the residuals from the individual models. Table 1. Lensing parameters of the 1L2S, 2L1S, and 2L2S models. Tab
Pa
χ2
t0 1 Fig. 3. Comparison of the four tested models, 1L2S,
2L1S, 2S2L, and 3L1S (close-close solution). The lower
panels show the residuals from the individual models. Table 1. Lensing parameters of the 1L2S, 2L1S, and 2L2S models. Parameter
2L1S
1L2S
2L2S
χ2
2244.3
4018.8
1949.1
t0,1 (HJD′)
8594.721 ± 0.024 8595.614 ± 0.013 8585.635 ± 0.124
u0,1
0.060 ± 0.003
0.0020 ± 0.0001
0.0084 ± 0.0014
t0,2 (HJD′)
–
8585.745 ± 0.149 8595.361 ± 0.022
u0,2
–
0.0070 ± 0.0009
0.0053 ± 0.0003
tE (days)
29.10 ± 1.68
297.36 ± 1.69
181.05 ± 9.90
s
0.623 ± 0.008
–
0.110 ± 0.004
q
0.067 ± 0.002
–
1.252 ± 0.307
α (rad)
2.257 ± 0.010
–
1.191 ± 0.014
ρ1 (10−3)
–
–
–
ρ2 (10−3)
–
–
–
qF
–
0.22 ± 0.02
7.15 ± 0.44
Notes. HJD′ ≡HJD −2 450 000. Notes. HJD′ ≡HJD −2 450 000. partial data. Figure 4 shows the ∆χ2 distribution in the log s3–
log q3 parameter plane obtained from this first-round modeling. We note that there exist two locals in the ∆χ2 map, indicating that
there are degenerate solutions: at (log s3, log q3) ∼(+0.15, −2.0)
and ∼(−0.15, −2.0). We discuss this degeneracy more below. 3.1. 2L1S, 1L2S, and 2L2S models The degeneracy between the close-close and
close-wide solutions is very severe, and the close-close solution
is preferred over the close-wide solution by merely ∆χ2 = 0.6. The fact that the M1–M3 separations of the two degenerate solu-
tions are in the relation of s3,close−close × s3,close−wide ∼1 indicates
that the degeneracy is caused by the close-wide degeneracy in s3
(Griest & Safizadeh 1998; Dominik 1999). (
)
Figure 5 shows the lens system configurations of the two
3L1S models. For each model, the caustic configuration (red fig-
ures) appears to be the superposition of the two sets of caustics
induced by two low-mass companions (blue dots labeled M2 and
M3 in the inset of each panel) of the primary (M1). We mark the
positions of the source at the three epochs of t1, t2, and t3 (small
empty circles drawn in magenta). The source position at t1 cor-
responds to the source passage through the positive deviation
region that extends from one of the sharp cusps of the caus-
tic induced by M2, and this produced a weak bump at around
t1. The source then crossed the tip of the caustic, producing a
sharp caustic-crossing feature, and the two data points at t2, one
from OGLE and the other from KMTC observations, correspond
to the time of the caustic entrance. Another caustic crossing
occurred when the source passed the tip of the caustic induced
by M3, and this produced a sharp peak around t3. The last peak
was covered by the KMTC data, both on the rising and falling
sides of the peak. Fig. 5. Lens system configurations of the close-close (left panel) and
close-wide (right panel) 3L1L models. The inset in each panel shows
the locations of the three lens components, denoted by blue dots labeled
M1, M2, and M3. The dashed circle in the inset represents the Einstein
ring. The line with an arrow represents the source trajectory. The three
small empty dots on the source trajectory represent the source positions
at the epochs of t1, t2, and t3 that are marked in Fig. 1. The size of the
dots is not scaled to the source size. The red figure represents the caus-
tic, and the gray curves around the caustic represent equi-magnification
contours. presented scale, and thus we arbitrarily set the circle size. 3.1. 2L1S, 1L2S, and 2L2S models Parameter
Close-close
Close-wide
Wide-close
Wide-wide
χ2
1268.8
1269.4
1282.7
1284.3
t0 (HJD′)
8594.422 ± 0.020
8594.411 ± 0.020
8594.304 ± 0.024
8594.301 ± 0.023
u0 (10−3)
11.98 ± 0.45
11.34 ± 0.53
12.73 ± 0.54
12.38 ± 0.77
tE (days)
75.24 ± 2.02
76.92 ± 2.89
76.24 ± 2.61
77.92 ± 3.56
s2
0.853 ± 0.003
0.854 ± 0.004
1.107 ± 0.005
1.090 ± 0.006
q2 (10−3)
3.54 ± 0.18
3.31 ± 0.20
4.26 ± 0.18
3.92 ± 0.28
α (rad)
2.633 ± 0.010
2.648 ± 0.010
2.563 ± 0.009
2.567 ± 0.009
s3
0.717 ± 0.007
1.379 ± 0.012
0.680 ± 0.006
1.479 ± 0.016
q3 (10−3)
10.56 ± 0.48
10.47 ± 0.51
13.39 ± 0.61
14.13 ± 0.96
ψ (rad)
5.445 ± 0.028
5.416 ± 0.023
5.590 ± 0.019
5.622 ± 0.019
ρ (10−3)
0.52 ± 0.03
0.50 ± 0.03
0.43 ± 0.04
0.43 ± 0.05
fs
0.029 ± 0.001
0.028 ± 0.001
0.030 ± 0.001
0.029 ± 0.002
fb
1.163 ± 0.0
1.164 ± 0.001
1.161 ± 0.001
1.162 ± 0.001 Fig. 5. Lens system configurations of the close-close (left panel) and
close-wide (right panel) 3L1L models. The inset in each panel shows
the locations of the three lens components, denoted by blue dots labeled
M1, M2, and M3. The dashed circle in the inset represents the Einstein
ring. The line with an arrow represents the source trajectory. The three
small empty dots on the source trajectory represent the source positions
at the epochs of t1, t2, and t3 that are marked in Fig. 1. The size of the
dots is not scaled to the source size. The red figure represents the caus-
tic, and the gray curves around the caustic represent equi-magnification
contours. fb ∼1.16, indicating that the observed flux is heavily blended. The estimated mass ratios of the companions to the primary lens
are q2 ∼(3.3−3.5) × 10−3 and q3 ∼10.5 × 10−3, both of which
correspond to the ratio between a giant planet and a star. This
indicates that the lens is a planetary system composed of two
giant planets. We note that the designation of q2 and q3 is not
arranged according to the mass, and it turns out that q2 < q3. We find that the fit of the 3L1S model is better than those of
the 1L2S, 2L1S, and 2L2S models by ∆χ2 = 2750.0, 974.5, and
680.3, respectively. 3.1. 2L1S, 1L2S, and 2L2S models In
the second round, we refined the local solutions found from the
first-round modeling using a downhill approach based on the
Markov chain Monte Carlo (MCMC) method. In this process,
we released all parameters as free parameters. Fig. 4. Distribution of ∆χ2 in the log s3–log q3 parameter plane
obtained from the preliminary 3L1S grid searches for the parameters
related to the third lens component. Red, yellow, green, cyan, and blue
are used to denote points with ∆χ2 < n(12), < n(22), < n(32), < n(42),
and < n(52), respectively, where n = 10. 1.0 for the other solution. We refer to the individual solutions as
“close-close” and “close-wide” solutions, respectively. We find that the anomaly features of the observed light curve
are well explained by a 3L1S model. The model curve is plotted
over the data points in Figs. 1 and 2, and the residual from the
model is compared to those of the other models (1L2S, 2L1S,
and 2L2S models) in Fig. 3. We find two solutions, in which
s2 < 1.0 and s3 < 1.0 for one solution and s2 < 1.0 and s3 > The lensing parameters of the two 3L1S solutions are listed
in Table 2. Also listed in the table are the χ2 values of the fits
and the flux parameters of the source, fs, and the blend, fb, as
measured on the OGLE flux scale. We note that the estimated
source flux, fs ∼0.03, is much smaller than the blend flux, A93, page 4 of 10 C. Han et al.: OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star Table 2. Lensing parameters of four degenerate 3L1S models. 3.1. 2L1S, 1L2S, and 2L2S models Magnification pattern around the caustics (for the close-wide
3L1S model) and the light curve around the time of the source approach
close to the planetary caustic induced by M2. The small empty circle
on the source trajectory represents the source position at the time of the
source approach, at tapproach ∼8581.7 in HJD′. The size of the circle is
arbitrarily set and is not scaled to the source size. Fig. 6. Magnification pattern around the caustics (for the close-wide
3L1S model) and the light curve around the time of the source approach
close to the planetary caustic induced by M2. The small empty circle
on the source trajectory represents the source position at the time of the
source approach, at tapproach ∼8581.7 in HJD′. The size of the circle is
arbitrarily set and is not scaled to the source size. Fig. 8. Cumulative distributions of the ∆χ2 difference from the best-
fit solution (close-close model). The light curve in the upper panel is
inserted to show the locations of the fit difference. come from the three data points (one from OGLE and two
from KMTC) taken at HJD′ ∼8594.7 and the seven data points
(from KMTC) covering the anomaly at t3. The superiority of the
close-xx solutions over the wide-xx solutions is found from the
comparison of the residuals of the models, presented in Fig. 7, as
well as the cumulative distributions of the χ2 difference from the
best-fit solution (close-close solution), presented in Fig. 8. The
degeneracy in s2 is resolvable because the separation (s2 ∼0.85
for the close solution and ∼1.1 for the wide solution) is close
to unity. In this case, the M1–M2 pair forms a resonant caustic,
for which the close-wide degeneracy is less severe (Chung et al. 2005). Fig. 7. Comparison of the residuals from the four degenerate 3L1S
solutions: close-close, close-wide, wide-close, and wide-wide solutions. As a representative model, the model curve of the close-close solution
is drawn over the data points in the top panel. We checked whether the parameters of the normalized source
radius, ρ, and the microlens-parallax, πE, can be measured. 3.1. 2L1S, 1L2S, and 2L2S models Mea-
surements of these parameters are important because the mass,
M, and distance to the lens, DL, can be uniquely determined with
these parameters by M = θE
κπE
;
DL =
AU
πEθE + πS
;
θE = θ∗
ρ ,
(1) (1) where κ = 4G/(c2AU) and πS denotes the annual parallax of the
source (Gould 2000). The value of the normalized source radius,
ρ ∼0.5 × 10−3, is firmly measured from the data points involved
with caustic crossings at around t2 and t3. On the other hand,
modeling that considers the microlens-parallax effect results in
a πE value with a considerable uncertainty due to the fact that
the photometry quality of the data is mediocre because of the
faintness of the source, together with the fact that all the main
features occur in an interval of 12 days, which is too short to
appreciate any deviations due to parallax effects. Figure 9 shows
the scatter plot of points in the MCMC chain on the πE,E–πE,N
plane, where πE,E and πE,N are the east and north components
of the microlens-parallax vector πE, respectively. The scatter
plot shows that the microlens-parallax is consistent with a zero-
parallax model within the 2σ level, and the uncertainty of πE,N is
big, although the uncertainty of πE,E is relatively small. Consid-
ering that the anomaly features in the light curve occurred within where κ = 4G/(c2AU) and πS denotes the annual parallax of the
source (Gould 2000). The value of the normalized source radius,
ρ ∼0.5 × 10−3, is firmly measured from the data points involved
with caustic crossings at around t2 and t3. On the other hand,
modeling that considers the microlens-parallax effect results in
a πE value with a considerable uncertainty due to the fact that
the photometry quality of the data is mediocre because of the
faintness of the source, together with the fact that all the main
features occur in an interval of 12 days, which is too short to
appreciate any deviations due to parallax effects. Figure 9 shows
the scatter plot of points in the MCMC chain on the πE,E–πE,N
plane, where πE,E and πE,N are the east and north components
of the microlens-parallax vector πE, respectively. 3.1. 2L1S, 1L2S, and 2L2S models The
lower panel shows the light curve around the time of the caustic
approach at tapproach ∼8581.7. Both the magnification map and
the light curve show that the deviation at around this approach is
too weak to emerge from the baseline. The insets of the panels in Fig. 5 show that the source passes
close to one of the triangular planetary caustics induced by M2. If the separation between the source and the caustic were very
close, this source approach would induce a low bump at the time
of the caustic approach. We checked this possibility by inspect-
ing the magnification pattern around the planetary caustic. The
magnification map around the caustics (for the close-wide 3L1S
model) is shown in Fig. 6, in which the source location at the
time of the caustic approach is marked by an empty magenta cir-
cle. The source size, ρ ∼0.5 × 10−3, is too small to be seen in the The fact that the caustic configuration of the 3L1S solutions
appears to be the superposition of two 2L1S caustics, together
with the fact that M3 is in the planetary-mass regime, suggests
that there may be additional degenerate solutions caused by the
close-wide degeneracy in s2. We, therefore, searched for addi-
tional solutions resulting from this degeneracy: solutions with
s2 > 1.0, s3 < 1.0 (“wide-close” solution), and s2 > 1.0, s3 > 1.0
(“wide-wide” solution). These degenerate solutions were found A93, page 5 of 10 A&A 658, A93 (2022) A93 (2022)
Fig. 8. Cumulative distributions of the ∆χ2 difference from the best-
fit solution (close-close model). The light curve in the upper panel is
inserted to show the locations of the fit difference. A&A 658, A93 (2022)
Fig. 6. Magnification pattern around the caustics (for the close-wide
3L1S model) and the light curve around the time of the source approach
close to the planetary caustic induced by M2. The small empty circle
on the source trajectory represents the source position at the time of the
source approach, at tapproach ∼8581.7 in HJD′. The size of the circle is
arbitrarily set and is not scaled to the source size
Fig. 8. Cumulative distributions of the ∆χ2 difference from the best-
fit solution (close-close model). The light curve in the upper panel is
inserted to show the locations of the fit difference. come from the three data points (one from OGLE and two Fig. 6. and µ = 4.41 ± 0.63 mas yr−1,
(5) µ = 4.41 ± 0.63 mas yr−1,
(5) (5) respectively. respectively. respectively. C. Han et al.: OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star Fig. 9. Scatter plot of points in the MCMC chain on the πE,E–πE,N
parameter plane for the close-close 3L1S solution. The color coding is
the same as in Fig. 4, except n = 1. Fig. 10. Locations of the source (empty blue dot with error bars),
RGC centroid (solid red dot), and blend (solid green dot) in the instru-
mental CMD of stars around the source constructed using the pyDIA
photometry of the KMTC data set Fig. 9. Scatter plot of points in the MCMC chain on the πE,E–πE,N
parameter plane for the close-close 3L1S solution. The color coding is
the same as in Fig. 4, except n = 1. Fig. 10. Locations of the source (empty blue dot with error bars),
RGC centroid (solid red dot), and blend (solid green dot) in the instru-
mental CMD of stars around the source constructed using the pyDIA
photometry of the KMTC data set. Fig. 9. Scatter plot of points in the MCMC chain on the πE,E–πE,N
parameter plane for the close-close 3L1S solution. The color coding is
the same as in Fig. 4, except n = 1. Fig. 10. Locations of the source (empty blue dot with error bars),
RGC centroid (solid red dot), and blend (solid green dot) in the instru-
mental CMD of stars around the source constructed using the pyDIA
photometry of the KMTC data set. a short time interval, the lens-orbital motion would not invoke
any significant effects. Nevertheless, we conducted an additional
modeling considering the lens-orbital model and found that the
fit improvement by the lens-orbital effect is negligible and that
the orbital motion was poorly constrained. This indicates that the source is a late G-type main-sequence star
located in the bulge. For the θ∗estimation from (V −I, I)0, we first converted the
measured V −I color into V −K color using the color-color rela-
tion of Bessell & Brett (1988) and second deduced the source
radius from the (V −K)–θ∗relation of Kervella et al. (2004). The estimated value is 4. Source star and angular Einstein radius For the determination of the angular Einstein radius, we needed
to estimate the angular radius of the source. We deduced θ∗from
the color and brightness of the source. To estimate the extinction-
and reddening-corrected (de-reddened) values, (V −I, I)0, from
the instrumental values, (V −I, I), we used the Yoo et al. (2004)
method, in which the centroid of a red giant clump (RGC), (V −
I, I)RGC, in the color-magnitude diagram (CMD) is used for the
calibration of the source color and magnitude. (3) θ∗= 0.47 ± 0.06 µas. (3) θ∗= 0.47 ± 0.06 µas. Together with the ρ and tE values measured from the modeling,
the angular Einstein radius and the relative lens-source proper
motion are estimated as θE = 0.91 ± 0.13 mas
(4) (4) g
Figure 10 shows the locations of the source (empty blue dot
with error bars) and the RGC centroid (solid red dot) in the
instrumental CMD constructed using the KMTC data processed
using the pyDIA software (Albrow 2017). The source position
in the CMD was determined via the regression of the I- and
V-band pyDIA data with the variation of the lensing magnifi-
cation. Also marked in the CMD is the blend position (solid
green dot), which lies on the main-sequence branch of fore-
ground disk stars. We show in Sect. 6 that the blended light is
due to the host star, a companion to the host star, or a combina-
tion of the two. The measured instrumental color and brightness
are (V −I, I) = (3.378 ± 0.099, 22.018 ± 0.004) for the source and
(V −I, I)RGC = (3.606, 16.856) for the RGC centroid. By measur-
ing the offsets in color, ∆(V −I), and magnitude, ∆I, between the
source and RGC centroid, together with the known de-reddened
values of the RGC centroid, (V −I, I)RGC,0 = (1.060, 14.268),
from Bensby et al. (2013) and Nataf et al. (2013), respectively,
we estimate that the de-reddened source color and magnitude
are and 3.1. 2L1S, 1L2S, and 2L2S models The scatter
plot shows that the microlens-parallax is consistent with a zero-
parallax model within the 2σ level, and the uncertainty of πE,N is
big, although the uncertainty of πE,E is relatively small. Consid-
ering that the anomaly features in the light curve occurred within Fig. 7. Comparison of the residuals from the four degenerate 3L1S
solutions: close-close, close-wide, wide-close, and wide-wide solutions. As a representative model, the model curve of the close-close solution
is drawn over the data points in the top panel. using the initial parameters of s2,wide,xx = 1/s2,close−xx. The lens-
ing parameters of these solutions are listed in Table 2. We find
that, although the wide-xx solutions approximately describe the
observed light curve, their fits are worse than the close-xx solu-
tions by ∆χ2 ∼14; the main contributions to this χ2 difference A93, page 6 of 10 A93, page 6 of 10 Han et al.: OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star 6. Baseline object From the position of the blend on the CMD (Fig. 10), it is found
that the flux of the baseline object is dominated by the light from
a main-sequence star in the foreground disk. Logically, there are
only four possibilities for this star: (1) the host of the planets,
(2) a companion to the host, (3) a companion to the source, or
(4) an ambient star that is unrelated to the event (or possibly a
combination of two or more of these possibilities). The position
of the blend on the CMD already rules out possibility (3) because
it is inconsistent with a bulge star. In the first step of the Bayesian analysis, we conducted a
Monte Carlo simulation to produce many (107) artificial lensing
events, for which lens and source locations, transverse lens-
source speeds, and lens masses are allocated following the
Galactic model. Then, the microlensing observables (tE, θE, πE)
of the individual events were computed using the relations in
Eqs. (1) and (6). In the second step, we constructed the distri-
butions of M and DL of events for which their observables are
consistent with the observed values. Because θE ∼0.9 mas, the lens must also be in the fore-
ground disk (unless it is a dark remnant). That is, if the lens
were in the bulge, with πrel <∼0.02 mas, then its mass would be
M = θ2
E/κπrel ≳5 M⊙, implying that it would be easily seen. This
suggests that the blended light may be due primarily to the host
of the planets. Figure 11 shows the posterior distributions of M and DL
obtained from the Bayesian analysis. In Table 3, we list the esti-
mated physical parameters of the masses of the individual lens
components (M1, M2, M3), the distance, and the projected physi-
cal separations (a⊥,2, a⊥,3) between M1–M2 and M1–M3 pairs for
the close-close and close-wide solutions. We adopted the median
values of the distributions as representative parameters, and the
uncertainties of the parameters were estimated as 16 and 84%
of the distributions. The two solutions result in similar physical
parameters, except for a⊥,3. The estimated mass of the host, To test this conjecture, we first measured the astrometric off-
set between the “baseline object” and the source (which has the
same position as the host at the time of the event). 5. Physical lens parameters Although the mass and distance to the lens cannot be uniquely
determined using the relation in Eq. (1) due to the insecure mea-
surement of the microlens parallax, the physical parameters can
still be constrained using the measured observables of tE and θE,
which are related to M and DL by tE = θE
µ ;
θE = (κMπrel)1/2. (6) (6) Here, πrel = AU(D−1
L −D−1
S ) represents the relative lens-source
parallax and DS denotes the distance to the source. For this con-
straint, we conducted a Bayesian analysis using a prior Galactic (V −I, I)0 = (V −I, I)RGC,0 + ∆(V −I, I)
= (0.832 ± 0.099, 19.431 ± 0.004). (V −I, I)0 = (V −I, I)RGC,0 + ∆(V −I, I)
= (0.832 ± 0.099, 19.431 ± 0.004). (2) (2) A93, page 7 of 10 A&A 658, A93 (2022) Fig. 11. Bayesian posterior distributions of the mass of (upper panel)
and distance to (lower panel) the lens. In each panel, the solid vertical
line indicates the median of the distribution, and the two dotted vertical
lines indicate the 1σ range of the distribution. The blue and red curves
represent the contributions by disk and bulge lenses, respectively. Table 3. Physical lens parameters. Table 3. Physical lens parameters. Parameter
Close-close
Close-wide
M1 (M⊙)
0.92+0.49
−0.32
←
M2 (MJ)
3.43+1.83
−1.17
3.21+1.71
−1.10
M3 (MJ)
10.22+5.46
−3.50
10.14+5.42
−3.47
DL (kpc)
4.40+1.15
−1.02
←
a⊥,2 (AU)
3.29+4.15
−2.52
3.29+4.16
−2.53
a⊥,3 (AU)
2.77+3.49
−2.12
5.32+6.71
−4.08
Notes. The arrows in the right column imply that the values are same
as those in the middle column. Parameter
Close-close
Close-wide Notes. The arrows in the right column imply that the values are same
as those in the middle column. Notes. The arrows in the right column imply that the values are same
as those in the middle column. indicates that the host is a G-type star, and the distance, indicates that the host is a G-type star, and the distance, DL = 4.40+1.15
−1.02 kpc,
(8) (8) indicates that the lens is likely to be in the disk. The two planets
have masses Fig. 11. Bayesian posterior distributions of the mass of (upper panel)
and distance to (lower panel) the lens. In each panel, the solid vertical
line indicates the median of the distribution, and the two dotted vertical
lines indicate the 1σ range of the distribution. 5. Physical lens parameters The blue and red curves
represent the contributions by disk and bulge lenses, respectively. (M2, M3) =
((3.43+1.83
−1.17 MJ, 10.22+5.46
−3.50 MJ),
for close-close,
(3.21+1.71
−1.10 MJ, 10.14+5.42
−3.47 MJ),
for close-wide,
(9) (M2, M3) =
((3.43+1.83
−1.17 MJ, 10.22+5.46
−3.50 MJ),
for close-close,
(3.21+1.71
−1.10 MJ, 10.14+5.42
−3.47 MJ),
for close-wide, (9) (9) and thus both planets are heavier than Jupiter. Considering
that the snow line distance from the host is dsl = 2.7(M/M⊙) ∼
2.5 AU, the estimated separations, model. Although the uncertainty of the measured πE is big, we
considered the measured πE by computing the parallax-ellipse
covariance matrix in the Bayesian estimation of the physical lens
parameters. (a⊥,2, a⊥,3) =
((3.29+4.15
−2.52 AU, 2.77+3.49
−2.12 AU),
for close-close,
(3.29+4.16
−2.53 AU, 5.32+6.71
−4.08 AU),
for close-wide,
(10) (a⊥,2, a⊥,3) =
((3.29+4.15
−2.52 AU, 2.77+3.49
−2.12 AU),
for close-close,
(3.29+4.16
−2.53 AU, 5.32+6.71
−4.08 AU),
for close-wide, The Galactic model defines the physical and dynamical dis-
tributions and the mass function of Galactic objects. We adopted
the Galactic model described in Jung et al. (2021). This model
uses the Robin et al. (2003) and Han & Gould (2003) phys-
ical matter distributions to specify the locations of disk and
bulge objects, respectively. It also uses the Han & Gould (1995)
and Jung et al. (2021) dynamical distributions to describe the
motions of disk and bulge objects, respectively. The mass func-
tion adopted in the model is described in Jung et al. (2018), and
it includes stellar remnants and brown dwarfs. 2.53
4.08
(10) (10) indicate that one planet lies near the snow line, and the other
planet lies beyond the snow line. M1 = 0.92+0.49
−0.32 M⊙, 6. Baseline object In brief, there are two channels for the baseline object to
be the host, with the astrometric measurement corrupted either
by an ambient star (p = 0.19) or by a less luminous compan-
ion widely separated from the planet host (p = 0.25/2 ∼0.12)
for a total of p = 0.31. By contrast, there is one channel for
the host to be a companion of the baseline object, with prob-
ability p = 0.25/2 ∼0.12. Therefore, the host of the planet is
likely responsible for the light of the baseline object, but the
host could also plausibly be a companion to the baseline object. Considering the inconclusive nature of the blend together with
the low quality of the spectra, we do not impose the constraint
from the spectra on the estimation of the physical parameters
presented in Table 3. Before considering these two remaining possibilities, we
examined the role of the key systematic effect that could cor-
rupt the position measurement of the baseline object at the
∆θ = [∆θ(N)2 + ∆θ(E)2]1/2 ∼80 mas level: namely, the possi-
bility that an ambient star lies within the point-spread function
of the baseline object. That is, if a fraction, f, of the blended
light were due to an ambient star with a separation from the
source of δθ, then the centroid of light (measured by DoPhot)
would be displaced by ∆θ = fδθ. For instance, for f = 0.2 and
δθ = 400 mas, ∆θ = 80 mas. In this example, the faint ambi-
ent star with a separation of 0.4′′ would not be identified as a
separate star by DoPhot, so the centroid would be shifted. p
y
,
We have just argued that ambient stars similar to or brighter
than the baseline object are rare. However, ambient stars that
could corrupt the astrometric measurement at this level are not
rare, for three reasons. First, such stars are not restricted to the
foreground main sequence, so the much larger population of
bulge stars is available. Second, there are more faint stars than
bright stars. Third, ambient stars can corrupt the astrometric
measurement with separations of up to δθ ∼1′′, whereas ambi-
ent stars that would explain the baseline object light must lie
within ∆θ = 80 mas. These three features also place limits on
which ambient stars can play this role. 6. Baseline object Fortunately, the baseline object
appears isolated in these images, so the statistical errors of this
measurement are not strongly impacted by the algorithm’s pro-
cedure for separating stars. We estimated this error to be 23 mas
in each direction from a comparison of two completely indepen-
dent reductions, that is, by reductions determined by different
people. We find the offset between the source and the baseline
object to be (11) ∆θ(E, N) = (36 ± 23, 73 ± 23) mas. (11) On the other hand, it is also possible that the baseline object
has a fainter companion that generated the main microlensing
event and so serves as the host for the planets. The main con-
straint on this scenario is that the blend, which is likely a G dwarf
at several kiloparsecs, should have a widely separated companion
at log(P/day) ∼4, with a mass ratio of qbase >∼0.5. Accord-
ing to Table 7 of Duquennoy & Mayor (1991), about 25% of
G dwarfs have companions with log(P/day) > 4 and a mass
ratio q ≥0.5. However, if the baseline object did have a com-
panion in this parameter range, it is about equally likely that the
baseline object served as the lens host for the event while the
companion generated the flux required to corrupt the astrometric
measurement. If we could ignore systematic effects and take the error distri-
butions to be a two-dimensional Gaussian, this would rule out
the identification of the host with baseline light at p = 0.002
probability. We note that this close alignment rules out possibility (4),
that is, that the blended light is due to an ambient star. We sim-
ply counted the number of stars on the foreground main sequence
in the 2 arcmin2 area of the OGLE finding chart that are brighter
than the baseline object, finding N = 64. This translates to a
surface density of n = 64/1202 = 4.4 × 10−3 arcsec−2. Thus, the
probability of such an alignment is p = π(∆θ)2n = 10−4. Hence,
the light from the baseline object must be due to either the planet
host or a companion to the host (or a combination of the two). 6. Baseline object The position
of the source is measured from difference images formed by sub-
tracting the reference image from a series of images taken at high
magnifications. This difference image essentially consists of an
isolated star on a blank background. Hence, its position can be M1 = 0.92+0.49
−0.32 M⊙, (7) A93, page 8 of 10 C. Han et al.: OGLE-2019-BLG-0468Lb,c: Two microlensing giant planets around a G-type star of sufficient brightness will be separately resolved by DoPhot
before the δθ = 1′′ limit is reached. accurately measured. We find a scatter among 15 images of just
(0.026, 0.037) pixels in the (east, north) directions, yielding a
standard error of the mean of (0.007, 0.010) pixels, that is, (3,
4) mas, given the 400 mas pixel size. These errors are far below
the other errors in the problem, which are discussed below. We
therefore ignore them in what follows. To make our evaluation, we first focused our attention on
the 2349 stars in the 2 arcmin2 OGLE-IV finding chart that
satisfy 0.7 < (I −Ibase) < 2.7, where Ibase = 17.8 is evaluated
in the OGLE-IV system. The faint limit is set by the require-
ment δθ < 1′′, while the bright limit is set by the requirement
that inclusion of the (usually) red ambient light does not drive
the baseline object off the foreground main sequence. Then,
for each of these 2349 stars we found the annulus of posi-
tions such that the star would induce an astrometric error of
>80 mas. The inner radius of the annulus is set by the flux ratio:
δθinner = ∆θ × 100.4(I−Ibase). We set δθouter = 0.2′′ [(I −Ibase) + 2.3]. We then find a total area subtended by these 2349 annuli to be
2800 arcsec2 (i.e., a fraction p = 19% of the 2 arcmin2) That is,
if the host were primarily responsible for the blended light, then
there is a p = 19% probability that an ambient star would cor-
rupt the astrometric measurement by enough to account for the
observed ∆θ ∼80 mas offset. By contrast, the problem of measuring the position of the
baseline object is far subtler. Its position is returned by the
DoPhot photometry package (Schechter et al. 1993), which
simultaneously fits for the positions and fluxes from possi-
bly overlapping stellar images. 6. Baseline object First, if the ambient star
were too bright (and came from the more populous, but redder,
bulge population), then it would also drive the combined color
of the resulting baseline object to the red, and so off the fore-
ground main-sequence feature, contrary to its actual location. Second, if the ambient star were too faint, its separation would
have to be δθ >∼1′′, at which point its light would no longer enter
into the DoPhot centroid. Third, as δθ is increased, ambient stars 7. Summary and conclusion 2021, AJ, 162, 163
Zhu, W., & Dong, S. 2021, ARA&A, 59, 291
Zhu, W., Penny, M., Mao, S., et al. 2014, ApJ, 788, 73 ∼3.4 MJ and ∼10.2 MJ, and they are orbiting around a G-type
star with a mass of ∼0.9 M⊙and a distance of ∼4.4 kpc. It was
found that the planet host was most likely responsible for the
light of the baseline object, although the possibility of the host
being a companion to the baseline object could not be ruled out. Acknowledgements. Work by C.H. was supported by the grants of National
Research Foundation of Korea (2020R1A4A2002885 and 2019R1A2C2085965). S.D. acknowledges the science research grants from the China Manned Space
Project with NO. CMS-CSST-2021-A11. This research has made use of the KMT-
Net system operated by the Korea Astronomy and Space Science Institute (KASI)
and the data were obtained at three host sites of CTIO in Chile, SAAO in South
Africa, and SSO in Australia. The observations using the SALT telescope were
conducted under the transients follow up programme 2018-2-LSP-001 (PI: D.B.),
which is supported by Poland under grant no. MNiSW DIR/WK/2016/07. D.B. also acknowledges research support from the National Research Foundation. M.G. is supported by the EU Horizon 2020 research and innovation programme
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|
https://openalex.org/W2901554198
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https://publikationen.bibliothek.kit.edu/1000089046/21266335
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English
| null |
Experimental Evaluation of a Membrane Micro Channel Reactor for Liquid Phase Direct Synthesis of Hydrogen Peroxide in Continuous Flow Using Nafion® Membranes for Safe Utilization of Undiluted Reactants
|
Catalysts
| 2,018
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cc-by
| 17,597
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Experimental Evaluation of a Membrane Micro
Channel Reactor for Liquid Phase Direct Synthesis of
Hydrogen Peroxide in Continuous Flow Using
Nafion® Membranes for Safe Utilization of
Undiluted Reactants Manuel Selinsek *, Manfred Kraut and Roland Dittmeyer * Manuel Selinsek *, Manfred Kraut and Roland Dittmeyer *
Institute for Micro Process Engineering, KIT, 76344 Eggenstein-Leopoldshafen, Germany;
manfred.kraut@kit.edu
* Correspondence: manuel.selinsek2@kit.edu (M.S.); roland.dittmeyer@kit.edu (R.D.);
Tel.: +49-721-608-23143 (M.K.R.D.) Institute for Micro Process Engineering, KIT, 76344 Eggenstein-Leopoldshafen, Germany;
manfred.kraut@kit.edu
* Correspondence: manuel.selinsek2@kit.edu (M.S.); roland.dittmeyer@kit.edu (R.D.);
Tel.: +49-721-608-23143 (M.K.R.D.) Received: 28 October 2018; Accepted: 13 November 2018; Published: 17 November 2018 Received: 28 October 2018; Accepted: 13 November 2018; Published: 17 November 2018 Abstract: In recent years, various modular micro channel reactors have been developed to overcome
limitations in challenging chemical reactions. Direct synthesis of hydrogen peroxide from hydrogen
and oxygen is a very interesting process in this regard. However, the complex triphasic process
(gaseous reactants, reaction in liquid solvent, solid catalyst) still holds challenges regarding safety,
selectivity and productivity. The membrane micro reactor system for continuous liquid phase H2O2
direct synthesis was designed to reduce safety issues by separate dosing of the gaseous reactants via
a membrane into a liquid-flow channel filled with a catalyst. Productivity is increased by enhanced
mass transport, attainable in micro channels and by multiple re-saturation of the liquid with the
reactants over the length of the reaction channel. Lastly, selectivity is optimized by controlling the
reactant distribution. The influence of crucial technical features of the design, such as micro channel
geometry, were studied experimentally in relationship with varying reaction conditions such as
residence time, pressure, reactant ratio and solvent flow rate. Successful continuous operation of
the reactor at pressures up to 50 bars showed the feasibility of this system. During the experiments,
control over the reactant ratio was found to be crucial in order to maximize product yield. Thereby,
yields above 80% were achieved. The results obtained are the key elements for future development
and optimization of this reactor system, which will hopefully lead to a breakthrough in decentralized
H2O2 production. Keywords:
multiphase reaction; Palladium catalyst; hydrogenation; multifunctional reactor;
membrane reactor catalysts catalysts 1. Introduction In recent years, several modular micro channel reactors have been developed at the Institute for
Micro Process Engineering at Karlsruhe Institute of Technology (KIT) to overcome kinetic, heat transfer
and mass transfer limitations in challenging chemical reactions and processes [1]. Target applications
are small scale and medium scale production of fuels and chemicals, energy storage, and utilization of
renewable feed stocks. Direct synthesis of hydrogen peroxide from hydrogen and oxygen is a very
interesting process in this regard. It is a promising green production route to enable decentralized
on-site hydrogen peroxide production for immediate use, e.g., as green oxidizing or bleaching agent
since water is the only oxidation by-product. Realization of a commercially viable process for direct Catalysts 2018, 8, 556; doi:10.3390/catal8110556 www.mdpi.com/journal/catalysts www.mdpi.com/journal/catalysts Catalysts 2018, 8, 556 2 of 19 H2O2 synthesis, therefore, has the potential to revolutionize the production from an environmental and
green chemistry perspective [2]. However, there are three major challenges connected with the direct
synthesis process: safety, selectivity and productivity. Most importantly, safety is an issue, because of
the wide concentration range where H2 and O2 form an explosive mixture in the gas phase. Right now hydrogen peroxide is produced on industrial scale by the anthraquinone (AQ) process. The AQ process is energy demanding and complex, consisting of hydrogenation, oxidation, recycling,
extraction and distillation, and is therefore only viable on a very large scale, i.e., typically well
beyond 50,000 tonnes per year. In addition, the large quantities of concentrated and highly reactive
H2O2 solutions produced have to be transported and stored, and they are usually diluted at the
point of use. On-site production for generating H2O2 solutions with the required concentration in
a specified reaction medium and concentration is therefore highly desirable [3]. The process for
direct synthesis of hydrogen peroxide, which was first patented in 1914 [4] is still not brought into
industrial practice. The suggested process which received the highest interest lately in research and
industry was developed and patented in 1985 by DuPont. The patent described the liquid phase
production of concentrated hydrogen peroxide solutions using a palladium on carbon catalyst in
aqueous solutions containing acids and halides at 25 ◦C and pressures up to 170 bars using an
explosive hydrogen-oxygen gas mixture [5]. The research on this direct synthesis process however
declined after explosion of a pilot-scale reactor at DuPont [6]. 1. Introduction In 2006, Degussa AG (nowadays Evonik
Industries AG) and Headwaters Incorporated patented a direct synthesis process utilizing a trickle
bed outside the explosive regime [7,8] and a nanocatalyst with controlled surface coordination [9,10]. A demonstration plant with a production capacity of several kilotons per year of hydrogen peroxide in
methanol was built in 2006 [11,12]. Even though successful operation of this demonstration unit [6]
and commercialization plans for 2009 [13] were reported, no further actions became known since
then [14]. In contrast, Evonik Industries AG opened a new plant in Jilin (China) with a capacity of
230,000 tons per year in 2014 [15] based on the conventional anthraquinone process [16]. Although the direct synthesis process was studied intensively in research [3,17–20] and
industry [4–16], there is still no commercial production process. One main issue of direct H2O2
synthesis is low selectivity. H2O2 is a thermodynamically unfavored and highly reactive intermediate
in the reaction network. Selectivity is diminished not only by competitive water formation as a side
reaction, but also due to consecutive hydrogenation and decomposition of H2O2 to water, as shown in
Equations 1–4. Several techniques were developed to increase the selectivity and yield and to stabilize
the product including addition of sodium bromide as a promoter, addition of phosphoric and/or
sulfuric acid to lower the pH value, passivation of steel equipment by acid treatment, and keeping the
temperature below 50 ◦C to prevent auto decomposition [18,19]. H2 + O2 →H2O2
(1)
2 H2 + O2 →2 H2O
(2)
H2O2 + H2 →2 H2O
(3)
H2O2 →H2O + 0.5 O2
(4) H2 + O2 →H2O2
(1)
2 H2 + O2 →2 H2O
(2)
H2O2 + H2 →2 H2O
(3)
H2O2 →H2O + 0.5 O2
(4) H2 + O2 →H2O2
(1)
2 H2 + O2 →2 H2O
(2)
H2O2 + H2 →2 H2O
(3)
H2O2 →H2O + 0.5 O2
(4) (1) (3) (4) Catalysts studied for direct synthesis are mainly noble metals or noble metal alloys on a variety of
inorganic supports, i.e., alumina, carbon, silica and titania [20]. Pd was found to be the most active for
direct synthesis of H2O2, but also very active for subsequent hydrogenation and decomposition. It was reported that alloying Pd with Au, Sn or Te gives better performance by increasing the
selectivity [21–23]. 1. Introduction However, it remains an open question whether metallic or oxidic Pd favors H2O2
formation and how exactly acids and promoters are involved in the reaction [19,24]. Recent studies
revealed that the reaction might take place at the interface between Pd and PdO, indicating the strong
dependence on both phases [25,26]. Surface and subsurface impurities such as C, N and O and surface
species such as PVA, CO2, SO42−were suggested to limit the uptake of hydrogen in Pd, thereby 3 of 19 Catalysts 2018, 8, 556 increasing the selectivity to H2O2 [27,28]. This also led to the conclusion that subsurface hydrogen
might play a crucial role in direct synthesis of H2O2 [29]. Recently, it was shown that alpha palladium
hydride is present during the reaction by operando X-ray Absorption Spectroscopy (XAS), proofing the
existence of subsurface hydrogen in the formation of H2O2 [30]. Furthermore, Pd nanoparticles with a
diameter of 1.4 nm achieved the best catalytic performance while single site Pd nanoparticles were
inactive, further indicating an important interaction between surface and subsurface domains [31]. Lee et al. recently studied catalysts using Pd supported on ordered mesoporous carbon doped
with heteropolyacids (HPA) and found increasing selectivity and productivity with increasing HPA
content/acidity [32]. Similarly, another group reported that catalytic performance increases with
acidity of the support with Pd supported on silica functionalized with aryl sulfonic, alkyl phosphonic
and alkyl carboxylic groups [33]. Lastly, structural modifications of the support material are known
to influence catalyst performance as well. Pd/TiO2 modified with heterogeneous carbon [34] and
core-shell structured Pd/SiO2 with a microporous Si shell on silica beads [35] showed an increased
catalytic performance with decreased adsorption of H2O2. Studying the reaction mechanism of the direct synthesis of hydrogen peroxide is a difficult task
because of the complex transport phenomena in the triphasic gas-liquid-solid system obscuring the
intrinsic kinetics [17]. Usually it is studied through activity tests in autoclaves in order to generate
defined reaction conditions. Thereby, it was proven that diatomic oxygen species adsorbed on Pd react
to H2O2, whereas water formation takes places via oxygen dissociation [17]. Density Functional Theory
(DFT) calculations on Pd clusters and Au clusters also showed that formation of adsorbed OOH* is the
rate limiting step [36,37]. Furthermore, it was shown that Au blocks the dissociation of O2 in Pd/Au
alloys leading to enhanced selectivity as observed experimentally [38]. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW The initial experiments were conducted using diluted Pd/C as catalyst in a fixed bed within the
micro channel, performing the reaction in water as solvent (0.15 mM NaBr, 0.15 mM H2SO4) at 20 bars
with undiluted reactant gases and solvent flow rates from 0.5 to 3 mLmin (Table 1, Exp.-Nr.: 1–12 and
empty squares in Figure 1a). The H2O2 concentration was found to be increasing with decreasing
solvent flow rate until a maximum concentration with a yield of 2% was reached. Further decrease
of solvent flow rate led to decreasing product concentrations. The increase in concentration with
decreasing flow rate at high solvent flow rates can be explained by a diffusion limitation introduced
by the gas transport through the membrane and by liquid phase diffusion from the membrane to the
bulk of the liquid. The decrease at lower solvent flow rates can only be caused by acceleration of H2O2
decomposition and hydrogenation since concentration of both reactants should increase equally. The
acceleration of consecutive reactions can be a result of an unfavorable reactant concentration ratio
caused by long residence times beneath the gas chambers and by longer catalyst contact times. For
comparison, the 1 wt.-% Pd/TiO2 catalyst was prepared and tested at similar conditions as it had
shown promising results in the literature [51]. Pd/TiO2 displayed the same behavior but was able
to achieve a higher maximum concentration at a lower solvent flow rate with a maximum yield of
5%. Hence, acceleration of the consecutive reactions was not as severe as with the Pd/C catalyst but
still limiting. In order to further stabilize the formed H2O2, the NaBr concentration was increased
from 0.15 mM to 4 mM. With the increased NaBr concentration, it was possible to lift the yield to
a maximum of 14% using the Pd/TiO2 catalyst. However, the behavior of accelerated consecutive
reactions with decreasing solvent flow rate was also observed in this set of experiments. The initial experiments were conducted using diluted Pd/C as catalyst in a fixed bed within the
micro channel, performing the reaction in water as solvent (0.15 mM NaBr, 0.15 mM H2SO4) at 20 bars
with undiluted reactant gases and solvent flow rates from 0.5 to 3 mLmin (Table 1, Exp.-Nr.: 1–12
and empty squares in Figure 1a). The H2O2 concentration was found to be increasing with decreasing
solvent flow rate until a maximum concentration with a yield of 2% was reached. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Further decrease
of solvent flow rate led to decreasing product concentrations. The increase in concentration with
decreasing flow rate at high solvent flow rates can be explained by a diffusion limitation introduced
by the gas transport through the membrane and by liquid phase diffusion from the membrane to the
bulk of the liquid. The decrease at lower solvent flow rates can only be caused by acceleration of H2O2
decomposition and hydrogenation since concentration of both reactants should increase equally. The
acceleration of consecutive reactions can be a result of an unfavorable reactant concentration ratio
caused by long residence times beneath the gas chambers and by longer catalyst contact times. For
comparison, the 1 wt.-% Pd/TiO2 catalyst was prepared and tested at similar conditions as it had
shown promising results in the literature [51]. Pd/TiO2 displayed the same behavior but was able to
achieve a higher maximum concentration at a lower solvent flow rate with a maximum yield of 5%. Hence, acceleration of the consecutive reactions was not as severe as with the Pd/C catalyst but still
limiting. In order to further stabilize the formed H2O2, the NaBr concentration was increased from
0.15 mM to 4 mM. With the increased NaBr concentration, it was possible to lift the yield to a
maximum of 14% using the Pd/TiO2 catalyst. However, the behavior of accelerated consecutive
reactions with decreasing solvent flow rate was also observed in this set of experiments. Figure 1. Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds in
a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 20 bars system pressure with water
as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 5–9),
diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). Figure 1. 1. Introduction It is common opinion in most
kinetic studies that hydrogen also reacts from an adsorbed state following a Langmuir-Hinshelwood
mechanism. However, Wilson et al. [39] recently showed that this mechanism was not able to describe
their experimental data for direct synthesis of H2O2 on Pd clusters. Hence, their conclusion was that
H2O2 is likely formed by reaction pathways that involve intermediates not present on the catalyst
surface. Their explanation fits very well with the observations from several groups as described in the
previous paragraph. Besides the chemical challenges, the most important engineering problem in the direct synthesis
of H2O2 is safety of the operation since H2 and O2 form explosive mixtures in the gas phase over a wide
concentration range [40]. Dilution as a precaution, however, reduces solubility of the gases and thereby
decreases productivity because of the triphasic gas-liquid-solid system. Hence, productivity is still a
primary concern for continuous flow operation [14]. Yet, the reaction has to be run continuously in
order to reduce contact time with the catalyst and thereby minimize hydrogenation and decomposition
reactions [40]. As a consequence of the challenging aspects of the reaction, novel reaction engineering
concepts aimed at enhancing process control, boosting H2O2 productivity, and offering safe operation
were proposed in the literature. Biasi et al. studied different catalytic systems in a trickle bed reactor
using diluted gas mixtures at a pressure of 10 bar [41–43]. Microchannel set ups were used by different
groups since the small inner dimensions on the one hand prevent explosions and thereby explosive
mixtures can be utilized, and on the other hand, they decrease liquid diffusion pathways by forcing
favorable flow patterns [44–53]. Membrane reactors are especially interesting since gas phase contact
of H2 and O2 can be prevented by introducing one or both reactants through a membrane into the
liquid solvent containing the catalyst [54–57]. Recently, our group developed a new approach, namely the concept of alternating reactant dosage
attempting to combine the benefits of reactant dosing through membranes and flow properties in
microchannels [57]. Simulation results indicated an optimized concentration profile that should lead to
high conversion and high selectivity in the liquid phase. An initial proof of concept has been achieved
previously [57]. This work describes the first experimental evaluations of the membrane micro channel
reactor with alternating reactant dosage. 4 of 19 Catalysts 2018, 8, 556 2. Results and Discussion 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds
in a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 20 bars system pressure with
water as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1
Exp.-Nr.: 1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
5–9), diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). Figure 1. Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds in
a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 20 bars system pressure with water
as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 5–9),
diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). Figure 1. Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds
in a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 20 bars system pressure with
water as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1
Exp.-Nr.: 1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
5–9), diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). Figure 1. Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds in
a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 20 bars system pressure with water
as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 5–9),
diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). Figure 1. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Hydrogen peroxide concentration over solvent flow rate using micro channel fixed beds
in a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 20 bars system pressure with
water as solvent: squares: Diluted Pd/C, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1
Exp.-Nr.: 1–4), circles: Pd/TiO2, solvent with 0.15 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.:
5–9), diamonds: Pd/TiO2, solvent with 4 mM NaBr and 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 10–12). 5 of 19 Catalysts 2018, 8, 556 Table 1. Reaction conditions of the experiments performed in this work. Table 1. Reaction conditions of the experiments performed in this work. Exp.-Nr. Micro
Channel
Cat. mcat, mg
Config. 1: mfb: microchannel fixed bed, fb: PVC-tube fixed bed. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW 1
Membrane
Solvent Flow
Rate, mL/min
p, bar
pO2/pH2
Additives
1–4
0.5 mm
Pd/C
200
mfb
180 µm
3/2/1/0.5
20
1
0.15 mM NaBr, 0.15 mM H2SO4
5–9
0.5 mm
Pd/TiO2
200
mfb
180 µm
1/0.75/0.5/0.4/0.25
20
1
0.15 mM NaBr, 0.15 mM H2SO4
10–12
0.5 mm
Pd/TiO2
200
mfb
180 µm
1/0.5/0.25
20
1
4 mM NaBr, 0.15 mM H2SO4
13–17
0.5 mm
Pd/TiO2
200
mfb
180 µm
2/1/0.5/0.25/0.1
10
1
4 mM NaBr, 0.15 mM H2SO4
18–24
0.5 mm
Pd/TiO2
200
mfb
180 µm
1
10
1/1.5/2/3/5/9/16
4 mM NaBr, 0.15 mM H2SO4
25–28
0.5 mm
Pd/TiO2
200
mfb
180 µm
1/0.5/0.25/0.1
10
9
4 mM NaBr, 0.15 mM H2SO4
29–33
0.5 mm
Pd/TiO2
100
coating
180 µm
0.5
10/20/30/40/50
1
4 mM NaBr, 0.15 mM H2SO4
34–37
0.5 mm
Pd/TiO2
100
coating
180 µm
0.5
10/20/30/40
1
no Additives
38–40
0.5 mm
Pd/TiO2
100
fb
180 µm
0.5/0.25/0.1
10
1
5 mM NaBr
41–43
0.5 mm
Pd/TiO2
200
fb
180 µm
0.5/0.25/0.1
10
1
5 mM NaBr
44–45
0.5 mm
Pd/TiO2
50
fb
180 µm
0.5/0.1
10
1
5 mM NaBr
46–48
3 × 0.125 mm
Pd/TiO2
100
fb
180 µm
1/0.4/0.2
10
1
5 mM NaBr
49–50
3 × 0.125 mm
Pd/TiO2
100
fb
180 µm
1/0.4/0.2
10
1
5 mM NaBr, Sodium acetate buffer, pH 3
51–53
3 × 0.125 mm
Pd/TiO2
100
fb
180 µm
1/0.4/0.2
10
1
5 mM NaBr
54–57
0.5 mm
Pd/TiO2
100
fb
15 µm
2/1/0.4/0.2
10
1
5 mM NaBr
58–59
0.5 mm
Pd/TiO2
100
coating
30 µm
1
10/40
1
5 mM NaBr
60–62
0.5 mm
Pd/TiO2
100
coating
30 µm
1/0.4/0.2
40
1
5 mM NaBr
63–64
0.5 mm
Pd/TiO2
100
fb
30 µm
1/0.4
10
1
5 mM NaBr
65–66
0.5 mm
Pd/TiO2
100
fb
30 µm
1/0.4
10
1
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4
67–69
0.5 mm
Pd/TiO2
100
fb
30 µm
1/0.4/0.2
40
1
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4
70
0.5 mm
Pd/TiO2
100
fb
30 µm
0.1
40
4
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4
1: mfb: microchannel fixed bed, fb: PVC-tube fixed bed. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Catalysts 2018, 8, 556
Catalysts 2018, 8, x FOR 6 of 19
6 of 19 The influence of catalyst mass was studied by using the membrane micro reactor just for gas
dosage and applying the catalyst as a fixed bed in a tube at the exit of the reactor (Table 1, Exp.-Nr.:
38–45 and Figure 2). This set of experiments was conducted with water as solvent (5 mM NaBr, no
acid) at 10 bars using undiluted reactant gases. A strong dependence on the amount of catalyst used
was found. The highest H2O2 concentration was achieved with 100 mg of catalyst, indicating that
the formation reaction was kinetically limited when 50 mg of catalyst was used and the consecutive
reactions were dominating in the case of the experiments with 200 mg of catalyst. For the solvent flow
rates tested, maximum yields of 12% for 100 mg, 8% for 50 mg and 4% for 200 mg were found. Only
the set of experiments with 200 mg of catalyst showed a maximum similar to the experiments with the
micro channel fixed bed discussed above. The influence of catalyst mass was studied by using the membrane micro reactor just for gas
dosage and applying the catalyst as a fixed bed in a tube at the exit of the reactor (Table 1, Exp.-Nr.:
38–45 and Figure 2). This set of experiments was conducted with water as solvent (5 mM NaBr, no
acid) at 10 bars using undiluted reactant gases. A strong dependence on the amount of catalyst used
was found. The highest H2O2 concentration was achieved with 100 mg of catalyst, indicating that the
formation reaction was kinetically limited when 50 mg of catalyst was used and the consecutive
reactions were dominating in the case of the experiments with 200 mg of catalyst. For the solvent flow
rates tested, maximum yields of 12% for 100 mg, 8% for 50 mg and 4% for 200 mg were found. Only
the set of experiments with 200 mg of catalyst showed a maximum similar to the experiments with
the micro channel fixed bed discussed above. Figure 2. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table
1 Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed bed
(Table 1 Exp.-Nr.: 13–17). Figure 2. Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table 1
Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed
bed (Table 1 Exp.-Nr.: 13–17). The fixed bed after the reactor showed, in comparison with the micro channel fixed bed (Exp.-
Nr.: 13–17), a lower H2O2 concentration at higher solvent flow rates, which could be a result from the
reactant refeeding in the latter case. For the lowest flow rate tested, the H2O2 concentration achieved
with the fixed bed after the reactor was higher for 50 and 100 mg of catalyst. Since acid was used in
the micro channel fixed bed experiments, the increased concentration at higher solvent flow rates can
not be solely attributed to the difference in catalyst arrangement. However, the maximum H2O2
concentration at 0.25 mL/min of both experiments with 200 mg of catalyst and the low product
concentration at low flow rates further shows the sensibility of catalyst mass. The fixed bed after the reactor showed, in comparison with the micro channel fixed bed (Exp.-Nr.:
13–17), a lower H2O2 concentration at higher solvent flow rates, which could be a result from the
reactant refeeding in the latter case. For the lowest flow rate tested, the H2O2 concentration achieved
with the fixed bed after the reactor was higher for 50 and 100 mg of catalyst. Since acid was used
in the micro channel fixed bed experiments, the increased concentration at higher solvent flow rates
can not be solely attributed to the difference in catalyst arrangement. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW However, the maximum H2O2
concentration at 0.25 mL/min of both experiments with 200 mg of catalyst and the low product
concentration at low flow rates further shows the sensibility of catalyst mass. 2.1. Influence of Residence Time and Catalyst
Catalysts 2018, 8, x FOR PEER REVIEW Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table
1 Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed bed
(Table 1 Exp.-Nr.: 13–17). Figure 2. Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table 1
Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed
bed (Table 1 Exp.-Nr.: 13–17). Figure 2. Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 μm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table
1 Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed bed
(Table 1 Exp.-Nr.: 13–17). Figure 2. Hydrogen peroxide concentration over solvent flow rate for varying catalyst masses and
catalyst arrangements using a 0.5 mm deep micro channel and a 180 µm Nafion® membrane at 10 bars
system pressure with water with additives as solvent: squares: 200 mg Pd/TiO2 in a PVC-tube (Table 1
Exp.-Nr.: 41–43), circles: 100 mg Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 38–40), diamonds: 50 mg
Pd/TiO2 in a PVC-tube (Table 1 Exp.-Nr.: 44–45), crosses: 200 mg Pd/TiO2 in a micro channel fixed
bed (Table 1 Exp.-Nr.: 13–17). Figure 2. 2.2. Influence of O2/H2-Ratio
2.2. Influence of O2/H2-Ratio This indicates
that a higher H2O2 concentration was achieved in comparison to the saturation concentration of H2 in
water calculated with the H2 partial pressure in the H2 gas compartment according to Henry’s law. The linear increase in yield and product concentration further indicates that selectivity was almost
unchanged even though the catalyst contact time was increased by a factor of 10. Therefore, excess O2
successfully slows down the consecutive reactions to water. Since excess O2 is present chemisorbed on
the Pd surface during the reaction [30], it seems to be beneficial to have the Pd covered in O2 in order
to prevent the catalyst from decomposing or hydrogenating the product. y
increase in yield to 20%. A residence time variation was then performed with a partial pressure ratio
of 1 and 9, respectively (Exp.-Nr.: 13–17, 25–28 and Figure 3b). The typical relationship between H2O2
concentration and residence time was observed for equal reactant partial pressures with a maximum
yield of 9%. In comparison, the H2O2 concentration and the yield are increasing linearly for a partial
pressure ratio of 9. The maximum yield was 1.2 at the highest residence time tested. This indicates
that a higher H2O2 concentration was achieved in comparison to the saturation concentration of H2 in
water calculated with the H2 partial pressure in the H2 gas compartment according to Henry’s law. The linear increase in yield and product concentration further indicates that selectivity was almost
unchanged even though the catalyst contact time was increased by a factor of 10. Therefore, excess
O2 successfully slows down the consecutive reactions to water. Since excess O2 is present chemisorbed
on the Pd surface during the reaction [30], it seems to be beneficial to have the Pd covered in O2 in
order to prevent the catalyst from decomposing or hydrogenating the product. (a)
(b)
Figure 3. (a) Hydrogen peroxide concentration (squares) and hydrogen peroxide yield (diamonds)
over partial pressure ratio of oxygen to hydrogen using Pd/TiO2 in a micro channel fixed bed in a 0.5
mm deep micro channel and a 180 μm Nafion® membrane at 10 bars system pressure with water with
additives as solvent (Table 1 Exp.-Nr.: 18–24). (b) Hydrogen peroxide concentration and hydrogen
peroxide yield over solvent flow rate using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep
Figure 3. 2.2. Influence of O2/H2-Ratio
2.2. Influence of O2/H2-Ratio f
f
The reactant concentration ratio is known to play a crucial role in the direct synthesis of H2O2. Even though the stoichiometry of the formation reaction should favor a reactant ratio of one, an
excess of O2 favors the formation of H2O2. With the membrane micro reactor, the influence of the
O2/H2-ratio on the synthesis was studied by diluting the H2 with N2 in the gas phase and thereby
lowering its concentration in the liquid phase. This set of experiments was performed with a Pd/TiO2
catalyst as a fixed bed in the micro channel, varying the partial pressure ratio pO2/pH2 between 1 and
16 (Exp.-Nr.: 18–24 and Figure 3a). The H2O2 concentration is decreasing with increasing pO2/pH2
since less hydrogen is available for the formation reaction. The yield is increasing strongly from 5%
The reactant concentration ratio is known to play a crucial role in the direct synthesis of H2O2. Even though the stoichiometry of the formation reaction should favor a reactant ratio of one, an excess
of O2 favors the formation of H2O2. With the membrane micro reactor, the influence of the O2/H2-ratio
on the synthesis was studied by diluting the H2 with N2 in the gas phase and thereby lowering its
concentration in the liquid phase. This set of experiments was performed with a Pd/TiO2 catalyst
as a fixed bed in the micro channel, varying the partial pressure ratio pO2/pH2 between 1 and 16
(Exp.-Nr.: 18–24 and Figure 3a). The H2O2 concentration is decreasing with increasing pO2/pH2 since
less hydrogen is available for the formation reaction. The yield is increasing strongly from 5% to 18% 7 of 19 Catalysts 2018, 8, 556 when increasing the ratio from 1 to 5. Increasing the ratio further to 16 resulted in a weaker increase
in yield to 20%. A residence time variation was then performed with a partial pressure ratio of 1
and 9, respectively (Exp.-Nr.: 13–17, 25–28 and Figure 3b). The typical relationship between H2O2
concentration and residence time was observed for equal reactant partial pressures with a maximum
yield of 9%. In comparison, the H2O2 concentration and the yield are increasing linearly for a partial
pressure ratio of 9. The maximum yield was 1.2 at the highest residence time tested. 2.2. Influence of O2/H2-Ratio
2.2. Influence of O2/H2-Ratio (a) Hydrogen peroxide concentration (squares) and hydrogen peroxide yield (diamonds)
over partial pressure ratio of oxygen to hydrogen using Pd/TiO2 in a micro channel fixed bed in a
0.5 mm deep micro channel and a 180 µm Nafion® membrane at 10 bars system pressure with water
with additives as solvent (Table 1 Exp.-Nr.: 18–24). (b) Hydrogen peroxide concentration and hydrogen
peroxide yield over solvent flow rate using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep
micro channel and a 180 µm Nafion® membrane at 10 bars system pressure with water with additives
as solvent: squares: pO2/pH2 = 1 (Table 1 Exp.-Nr.: 13–17), diamonds: pO2/pH2 = 9 (Table 1 Exp.-Nr.:
25–28). (b) (a) (a) (b) Figure 3. (a) Hydrogen peroxide concentration (squares) and hydrogen peroxide yield (diamonds)
over partial pressure ratio of oxygen to hydrogen using Pd/TiO2 in a micro channel fixed bed in a 0.5
mm deep micro channel and a 180 μm Nafion® membrane at 10 bars system pressure with water with
additives as solvent (Table 1 Exp.-Nr.: 18–24). (b) Hydrogen peroxide concentration and hydrogen
peroxide yield over solvent flow rate using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep
Figure 3. (a) Hydrogen peroxide concentration (squares) and hydrogen peroxide yield (diamonds)
over partial pressure ratio of oxygen to hydrogen using Pd/TiO2 in a micro channel fixed bed in a
0.5 mm deep micro channel and a 180 µm Nafion® membrane at 10 bars system pressure with water
with additives as solvent (Table 1 Exp.-Nr.: 18–24). (b) Hydrogen peroxide concentration and hydrogen
peroxide yield over solvent flow rate using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep
micro channel and a 180 µm Nafion® membrane at 10 bars system pressure with water with additives
as solvent: squares: pO2/pH2 = 1 (Table 1 Exp.-Nr.: 13–17), diamonds: pO2/pH2 = 9 (Table 1 Exp.-Nr.:
25–28). 2.4. Influence of Micro Channel Depth and Membrane Thickness
2.4. Influence of Micro Channel Depth and Membrane Thickness 2.4. Influence of Micro Channel Depth and Membrane Thickness
2.4. Influence of Micro Channel Depth and Membrane Thickness At high flow rates it was shown that mass transfer through the membrane and transport within
the channel are the limiting steps in the membrane micro reactor. In order to potentially increase
mass transport, a plate with three parallel channels (depth of 0.125 mm) was manufactured and tested
using Pd/TiO2 in a tubular fixed bed after the membrane micro device, equipped with the 180 μm
thick membrane (Exp.-Nr.:46–48). In these experiments, the membrane micro reactor again only
served as a saturator. The H2O2 concentration downstream the fixed bed was greatly increased in
comparison to the micro channel with a depth of 0.5 mm (Exp.-Nr.: 38–40) at comparable conditions
(Figure 5). This means that at the stated conditions saturation was not achieved at low residence times. The associated increase in yield from 8% for the 0.5 mm channel to 20% for the 3 × 0.125 mm channel
at a similar flow rate of about 0.25 mL/min further indicates the limitations by mass transport within
the channel since reactant partial pressures were maintained constant. The influence of mass transfer
was studied by performing a set of experiments with two thinner Nafion® membranes with
thicknesses of 15 and 30 μm in comparison to the 180 μm thick membrane with the 0.5 mm micro
channel (Exp.-Nr.: 54–57, 63–64). As expected, the reduced thickness of the membrane also increased
the H2O2 concentration and yield by increasing the mass transfer from the gas phase to the liquid
phase. Therefore, the diffusivity of the reactant gases hydrogen and oxygen in the membrane material
must be lower than in the free liquid phase. In the literature, permeabilities for hydrogen and oxygen
through Nafion® membranes similar to the membrane used here are reported [59–61]. The
permeability through Nafion® was found to increase linearly with humidity/water uptake,
temperature and pressure [59]. Calculation of diffusion coefficients for hydrogen and oxygen for the
membrane used based on the literature permeabilities leads to values between 1e−11 to 1e−10 m²/s at a
pressure of 20 bars. Since liquid-phase diffusivity of gases is usually in the range of 1e−9 m²/s, diffusion
through Nafion® is about one to two orders of magnitude slower in comparison with the free liquid
phase. Theoretically, diffusion through Nafion® becomes faster than liquid phase diffusion at
pressures above 100 bars. 2.4. Influence of Micro Channel Depth and Membrane Thickness
2.4. Influence of Micro Channel Depth and Membrane Thickness This indicates the feasibility of Nafion® in being able to improve the safety
of H2O2 direct synthesis while delivering promising mass transfer properties for a membrane under
At high flow rates it was shown that mass transfer through the membrane and transport within
the channel are the limiting steps in the membrane micro reactor. In order to potentially increase mass
transport, a plate with three parallel channels (depth of 0.125 mm) was manufactured and tested using
Pd/TiO2 in a tubular fixed bed after the membrane micro device, equipped with the 180 µm thick
membrane (Exp.-Nr.:46–48). In these experiments, the membrane micro reactor again only served as a
saturator. The H2O2 concentration downstream the fixed bed was greatly increased in comparison to
the micro channel with a depth of 0.5 mm (Exp.-Nr.: 38–40) at comparable conditions (Figure 5). This
means that at the stated conditions saturation was not achieved at low residence times. The associated
increase in yield from 8% for the 0.5 mm channel to 20% for the 3 × 0.125 mm channel at a similar
flow rate of about 0.25 mL/min further indicates the limitations by mass transport within the channel
since reactant partial pressures were maintained constant. The influence of mass transfer was studied
by performing a set of experiments with two thinner Nafion® membranes with thicknesses of 15 and
30 µm in comparison to the 180 µm thick membrane with the 0.5 mm micro channel (Exp.-Nr.: 54–57,
63–64). As expected, the reduced thickness of the membrane also increased the H2O2 concentration and
yield by increasing the mass transfer from the gas phase to the liquid phase. Therefore, the diffusivity
of the reactant gases hydrogen and oxygen in the membrane material must be lower than in the free
liquid phase. In the literature, permeabilities for hydrogen and oxygen through Nafion® membranes
similar to the membrane used here are reported [59–61]. The permeability through Nafion® was
found to increase linearly with humidity/water uptake, temperature and pressure [59]. Calculation
of diffusion coefficients for hydrogen and oxygen for the membrane used based on the literature
permeabilities leads to values between 1e−11 to 1e−10 m2/s at a pressure of 20 bars. Since liquid-phase
diffusivity of gases is usually in the range of 1e−9 m2/s, diffusion through Nafion® is about one to two
orders of magnitude slower in comparison with the free liquid phase. micro channel and a 180 μm N
as solvent: squares: pO2/pH2 =
2.3. Influence of System Pressure 25–28). 2.3. Influence of System Pressure
The influence of the system pressure was studied using a Pd/TiO2 coating and by varying the
pressure in the overall system between 10 and 50 bars at a partial pressure ratio of 1 (Exp.-Nr.: 29–33
and Figure 4). The Pd/TiO2 coating was used because of the easier implementation in the micro
channel and the easier pressure control since pressure drop is lower in comparison to a fixed bed. The H2O2 concentration is increasing linearly with increasing pressure to a maximum of 6.5 mM at
50 bars. A yield between 15 and 18% for all pressures was observed. After 50 hours of operation, the
activity of the catalyst started to decline until no more H2O2 was produced after 90 hours. To check
for possible leaching or mechanical removal of particles, the experiment was repeated without
additives (Exp.-Nr.: 34–37 and Figure 4). A linear dependence on the pressure and a stable yield was
observed, similar to the experiment using stabilizers. A lower product concentration and a yield of 5
The influence of the system pressure was studied using a Pd/TiO2 coating and by varying the
pressure in the overall system between 10 and 50 bars at a partial pressure ratio of 1 (Exp.-Nr.: 29–33
and Figure 4). The Pd/TiO2 coating was used because of the easier implementation in the micro
channel and the easier pressure control since pressure drop is lower in comparison to a fixed bed. The H2O2 concentration is increasing linearly with increasing pressure to a maximum of 6.5 mM at
50 bars. A yield between 15 and 18% for all pressures was observed. After 50 hours of operation,
the activity of the catalyst started to decline until no more H2O2 was produced after 90 hours. To
check for possible leaching or mechanical removal of particles, the experiment was repeated without
additives (Exp.-Nr.: 34–37 and Figure 4). A linear dependence on the pressure and a stable yield was
observed, similar to the experiment using stabilizers. A lower product concentration and a yield of 5
to 6% indicate a lower selectivity when no stabilizers are added to the solvent. Still, it was shown that
keeping the partial pressure ratios constant and increasing the overall system pressure does not affect
the catalyst selectivity as claimed in the literature by several research groups [40,47,49,58]. micro channel and a 180 μm N
as solvent: squares: pO2/pH2 =
2.3. Influence of System Pressure This shows 8 of 19 Catalysts 2018, 8, 556 that the productivity of our reactor can be scaled linearly with overall pressure and partial pressure of
the reactants. Catalysts 2018, 8, x FOR PEER REVIEW
8 of 19 Figure 4. Hydrogen peroxide concentration (empty symbols) and hydrogen peroxide yield (filled
symbols) over system pressure using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep micro
channel and a 180 μm Nafion® membrane with a solvent flow rate of 0.5 mL/min and water as solvent:
squares: 4 mM NaBr, 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 29–33), diamonds: no additives (Table 1 Exp.-
Nr.: 34–37). Figure 4. Hydrogen peroxide concentration (empty symbols) and hydrogen peroxide yield (filled
symbols) over system pressure using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep micro
channel and a 180 µm Nafion® membrane with a solvent flow rate of 0.5 mL/min and water as solvent:
squares: 4 mM NaBr, 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 29–33), diamonds: no additives (Table 1
Exp.-Nr.: 34–37). Figure 4. Hydrogen peroxide concentration (empty symbols) and hydrogen peroxide yield (filled
symbols) over system pressure using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep micro
channel and a 180 μm Nafion® membrane with a solvent flow rate of 0.5 mL/min and water as solvent:
squares: 4 mM NaBr, 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 29–33), diamonds: no additives (Table 1 Exp.-
Nr.: 34–37). Figure 4. Hydrogen peroxide concentration (empty symbols) and hydrogen peroxide yield (filled
symbols) over system pressure using Pd/TiO2 in a micro channel fixed bed in a 0.5 mm deep micro
channel and a 180 µm Nafion® membrane with a solvent flow rate of 0.5 mL/min and water as solvent:
squares: 4 mM NaBr, 0.15 mM H2SO4 (Table 1 Exp.-Nr.: 29–33), diamonds: no additives (Table 1
Exp.-Nr.: 34–37). 2.5. Influence of Additives
2.5. Influence of Additives Additives were found to play a crucial role in the direct synthesis reaction especially in a
continuous system. Adding 5 mM NaBr to the solvent was found to be necessary to prevent
hydrogenation of H2O2 in the presence of H2 in stainless steel equipment even after passivation with
HNO3. Acids like H2SO4 and H3PO4 are also often used to further stabilize H2O2 and increase the yield
of the reaction. The salts of sulfuric- and phosphoric acid certainly had a beneficial effect similar to
NaBr. Furthermore, it is common belief that H+ has a positive effect in the formation of H2O2 [39]
which, e.g., also motivated research on solid acids as catalyst supports to enhance selectivity [32,33]. To study the influence on our system, a set of experiments have been performed by controlling the
pH value in the solvent via a sodium acetate buffer at pH 3 and comparing it to a solution with 5 mM
H3PO4 and 25 mM H2SO4 to study the influence of H+ (Exp.-Nr. 46–53 and Figure 6). Addition of the
sodium acetate buffer basically had no effect but addition of phosphoric and sulfuric acid showed an
almost doubled H2O2 concentration (Exp.-Nr. 63–66 and Figure 6). From that experiment it is quite
clear that H+ is less important for the selectivity of the reaction than the presence of stabilizing
molecules. For further study of the addition of acids, a set of experiments was conducted; 5 mM
H3PO4 and 25 mM H2SO4 were tested at 40 bars, and a stable H2O2 concentration of 6.5 mmol/L was
measured at a H2 partial pressure of 10 bars over 4 hours resulting in a yield of 80%. Additives were found to play a crucial role in the direct synthesis reaction especially in a
continuous system. Adding 5 mM NaBr to the solvent was found to be necessary to prevent
hydrogenation of H2O2 in the presence of H2 in stainless steel equipment even after passivation
with HNO3. Acids like H2SO4 and H3PO4 are also often used to further stabilize H2O2 and increase
the yield of the reaction. The salts of sulfuric- and phosphoric acid certainly had a beneficial effect
similar to NaBr. Furthermore, it is common belief that H+ has a positive effect in the formation
of H2O2 [39] which, e.g., also motivated research on solid acids as catalyst supports to enhance
selectivity [32,33]. 2.4. Influence of Micro Channel Depth and Membrane Thickness
2.4. Influence of Micro Channel Depth and Membrane Thickness Theoretically, diffusion through
Nafion® becomes faster than liquid phase diffusion at pressures above 100 bars. This indicates the
feasibility of Nafion® in being able to improve the safety of H2O2 direct synthesis while delivering
promising mass transfer properties for a membrane under these conditions. Catalysts 2018, 8, 556
Catalysts 2018, 8, x FOR 9 of 19
9 of 19 (a)
(b)
Figure 5. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in a fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 0.5 mm deep microchannel, 30 μm Nafion® membrane (Table 1 Exp.-Nr.: 63–64), circles: 0.5
mm deep microchannel, 15 μm Nafion® membrane (Table 1 Exp.-Nr.: 54–57), diamonds: 3 × 0.125 mm
deep microchannel, 180 μm Nafion® membrane (Table 1 Exp.-Nr.: 46–48), crosses: 0.5 mm deep
microchannel, 180 μm Nafion® membrane (Table 1 Exp.-Nr.: 38–40). Figure 5. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in a fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 0.5 mm deep microchannel, 30 µm Nafion® membrane (Table 1 Exp.-Nr.: 63–64), circles: 0.5 mm
deep microchannel, 15 µm Nafion® membrane (Table 1 Exp.-Nr.: 54–57), diamonds: 3 × 0.125 mm
deep microchannel, 180 µm Nafion® membrane (Table 1 Exp.-Nr.: 46–48), crosses: 0.5 mm deep
microchannel, 180 µm Nafion® membrane (Table 1 Exp.-Nr.: 38–40). (b) (a) (b) (a) Figure 5. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in a fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 0.5 mm deep microchannel, 30 μm Nafion® membrane (Table 1 Exp.-Nr.: 63–64), circles: 0.5
mm deep microchannel, 15 μm Nafion® membrane (Table 1 Exp.-Nr.: 54–57), diamonds: 3 × 0.125 mm
deep microchannel, 180 μm Nafion® membrane (Table 1 Exp.-Nr.: 46–48), crosses: 0.5 mm deep
microchannel, 180 μm Nafion® membrane (Table 1 Exp.-Nr.: 38–40). Figure 5. 2.4. Influence of Micro Channel Depth and Membrane Thickness
2.4. Influence of Micro Channel Depth and Membrane Thickness (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in a fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 0.5 mm deep microchannel, 30 µm Nafion® membrane (Table 1 Exp.-Nr.: 63–64), circles: 0.5 mm
deep microchannel, 15 µm Nafion® membrane (Table 1 Exp.-Nr.: 54–57), diamonds: 3 × 0.125 mm
deep microchannel, 180 µm Nafion® membrane (Table 1 Exp.-Nr.: 46–48), crosses: 0.5 mm deep
microchannel, 180 µm Nafion® membrane (Table 1 Exp.-Nr.: 38–40). 2.6. System Productivity
2.6. System Productivity Comparing the two catalysts used for the reaction, the productivity of Pd/TiO2 was only slightly
higher than of Pd/C, if production per mass of Pd is considered (Table 2 Exp.-Nr.: 1–9). Comparing
xperiments at the same conditions, both catalysts showed linearly increasing productivity with
ncreasing solvent flow rate as also described by Biasi et al. [41,43], indicating the mass transfer
imitation introduced by the membrane as found by other indicators in this work. A similar behavior
was found for the PVC-tube fixed beds and the coating experiments. The productivity was also found
o increase linearly with increasing system pressure. Hence, the highest productivity of 189
molH2O2/kgPd/h of the experiments conducted in this work was measured at 50 bar using a Pd/TiO2
oating (Table 2 Exp.-Nr.: 33). The second highest productivity of 110 molH2O2/kgPd/h of was achieved
at 40 bars using a PVC-tube fixed bed and water with added H2SO4 and H3PO4 as solvent (Table 2
Exp.-Nr.: 67). In comparison with the literature, only Biasi et al. found similarly low productivities of
80 molH2O2/kgPd/h using methanol without additives as solvent in a trickle bed reactor. In continuous
low experiments, most of the groups found productivities between 1500 and 6000 molH2O2/kgPd/h
41,43,44,48,52,54,55,62]; only Kanungo et al. [46] stood out with achieving a productivity of 1,4300
molH2O2/kgPd/h by using coated microchannel capillaries. The reactors used in the literature were
mostly trickle bed reactors, stirred autoclaves or microchannel trickle beds, and hence tri-phasic
ystems with the related safety issues. The increased gas-liquid mass transfer of these systems led to
high productivities. Whereas, in this work, the gas-liquid mass transfer was limited by the Nafion®
membranes used, which in return offered increased safety. Still, promising methods to further
ncrease productivity of the system presented here were presented and discussed. Comparing the two catalysts used for the reaction, the productivity of Pd/TiO2 was only slightly
higher than of Pd/C, if production per mass of Pd is considered (Table 1 Exp.-Nr.: 1–9). Comparing
experiments at the same conditions, both catalysts showed linearly increasing productivity with
increasing solvent flow rate as also described by Biasi et al. [41,43], indicating the mass transfer
limitation introduced by the membrane as found by other indicators in this work. A similar behavior
was found for the PVC-tube fixed beds and the coating experiments. The productivity was also
found to increase linearly with increasing system pressure. 2.5. Influence of Additives
2.5. Influence of Additives To study the influence on our system, a set of experiments have been performed
by controlling the pH value in the solvent via a sodium acetate buffer at pH 3 and comparing it to
a solution with 5 mM H3PO4 and 25 mM H2SO4 to study the influence of H+ (Exp.-Nr. 46–53 and
Figure 6). Addition of the sodium acetate buffer basically had no effect but addition of phosphoric
and sulfuric acid showed an almost doubled H2O2 concentration (Exp.-Nr. 63–66 and Figure 6). From
that experiment it is quite clear that H+ is less important for the selectivity of the reaction than the
presence of stabilizing molecules. For further study of the addition of acids, a set of experiments was
conducted; 5 mM H3PO4 and 25 mM H2SO4 were tested at 40 bars, and a stable H2O2 concentration of
6.5 mmol/L was measured at a H2 partial pressure of 10 bars over 4 hours resulting in a yield of 80%. Catalysts 2018, 8, 556
Catalysts 2018, 8, x FOR 10 of 19
10 of 19 10 of 19
10 of 19 (a)
(b)
Figure 6. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 3 × 0.125 mm deep microchannel, 180 μm Nafion® membrane, 5 mM NaBr, NaAc buffer, pH
3 (Table 1 Exp.-Nr.: 49–50), diamonds: 3 × 0.125 mm deep microchannel, 180 μm Nafion® membrane,
5 mM NaBr (Table 1 Exp.-Nr.: 46–48), circles: 0.5 mm deep microchannel, 30 μm Nafion® membrane,
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4 (Table 1 Exp.-Nr.: 65–66), crosses: 0.5 mm deep microchannel,
30 μm Nafion® membrane, 5 mM NaBr (Table 1 Exp.-Nr.: 63–64). Figure 6. 2.6. System Productivity
2.6. System Productivity Hence, the highest productivity of
189 molH2O2/kgPd/h of the experiments conducted in this work was measured at 50 bar using a
Pd/TiO2 coating (Table 1 Exp.-Nr.: 33). The second highest productivity of 110 molH2O2/kgPd/h of
was achieved at 40 bars using a PVC-tube fixed bed and water with added H2SO4 and H3PO4 as
solvent (Table 1 Exp.-Nr.: 67). In comparison with the literature, only Biasi et al. found similarly low
productivities of 180 molH2O2/kgPd/h using methanol without additives as solvent in a trickle bed
reactor. In continuous flow experiments, most of the groups found productivities between 1500 and
6000 molH2O2/kgPd/h [41,43,44,48,52,54,55,62]; only Kanungo et al. [46] stood out with achieving a
productivity of 1,4300 molH2O2/kgPd/h by using coated microchannel capillaries. The reactors used in
the literature were mostly trickle bed reactors, stirred autoclaves or microchannel trickle beds, and
hence tri-phasic systems with the related safety issues. The increased gas-liquid mass transfer of these
systems led to high productivities. Whereas, in this work, the gas-liquid mass transfer was limited by
the Nafion® membranes used, which in return offered increased safety. Still, promising methods to
further increase productivity of the system presented here were presented and discussed. 2.5. Influence of Additives
2.5. Influence of Additives (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 3 × 0.125 mm deep microchannel, 180 µm Nafion® membrane, 5 mM NaBr, NaAc buffer, pH 3
(Table 1 Exp.-Nr.: 49–50), diamonds: 3 × 0.125 mm deep microchannel, 180 µm Nafion® membrane,
5 mM NaBr (Table 1 Exp.-Nr.: 46–48), circles: 0.5 mm deep microchannel, 30 µm Nafion® membrane,
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4 (Table 1 Exp.-Nr.: 65–66), crosses: 0.5 mm deep microchannel,
30 µm Nafion® membrane, 5 mM NaBr (Table 1 Exp.-Nr.: 63–64). (a) (b) (a) (b) (a) Figure 6. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 3 × 0.125 mm deep microchannel, 180 μm Nafion® membrane, 5 mM NaBr, NaAc buffer, pH
3 (Table 1 Exp.-Nr.: 49–50), diamonds: 3 × 0.125 mm deep microchannel, 180 μm Nafion® membrane,
5 mM NaBr (Table 1 Exp.-Nr.: 46–48), circles: 0.5 mm deep microchannel, 30 μm Nafion® membrane,
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4 (Table 1 Exp.-Nr.: 65–66), crosses: 0.5 mm deep microchannel,
30 μm Nafion® membrane, 5 mM NaBr (Table 1 Exp.-Nr.: 63–64). Figure 6. (a) Hydrogen peroxide concentration and (b) yield over solvent flow rate using 100 mg
Pd/TiO2 in fixed bed in a PVC-tube at 10 bars system pressure with water with additives as solvent:
squares: 3 × 0.125 mm deep microchannel, 180 µm Nafion® membrane, 5 mM NaBr, NaAc buffer, pH 3
(Table 1 Exp.-Nr.: 49–50), diamonds: 3 × 0.125 mm deep microchannel, 180 µm Nafion® membrane,
5 mM NaBr (Table 1 Exp.-Nr.: 46–48), circles: 0.5 mm deep microchannel, 30 µm Nafion® membrane,
5 mM NaBr, 4 mM H3PO4, 25 mM H2SO4 (Table 1 Exp.-Nr.: 65–66), crosses: 0.5 mm deep microchannel,
30 µm Nafion® membrane, 5 mM NaBr (Table 1 Exp.-Nr.: 63–64). 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor e
e
a e
i o
a
e
ea o
The reactor used in this work was designed to carry out the liquid phase direct synthesis of
hydrogen peroxide in continuous mode. To achieve this, hydrogen and oxygen are fed separately
and in an alternating fashion to the liquid solvent through a membrane along the reaction zone. The
dea is to restore the concentration of the reactants while they are consumed by the reaction as well
The reactor used in this work was designed to carry out the liquid phase direct synthesis of
hydrogen peroxide in continuous mode. To achieve this, hydrogen and oxygen are fed separately
and in an alternating fashion to the liquid solvent through a membrane along the reaction zone. The
idea is to restore the concentration of the reactants while they are consumed by the reaction as well 11 of 19 Catalysts 2018, 8, 556 as to maintain the concentration ratio of the two reactants within a desired window even if they are
not consumed according to the 1:1 stoichiometry of the target reaction. The gases are fed from two
different gas chambers in the device to enable the usage of undiluted reactants at high pressures in
order to increase their concentration in the liquid phase. The dissolved gases can then react with a
catalyst present either in the micro channel or downstream. A scheme and a picture of the device
have already been presented in Ref. [57] and are reproduced here in Figure 7. A frame holding the
membrane is placed on top of a plate with a milled micro channel. The membrane frame and the gas
chamber plate both have two longitudinal channels, one for each reactant gas. The frame enables
thin and flexible membranes to be pressed against the micro channel plate. The meander-like form
of the micro channel allows the liquid to switch the gas chambers beneath the membrane in order
to alternately and repeatedly pick up both reactants on its way through the device. The liquid entry
and exit is realized by holes at the ends of the channel leading to a bottom adapter plate. This design
allows evaluation of different shapes of the micro channel to influence the residence time in the device
and beneath each reactant gas chamber. Catalysts 2018, 8, x FOR PEER REVIEW
11 of 19
not consumed according to the 1:1 stoichiometry of the target reaction. 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor The gases are fed from two
different gas chambers in the device to enable the usage of undiluted reactants at high pressures in
order to increase their concentration in the liquid phase. The dissolved gases can then react with a
catalyst present either in the micro channel or downstream. A scheme and a picture of the device
have already been presented in Ref. [57] and are reproduced here in Figure 7. A frame holding the
membrane is placed on top of a plate with a milled micro channel. The membrane frame and the gas
chamber plate both have two longitudinal channels, one for each reactant gas. The frame enables thin
and flexible membranes to be pressed against the micro channel plate. The meander-like form of the
micro channel allows the liquid to switch the gas chambers beneath the membrane in order to
alternately and repeatedly pick up both reactants on its way through the device. The liquid entry and
exit is realized by holes at the ends of the channel leading to a bottom adapter plate. This design
allows evaluation of different shapes of the micro channel to influence the residence time in the device
and beneath each reactant gas chamber
Catalysts 2018, 8, x FOR PEER REVIEW
11 of 19
not consumed according to the 1:1 stoichiometry of the target reaction. The gases are fed from two
different gas chambers in the device to enable the usage of undiluted reactants at high pressures in
order to increase their concentration in the liquid phase. The dissolved gases can then react with a
catalyst present either in the micro channel or downstream. A scheme and a picture of the device
have already been presented in Ref. [57] and are reproduced here in Figure 7. A frame holding the
membrane is placed on top of a plate with a milled micro channel. The membrane frame and the gas
chamber plate both have two longitudinal channels, one for each reactant gas. The frame enables thin
and flexible membranes to be pressed against the micro channel plate. The meander-like form of the
micro channel allows the liquid to switch the gas chambers beneath the membrane in order to
alternately and repeatedly pick up both reactants on its way through the device. The liquid entry and
exit is realized by holes at the ends of the channel leading to a bottom adapter plate. 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor Channels with varying depth were used in this work: a single
channel with a depth of 0.5 mm, a single channel with a depth of 0.125 mm and three parallel channels
with depths of 0.125 mm. All channels had a total length of 768 mm and a width of 1 mm. The
geometry is illustrated in Figure 8. The meander shape was constructed by connecting twenty 275◦circle segments with a diameter of
16 mm in the channel center to form a channel with a total length of 768 mm. From each circle segment,
180◦is in contact with the membrane resulting in a membrane contact length per segment of 25 mm. Due to the wall between the gas chambers, a length of 13 mm is not in contact with the membrane
between the circle segments. Channels with varying depth were used in this work: a single channel
with a depth of 0.5 mm, a single channel with a depth of 0.125 mm and three parallel channels with
depths of 0.125 mm. All channels had a total length of 768 mm and a width of 1 mm. The geometry is
illustrated in Figure 8. The meander shape was constructed by connecting twenty 275° circle segments with a diameter
of 16 mm in the channel center to form a channel with a total length of 768 mm. From each circle
segment, 180° is in contact with the membrane resulting in a membrane contact length per segment
of 25 mm. Due to the wall between the gas chambers, a length of 13 mm is not in contact with the
membrane between the circle segments. Channels with varying depth were used in this work: a single
channel with a depth of 0.5 mm, a single channel with a depth of 0.125 mm and three parallel channels
with depths of 0.125 mm. All channels had a total length of 768 mm and a width of 1 mm. The
geometry is illustrated in Figure 8. Figure 8. Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. Figure 8. Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. Figure 8. Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. Figure 8. 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor This design
allows evaluation of different shapes of the micro channel to influence the residence time in the device
d b
h
h
h
b g
(a)
(b)
Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the actual
device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the
actual device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. d beneath each reactant gas chamber. (a)
(b)
Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the actual
device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. (a)
(a) (b)
(b) (b)
(b) (a)
(a) Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the actual
device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the
actual device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. Figure 7. Scheme of the microchannel membrane reactor (a) and photo of the plates forming the actual
device (from left to right): liquid adapter plate, micro channel plate, membrane frame bottom,
membrane frame top including gas chambers and gas adapter plate (b). Adapted from Ref. [57]. The meander shape was constructed by connecting twenty 275° circle segments with a diameter
of 16 mm in the channel center to form a channel with a total length of 768 mm. From each circle
segment, 180° is in contact with the membrane resulting in a membrane contact length per segment
of 25 mm. Due to the wall between the gas chambers, a length of 13 mm is not in contact with the
membrane between the circle segments. 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. Figure 8. Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. Figure 8. Top view scheme of the geometry of the meander-shaped micro channel and the relative
position of the two membrane areas. 12 of 19 Catalysts 2018, 8, 556 Membrane integration into the system is a crucial part of the reactor design. The requirements
for the membrane are: chemical resistance, high resistance against liquid water breakthrough into
the gas phase and high resistance against gas bubble breakthrough into the liquid phase. Nafion®
was found to be suitable for this purpose. Nafion® is a sulfonated tetrafluoroethylene based on
PTFE and is usually available as thin films or sheets. Integrating thin flexible sheets into the
reactor design was solved by having two metal frames sandwiching one membrane sheet. Through
the geometry of the metal frames, the membrane is pressed against the micro channel over two
larger areas with a labyrinth seal (Figure 9a). The assembled membrane frame with a 15 µm
thick Nafion® membrane after a test run in the system is shown in Figure 9b. Three membranes
were used for the reaction experiments: a 180 µm thick reinforced Nafion® membrane (Fumasep
F-101180-PTFE, Fumatech, Bietigheim-Bissingen, Germany), a 30 µm reinforced Nafion® membrane
(FUMAPEM® FS-930-RFS, Fumatech, Bietigheim-Bissingen, Germany) and a 15 µm non reinforced
Nafion® membrane (FUMAPEM® FS-715-RFS, Fumatech, Bietigheim-Bissingen, Germany). Catalysts 2018, 8, x FOR PEER REVIEW
12 of 19
found to be suitable for this purpose. Nafion® is a sulfonated tetrafluoroethylene based on PTFE and
is usually available as thin films or sheets. Integrating thin flexible sheets into the reactor design was
solved by having two metal frames sandwiching one membrane sheet. Through the geometry of the
metal frames, the membrane is pressed against the micro channel over two larger areas with a
labyrinth seal (Figure 9a). The assembled membrane frame with a 15 μm thick Nafion® membrane
after a test run in the system is shown in Figure 9b. 3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide
3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide The experimental setup to conduct the direct synthesis of hydrogen peroxide in continuous
mode is schematically shown in Figure 10. The gaseous components, H2, O2 and N2 needed for start-
up of the reactor as well as an optional diluent, were fed via mass flow controllers (EL-FLOW,
Bronkhorst, Ruurlo, Netherlands) and the liquid solvent was introduced via a double syringe pump
(Syrdos 2, Hitec Zang, Herzogenrath, Germany). System pressure was controlled with three
automated backpressure controllers (EL-PRESS, Bronkhorst, Ruurlo, Netherlands), enabling a
uniform and simultaneous pressure buildup of two gas streams and one liquid stream. At the exit of
the system, the liquid solvent was analyzed for its H2O2 content. Therefore, it was either collected for
cerimetric titration or fed to an automated flow injection analysis (FIA) in the later stages of the work. Stainless steel was used as tubing material in the setup and PEEK tubing was used for the FIA. The experimental setup to conduct the direct synthesis of hydrogen peroxide in continuous mode
is schematically shown in Figure 10. The gaseous components, H2, O2 and N2 needed for start-up of
the reactor as well as an optional diluent, were fed via mass flow controllers (EL-FLOW, Bronkhorst,
Ruurlo, Netherlands) and the liquid solvent was introduced via a double syringe pump (Syrdos 2, Hitec
Zang, Herzogenrath, Germany). System pressure was controlled with three automated backpressure
controllers (EL-PRESS, Bronkhorst, Ruurlo, Netherlands), enabling a uniform and simultaneous
pressure buildup of two gas streams and one liquid stream. At the exit of the system, the liquid solvent
was analyzed for its H2O2 content. Therefore, it was either collected for cerimetric titration or fed to
an automated flow injection analysis (FIA) in the later stages of the work. Stainless steel was used as
tubing material in the setup and PEEK tubing was used for the FIA. S ai
e
ee
a u e
a
u i g
a e ia i
e e up a
EE
u i g
a u e
o
e
IA
For cerimetric titration, several drops of Ferroin solution were added to acidified water (1:19
H2SO4/H2O wt.-%). The red solution was then titrated using a standardized 0.01 N cerium sulfate
solution (prepared by dilution of 0.1 N CeSO4 solution (35066, Honeywell, Morristown, NJ, USA)
with 1 mol/L H2SO4 in H2O) until the color changed to pale blue. At this point, the red Fe(II) was
fully oxidized to blue Fe(III). 3 1 The Membrane Micro Channel Reactor
3.1. The Membrane Micro Channel Reactor Three membranes were used for the reaction
experiments: a 180 μm thick reinforced Nafion® membrane (Fumasep F-101180-PTFE, Fumatech,
Bietigheim-Bissingen, Germany), a 30 μm reinforced Nafion® membrane (FUMAPEM® FS-930-RFS,
Fumatech, Bietigheim-Bissingen, Germany) and a 15 μm non reinforced Nafion® membrane
(FUMAPEM® FS 715 RFS F
t
h Bi ti h i
Bi
i
G
) g
g
y)
(a)
(b)
Figure 9. Schematic cross section of the membrane frame (a) and photo showing the assembled
membrane frame with a 15 μm thick Nafion® membrane after a test run (an imprint of the meander-
like micro channel can be seen on the surface of the clear membrane) (b). Figure 9. Schematic cross section of the membrane frame (a) and photo showing the assembled
membrane frame with a 15 µm thick Nafion® membrane after a test run (an imprint of the meander-like
micro channel can be seen on the surface of the clear membrane) (b). (a) (b) (b) (a) Figure 9. Schematic cross section of the membrane frame (a) and photo showing the assembled
membrane frame with a 15 μm thick Nafion® membrane after a test run (an imprint of the meander-
like micro channel can be seen on the surface of the clear membrane) (b). Figure 9. Schematic cross section of the membrane frame (a) and photo showing the assembled
membrane frame with a 15 µm thick Nafion® membrane after a test run (an imprint of the meander-like
micro channel can be seen on the surface of the clear membrane) (b). 3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide
3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide In the next step, the hydrogen peroxide-containing solution was added. Subsequently, the color changed to purple or light red depending on the H2O2 concentration. By
addition of the standardized cerium sulfate solution, the color changed first to a deeper red and then
again to blue since 2 moles of Ce(IV) are needed to react H2O2 to oxygen and Ce(III). The reaction
equation is shown in Equation 5 and the H2O2 concentration was calculated by Equation (6). 2 Ce(IV) + H2O2 →2 Ce(III) + 2 H+ + O2
(5) 2 Ce(IV) + H2O2 →2 Ce(III) + 2 H+ + O2 (5) 13 of 19
ge pump
th three Catalysts 2018, 8, 556
(Syrdos 2, Hitec cH2O2 =
cCeSO4,sol.·msample·ρCeSO4,sol. 2·mCeSO4,sol.·ρsample
(
ith: ρCeSO4,sol. = 1.11 kg/m3; ρsample = 1 kg/m3. p
(
)
g
niform and simultaneous pressure buildup of two gas streams and one liquid stream. At the exit
e system, the liquid solvent was analyzed for its H2O2 content. Therefore, it was either collected f
rimetric titration or fed to an automated flow injection analysis (FIA) in the later stages of the wor
ainless steel was used as tubing material in the setup and PEEK tubing was used for the FIA. Figure 10. Overview of the experimental setup for continuous direct synthesis of H2O2 with mass flow
controller (MFC), pressure controller (PIC) and flow injection analysis (FIA). Figure 10. Overview of the experimental setup for continuous direct synthesis of H2O2 with mass flow
controller (MFC), pressure controller (PIC) and flow injection analysis (FIA). (6)
xit of
d for 2·mCeSO4,sol.·ρsample
for its H2O2 content. Th
i j
ti
l
i (F with: ρCeSO4,sol. = 1.11 kg/m3; ρsample = 1 kg/m3. cerimetric titration or fed to an automated flow injec
Stainless steel was used as tubing material in the set Figure 10. Overview of the experimental setup for continuous direct synthesis of H2O2 with mass flow
controller (MFC), pressure controller (PIC) and flow injection analysis (FIA). Figure 10. Overview of the experimental setup for continuous direct synthesis of H2O2 with mass flow
controller (MFC), pressure controller (PIC) and flow injection analysis (FIA). The automated flow injection system (FIA) was based on a system first described by
Pashkova et al. in 2009 [63]. 3.3. Experimental Procedure First, the membrane micro channel reactor was prepared and introduced into the laboratory set
up without connecting the tubes for the liquid stream to prevent any unintentional liquid entry. Then
nitrogen was fed to both gas chambers with a flow rate of 100 NmL/min and pressure was built up
to 2 bars gauge. This forced the flexible membrane to be pressed against the micro channel plate, to
ensure that the liquid followed the path of the micro channel. Afterwards, the tubes for the liquid
flow were connected and the liquid feed was started, usually demineralized water with additives at a
flow rate of 1 mL/min. After ensuring a constant liquid flow at the exit of the liquid pressure control
valve, the pressure was increased in both gas phases and in the liquid phase, while always maintaining
2 bars differential pressure between gas and liquid. When the desired pressure was reached in the
liquid phase, e.g., 10 bars, the gas phase pressures were reduced to give a differential pressure of
100 mbar between gas and liquid, e.g., 10.1 bar in both gas phases and 10 bar in the liquid phase. In
order to always ensure a higher pressure in the gas phases, the liquid pressure was controlled using
the pressure at the reactor inlet. If liquid pressure became greater than the gas phase pressure, the
membrane was lifted off the channel top and the liquid was no longer following the micro channel. Finally, the gas feed was changed to hydrogen and oxygen in order to start the reaction. Downstream of the reactor, the hydrogen peroxide concentration was determined. Cerimetric
titration was applied for the earlier experiments (Table 1, Exp.-Nr.: 1–28). Samples of about 2 mL were
collected and analyzed every 5 to 20 minutes and this was repeated until the titrated concentration
was found to be stable over a period of 30 to 90 minutes depending on the flow rate of the liquid. For
the latter experiments (Table 1, Exp.-Nr.: 29–66), each experiment was run for several hours, usually
between 4 and 8 h, and one measurement was performed every 5 min with the FIA. The stable H2O2
concentration was then averaged over the time of the experimental run. g
p
Commercial 5 wt.-% Pd/C (75992, Sigma-Aldrich, St. 3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide
3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide 14 of 19 Catalysts 2018, 8, 556 Consequently, if re-saturation of the liquid occurs, then the hydrogen peroxide yield may reach values
well above one. Consequently, if re-saturation of the liquid occurs, then the hydrogen peroxide yield may reach values
well above one. 3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide
3.2. Experimental Setup for Direct Synthesis of Hydrogen Peroxide In the FIA, H2O2 is contacted with titanium(IV) oxysulfate to form
an orange-colored complex which is then detected by a UV-Vis spectrometer (“DT-MINI-2-GS” light
source with “USB4000-UV-VIS” detector, Ocean Optics, Largo, FL, USA) measuring the absorbance at
409 nm. H2O2 concentration in the effluent can then be calculated based on a calibration curve. The yield of hydrogen peroxide is defined as the ratio of the overall rate of hydrogen peroxide
production delivered by the reactor divided by the hydrogen or the oxygen feed rate. In the present
system, however, with separate dosage of the two gases and the solvent, a balance over the liquid phase
only appears more meaningful. Unfortunately, this suffers from the difficulty of assessing the amount
of the respective gas effectively introduced in the liquid via the membrane, e.g., from measurements of
the gas flows into and out of the two gas compartments or from simultaneous measurements of the
hydrogen peroxide and water production rates. The former is impeded by the fact that only a small
amount of the gas supplied to the gas chamber enters into the liquid, whereas the latter is obviously
not possible for aqueous solvents. For the same reasons, neither the conversion nor the selectivity can
be assessed. Therefore, a simplified hydrogen-based yield of hydrogen peroxide YH2O2 is introduced here by
division of the H2O2 concentration measured at the reactor outlet cH2O2 FIA by the H2 concentration in
the liquid phase in contact with the gas via the membrane cH2 sat, assuming saturation of the liquid at
the given partial pressure of hydrogen in the H2 gas chamber (Equation 7). The H2 concentration in
the liquid was calculated from Henry’s law (Equation 8). YH2O2 = cH2O2
cH2 sat
(7)
cH2 sat = pH2 ·Hcp
H2,H2O
(8) (7) cH2 sat = pH2 ·Hcp
H2,H2O
(8) (8) with: Henry constant in water Hcp
H2,H2O (taken from the DETHERM database [64]). with: Henry constant in water Hcp
H2,H2O (taken from the DETHERM database [64]). 2
2
Note, that according to this definition, a hydrogen peroxide yield of one indicates that an amount
of hydrogen peroxide has been formed which corresponds to saturation of the liquid phase under the
conditions prevailing at the membrane interface between the hydrogen gas chamber and the liquid. 3.3. Experimental Procedure Louis, MO, USA) and self-prepared 1
wt.-% Pd/TiO2 were chosen as catalysts for the reaction since both had shown high selectivity and
reaction rates according to the literature [51,55,65]. Pd/TiO2 was prepared according to a reported
wet impregnation method [51]. The procedure as well as catalyst characterization was reported
previously [30]. Three different catalyst arrangements were tested. The 5 wt.-% Pd/C was diluted with
active carbon (242276 Sigma-Aldrich, St. Louis, MO, USA) in a ratio of 1:9, pressed, crushed and sieved
to a fraction of 100 to 250 µm, and finally 200 mg of this mixture was filled in the micro channel to
form a fixed bed. Also, 200 mg of Pd/TiO2 was sieved to 100 to 250 µm and tested as a fixed bed in the
channel. Glass wool was placed in the tube leading to and from the micro channel to hold the catalysts
in place. Pd/TiO2 was also tested as a coating. For this, Pd/TiO2 was ground, stirred in acetone, and
then added to the micro channel using a pipette. After drying, the particles were stuck to the walls of
the micro channel. Under experimental conditions, the layer was found to give stable reaction rates
over a time period of 2 days, afterwards, particle discharge led to rapidly declining activity. Lastly, a
tubular fixed bed was prepared by sandwiching 50, 100 and 200 mg of 1 wt.-% Pd/TiO2 sieved to 100
to 250 µm with glass wool in a 6 mm PVC tube. The tubular fixed bed was applied as a third option for
catalyst integration since pressure control of the micro channel fixed bed was challenging and led to
numerous membrane lift offs due to the high pressure drop, and catalyst discharge was found to occur
when the catalyst coating was used. A photo or illustration of each catalyst arrangement is shown in
Figure 11. 15 of 19
catalyst
of each Catalysts 2018, 8, 556
was challenging a
discharge was fou alyst arrangement is shown in Figure 11. (a)
(b)
ysts 2018, 8, x FOR PEER REVIEW
15 of
(c)
(d)
Figure 11. Photos and illustrations of the different catalyst arrangements tested: Pd/C micro channel
fixed bed (a), Pd/TiO2 micro channel fixed bed (b), catalyst arrangement with a tubular fixed bed (c)
and Pd/TiO2 micro channel coating (d). Figure 11. 3.3. Experimental Procedure Photos and illustrations of the different catalyst arrangements tested: Pd/C micro channel
fixed bed (a), Pd/TiO2 micro channel fixed bed (b), catalyst arrangement with a tubular fixed bed (c)
and Pd/TiO2 micro channel coating (d). (b)
15 (a)
s 2018, 8, x FOR PEER REVIEW (b) (a)
, ,
(c) (a) (d) (d) (c) Figure 11. Photos and illustrations of the different catalyst arrangements tested: Pd/C micro channel
fixed bed (a), Pd/TiO2 micro channel fixed bed (b), catalyst arrangement with a tubular fixed bed (c)
and Pd/TiO2 micro channel coating (d). Figure 11. Photos and illustrations of the different catalyst arrangements tested: Pd/C micro channel
fixed bed (a), Pd/TiO2 micro channel fixed bed (b), catalyst arrangement with a tubular fixed bed (c)
and Pd/TiO2 micro channel coating (d). Residence time variations were performed by varying the flow rate of the liquid solvent between
0.1 and 3 mL/min. Pressure variations have been examined by varying the total system pressure
between 10 and 50 bars. Furthermore, the influence of the O2/H2-ratio on the reaction has been
examined by diluting H2 with N2 in the gas phase and thereby, varying the liquid phase concentration
through the H2 partial pressure. The O2/H2-ratio was varied between 1 and 16. The influence of the
permeability was studied by performing the reaction applying reinforced Nafion® membranes with
a thickness of 180 μm and 30 μm. The influence of acids, their salts, and the pH value on the reaction
was studied by performing the reaction with different additives in demineralized water: a sodium
acetate buffer at pH 3 (60 mM CH3COOH, 1 mM CH3COONa) and a solution with 5 mM H3PO4 and
25 mM H2SO4. The reaction experiments performed in this work are summarized in Table 1 for an
easier overview. Residence time variations were performed by varying the flow rate of the liquid solvent between
0.1 and 3 mL/min. Pressure variations have been examined by varying the total system pressure
between 10 and 50 bars. Furthermore, the influence of the O2/H2-ratio on the reaction has been
examined by diluting H2 with N2 in the gas phase and thereby, varying the liquid phase concentration
through the H2 partial pressure. The O2/H2-ratio was varied between 1 and 16. The influence of the
permeability was studied by performing the reaction applying reinforced Nafion® membranes with a
thickness of 180 µm and 30 µm. 3.3. Experimental Procedure The influence of acids, their salts, and the pH value on the reaction
was studied by performing the reaction with different additives in demineralized water: a sodium
acetate buffer at pH 3 (60 mM CH3COOH, 1 mM CH3COONa) and a solution with 5 mM H3PO4 and
25 mM H2SO4. The reaction experiments performed in this work are summarized in Table 1 for an
easier overview. During the course of this work, it was found that stainless steel equipment and traces of catalyst
can easily decompose H2O2. This was noted when feeding a solution of 7 mM H2O2 in water to the
system and recording the H2O2 concentration at the exit of the system. Therefore, the setup was
passivated by flushing all stainless steel parts with a HNO3 solution [66]. After passivation, a
concentration of 5 mM NaBr in the feed was found to be sufficient to prevent any decomposition in
stainless steel equipment even when H2 is present. The passivation procedure was applied and the
setup was thoroughly cleaned once it had been found that catalyst traces were present after the
coating experiments (Table 1, Exp.-Nr.: 29–37). Experiments affected by catalyst traces are obviously
not shown in this work and are neither part of the experimental discussion. During the course of this work, it was found that stainless steel equipment and traces of catalyst
can easily decompose H2O2. This was noted when feeding a solution of 7 mM H2O2 in water to
the system and recording the H2O2 concentration at the exit of the system. Therefore, the setup
was passivated by flushing all stainless steel parts with a HNO3 solution [66]. After passivation, a
concentration of 5 mM NaBr in the feed was found to be sufficient to prevent any decomposition
in stainless steel equipment even when H2 is present. The passivation procedure was applied and
the setup was thoroughly cleaned once it had been found that catalyst traces were present after the
coating experiments (Table 1, Exp.-Nr.: 29–37). Experiments affected by catalyst traces are obviously
not shown in this work and are neither part of the experimental discussion. 4. Conclusions
4. Conclusions This work reports for the first time an experimental evaluation of a membrane micro channel
reactor designed to safely perform the direct synthesis of hydrogen peroxide at elevated pressure
with undiluted reactants in a liquid solvent. The results revealed unknown relationships between
This work reports for the first time an experimental evaluation of a membrane micro channel
reactor designed to safely perform the direct synthesis of hydrogen peroxide at elevated pressure
with undiluted reactants in a liquid solvent. The results revealed unknown relationships between Catalysts 2018, 8, 556 16 of 19 16 of 19 reactor design and reaction conditions in a system of this kind. The Nafion® membranes used in this
work were found to introduce a mass transfer resistance as shown by solvent flow rate variations
and the influence on product concentration and productivity. Usage of Nafion® membranes with
reduced thickness showed an increased mass transfer. In theory, by increasing system pressure, the
mass transport can be increased to a point were liquid-phase diffusion becomes the rate-limiting
step. Therefore, Nafion® can be considered suitable, as it performed great in regards to its technical
properties. Even though Nafion® was found to limit the reaction, it was shown that reducing the depth
of the micro channel still was able to further increase product formation. The yield of the system was found to be greatly dependent on two factors, stabilizers and reactant
ratio. A combination of NaBr, H2SO4 and H3PO4 was found to greatly increase product yield in
comparison to other combinations. In contrast, addition of a sodium acetate buffer at pH 3 to the
solvent containing 5 mM NaBr showed only minimal benefits. Hence, the salts of the stabilizers seem
to be more relevant than the acidic function. A similar effect was found by varying the reactant ratio. Increasing the pO2/pH2 ratio to a value of 5 to 10 vastly increased the yield of the system. If hydrogen
is the limiting reagent, then the Pd surface must be covered with oxygen. In conclusion, excess oxygen
and stabilizers both prevent hydrogenation of the product, most probably by hindering the adsorption
of hydrogen peroxide on the Pd surface. Even with stabilizers, without increased pO2/pH2 ratio, the yield was limited to about 20%. 4. Conclusions
4. Conclusions With
the introduction of excess oxygen it was possible to increase the yield to 80% in an experiment where
the reactor was used only for gas dosage and reaction was performed downstream. In experiments
where the catalyst was present within the micro channel it was possible to achieve hydrogen peroxide
concentrations above the saturation concentration of hydrogen. This proves the ability of the reactor to
feed the reactants continuously over the length of the micro channel without having the reactants in
contact in the gas phase. Furthermore, linear dependency of productivity on pressure, adjustability of
reactant ratio, several degrees of freedom in regards to the micro channel design, and exploitation of
high pressures further proves the feasibility of the membrane microchannel reactor. The knowledge
gained in this work provides the basis to enhance productivity and product concentration of this
system in future studies. Author Contributions: Conceptualization, M.S.; methodology, M.S., M.K. and R.D.; validation, M.S. and M.K.;
investigation, M.S.; writing—original draft preparation, M.S.; writing—review and editing, M.K. and R.D.;
visualization, M.S.; supervision, M.K. and R.D.; project administration, R.D. Funding: We would like to thank the Helmholtz Association for funding within the program Science and
Technology of Nanosystems (STN). Acknowledgments: We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing
Fund of Karlsruhe Institute of Technology. Acknowledgments: We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing
Fund of Karlsruhe Institute of Technology. Conflicts of Interest: The authors declare no conflict of interest Conflicts of Interest: The authors declare no conflict of interest 6.
Centi, G.; Perathoner, S.; Abate, S. Direct synthesis of hydrogen peroxide: Recent advances. Mod. Heterog.
Oxid. Catal. Des. React. Charact. 2009, 253–287. [CrossRef] Anastas, P.T.; Warner, J.C. Green Chemistry: Theory and Practice; Oxford University Press: Oxford, UK, 2000
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g
p
y
g
yg
y
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Henkel, H.; Weber, W. Manufacture of Hydrogen Peroxide. U.S. Patent 1108752, 25 August 1914. 1.
Dittmeyer, R.; Boeltken, T.; Piermartini, P.; Selinsek, M.; Loewert, M.; Dallmann, F.; Kreuder, H.; Cholewa, M.;
Wunsch, A.; Belimov, M.; et al. Micro and micro membrane reactors for advanced applications in chemical
energy conversion. Curr. Opin. Chem. Eng. 2017, 17, 108–125. [CrossRef] Gosser, L.W. Catalytic Process for Making H2O2 from Hydrogen and Oxygen.
U.S. Patent 4681751
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236794.pdf (accessed on 25 October 2018). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Endogenous<i> Candida</i> Endophthalmitis as a Rare Complication of Trans-Urethral Lithotripsy in a Healthy Woman: A Case Report
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Case Report
DOI: 10.4274/tjo.galenos.2019.02328
Turk J Ophthalmol 2019;49:294-296 Case Report
DOI: 10.4274/tjo.galenos.2019.02328
Turk J Ophthalmol 2019;49:294-296 DOI: 10.4274/tjo.galenos.2019.02328
Turk J Ophthalmol 2019;49:294-296 Case Report ©Copyright 2019 by Turkish Ophthalmological Association
Turkish Journal of Ophthalmology, published by Galenos Publishing House.
Cite this article as: Shirvani M, Hosseini S, Maalhagh M, Mohaghegh S. Endogenous Candida Endophthalmitis as a Rare Complication of Trans-Urethral
Lithotripsy in a Healthy Woman: A Case Report. Turk J Ophthalmol. 2019;49:294-296
Address for Correspondence: Sahar Mohaghegh MD, Shiraz University of Medical Sciences, Department of Ophthalmology, Shiraz, Iran
Phone:+989173056454 E-mail: mohaghegh_sahar@yahoo.com ORCID-ID: orcid.org/0000-0002-5953-3085
Received: 14.12.2018 Accepted: 07.05.2019 Introduction Endogenous endophthalmitis is an ocular emergency that
can lead to catastrophic ophthalmic complications. Endogenous
fungal endophthalmitis (EFE) results from dissemination of
fungal organisms from infected organs to the ocular vascular
network following fungus seeding in the choroid and retina.1,2,3
The organisms responsible for EFE are Candida, Aspergillus, and
Coccidious.2,3 Trans-urethral lithotripsy (TUL) is a minimally
invasive endoscopic procedure performed using a rigid or flexible
uretroscope.4 Here, we report a rare case of endogenous Candida
endophthalmitis (ECE) after TUL in a healthy woman. Abstract Endogenous endophthalmitis is a serious sight-threatening ocular emergency that usually occurs in patients with serious underlying
risk factors. In this report, we describe a case of endogenous Candida endophthalmitis following trans-urethral lithotripsy in an
immunocompetent woman. In our case, the retinal lesion regressed completely and vision was restored. We discuss diagnostic procedures
and management strategies in this article. Keywords: Endogenous endophthalmitis, Candida endophthalmitis, lithotripsy, ureteral stone 19-mm proximal left ureteral stone 2 weeks before presentation
to the ophthalmology clinic. Past medical and drug history
was negative. Pre- and postoperative urine and blood cultures
were negative and urine analysis was unremarkable. Upon
examination, her best-corrected visual acuity (BCVA) in the left
eye was 1/10. Intraocular pressure was 11 mmHg. Slit-lamp
examination revealed +1 ciliary injection with no signs of keratic
precipitate (KP), and hypopyon and +1 cells in the anterior
chamber. The iris and lens were normal. Mild vitritis was seen in
the vitreous cavity. On fundus examination, media was clear and
a creamy, mildly elevated lesion 1/4 disc diameter in size with
indistinct borders was observed in the inferior parafoveal region
(Figure 1a). Spectral-domain optical coherence tomography
showed subretinal fluid aggregation and macular edema (Figure
1b,c). Examination of the right eye was unremarkable. Mohammad Shirvani, Shahla Hosseini, Mehrnoosh Maalhagh, Sahar Mohaghegh Mohammad Shirvani, Shahla Hosseini, Mehrnoosh Maalhagh, Sahar Mohagheg Shiraz University of Medical Sciences, Department of Ophthalmology, Shiraz, Iran Shiraz University of Medical Sciences, Department of Ophthalmology, Shiraz, Iran Case Report A 31-year-old woman presented to the ophthalmology
emergency room complaining of painless, gradual reduction
in visual acuity in her left eye starting 1 week earlier. The
patient had undergone TUL with double-J stent placement for a Following hospital admission, a diagnostic vitreous tap
was performed and a sample was sent for smear, culture, and
real-time polymerase chains reaction (RT-PCR) analysis. The ani M, Hosseini S, Maalhagh M, Mohaghegh S. Endogenous Candida Endophthalmitis as a Rare Complication of Trans-Urethral
Lithotripsy in a Healthy Woman: A Case Report. Turk J Ophthalmol. 2019;49:294-296 ©Copyright 2019 by Turkish Ophthalmological Association
Turkish Journal of Ophthalmology, published by Galenos Publishing House. 294 Shirvani et al, Endogenous Candida Endophthalmitis After Trans-Urethral Lithotripsy smear was unremarkable, but RT-PCR was positive for Candida
albicans. Therefore, intravitreal injection of amphotericin-B (0.5
µg/0.1 mL) was performed. Treatment with topical levofloxacin,
hematropin, and prednisone acetate 1% every 6 hours and oral
fluconazole 200 mg every 12 hours was initiated. Blood and
urine culture at the time of presentation were negative and
urine analyses were unremarkable. Viral markers including
hepatitis B virus surface antigen and core antibody, hepatitis C
virus antibody, and human immunodeficiency virus antibody
(HIV Ab) were negative. Serology was negative for Toxoplasma
(IgM and IgG), Borrelia, and Bartonella. In systemic workup,
antinuclear antibody, antineutrophil cytoplasmic antibody,
antimitochondrial antibody, venereal disease research laboratory,
fluorescent treponemal antibody absorption, Mantoux, and
interferon-γ tests were all negative. Erythrocyte sedimentation
rate, C-reactive protein, complete blood count, platelet count,
fasting blood glucose, aspartate aminotransferase, alanine
aminotransferase, blood urea nitrogen, creatinine, angiotensin
converting enzyme, and immunoglobulin G, M, and A levels
were within normal limits. Peripheral blood smear and paranasal
sinus and chest x-rays were normal. Discussion ECE is a devastating ocular infection. Predisposing conditions
include long-term systemic antibiotic usage, hospitalization,
indwelling catheters, candiduria, major gastrointestinal
intervention, prolonged intravenous line, hemodialysis,
liver cirrhosis, intravenous drug abuser, immunomodulatory
therapy, chemotherapy, diabetes mellitus, hematopoietic, organ
transplantation, abortion, and HIV.1,2,3,5 Fungi may enter bloodstream during urinary tract
interventions due to mechanical abrasion and epithelial trauma,
leading to candidemia and intraocular candidiasis. Some reported
infectious complications after urinary tract procedures include
urinary tract infection, urosepsis and candidemia, perinephric
and renal abscesses, urinoma, Klebsiella endophthalmitis, and
retroperitoneal abscess.6 We found 5 case reports of ECE following
urinary tract lithotripsy in our literature review.7,8,9,10,11 In 3 cases,
ECE occurred after ESWL and uretroscopy for double-J stent
placement.7,8,9 In one case, ECE occurred following TUL and
ureteral stent placement10 and in the last case report it occurred
after decompressive nephrostomy.11 In 4 cases, preoperative urine
culture was positive for C. albicans and the patients suffered
from debilitating diseases (liver cirrhosis, rheumatic arthritis,
alcoholic liver disease, or diabetes mellitus).8,9,10,11 In our case,
ECE occurred in an immunocompetent woman after TUL
double-J stent placement while pre- and postoperative urine and
blood cultures were negative and there were no underlying risk
factors. Forty-eight hours after initiating treatment, the patient’s
BCVA increased to 3/10. Conjunctival injection and vitritis
disappeared, and the borders of the infiltrative lesion became
sharp. She was discharged with oral fluconazole 200 mg every
12 hours for 6 weeks. Vitreous tap culture was negative after
72 hours. The diagnosis of ECE is difficult due to its various ocular
manifestations and low positive culture rate, especially in cases
with minimal vitreous involvement. The condition does not only
occur in patients with underlying risk factors, but also in healthy
individuals. Thus, there is the risk of misdiagnosis, leading to
delay in initiating appropriate treatment. For more accurate
diagnosis, vitreous tap sampling or diagnostic vitrectomy is
recommended in suspicious cases, since diagnostic vitrectomy
shows a higher positive culture rate and intravitreal injection
can be performed simultaneously.1,2,3,5,8 Moreover, RT-PCR is
more sensitive than culture, but more expensive and might
be unavailable.1,2,3 In this case report, RT-PCR analysis of the
vitreous sample was positive for C. albicans, but vitreous smear
and culture were negative. After 6 weeks, her BCVA was 9/10 and the fungal infiltrative
lesion had completely disappeared. Macular edema was resolved
with no scarring or epiretinal membrane formation (Figure 2a,
b, c). Discussion The final BCVA outcome was 10/10 and there was no
recurrence in 3-year follow-up. Figure 1. Initial appearance at time of presentation. (a) Color fundus photo
showed a creamy lesion in the parafoveal area; (b) Spectral-domain optical coherence
tomography revealed macular edema and micro-abscess formation in the sensory
retina; (c) Topographic macular map displayed an elevated lesion on the macula Figure 1. Initial appearance at time of presentation. (a) Color fundus photo
showed a creamy lesion in the parafoveal area; (b) Spectral-domain optical coherence
tomography revealed macular edema and micro-abscess formation in the sensory
retina; (c) Topographic macular map displayed an elevated lesion on the macula Timely diagnosis and rapid antifungal therapy are
associated with better visual outcomes.2,3 ECE treatment
depends on the severity of inflammation and the patient’s
visual acuity. Appropriate treatment in patients with isolated
choroidoretinitis is systemic medication with good intravitreal
penetration, such as voriconazole and fluconazole. When a
patient presents with choroidoretinitis and mild to moderate
vitritis, systemic therapy accompanied by intravitreal injection
of amphotericin-B or voriconazole is appropriate. In sight-
threatening conditions and severe vitritis, pars plana vitrectomy
with intravitreal medication during vitrectomy and systemic
medication are recommended.1,2,3 Although intravitreal injection
of amphotericin-B is very effective, intravenous injection of Figure 2. Regression of the fungal lesion 6 weeks after antifungal treatment. (a) Infrared fundus image showed complete disappearance of the fungal lesion;
spectral-domain optical coherence tomography (b) and topographic macular map
(b) revealed resolution of the macular edema without scarring or traction formation Figure 2. Regression of the fungal lesion 6 weeks after antifungal treatment. (a) Infrared fundus image showed complete disappearance of the fungal lesion;
spectral-domain optical coherence tomography (b) and topographic macular map
(b) revealed resolution of the macular edema without scarring or traction formation 295 Turk J Ophthalmol 49; 5: 2019 References amphotericin-B is not recommended due to poor intravitreal
penetration and systemic complications such as nephrotoxicity.1
In our case, swift diagnosis and appropriate antifungal treatment
(systemic fluconazole + intravitreal amphotericin-B) led to good
visual outcome. 1. Sadiq MA, Hassan M, Agarwal A, Sarwar S, Toufeeq S, Soliman MK. Hanout
M, Sepah YJ, Do DV, Nguyen QD. Endogenous endophthalmitis: diagnosis,
management, and prognosis. J Ophthalmic Inflamm Infect. 2015;5:32. 2. Sallam A, Lynn W, McCluskey P, Lightman S. Endogenous Candida
endophthalmitis. Expert Rev Anti Infect Ther. 2006;4:675-685. ECE after urinary tract interventions is a rare but vision-
threatening infection that may occur in immunocompetent
individuals. Early detection and timely treatment can lead to
better visual prognosis. 3. Lingappan A, Wykoff CC, Albini TA, Miller D, Pathengay A, Davis JL,
Flynn HW JR. Endogenous fungal endophthalmitis: causative organisms,
management strategies, and visual acuity outcomes. Am J Ophthalmol. 2012;153:162-166. Acknowledgments 4. Mehrabi S, Rahmani A, Mehrabi A, Motlagh A. Extracorporeal shockwave
lithotripsy versus laser lithotripsy by semirigid ureteroscope in treatment of
upper ureteral stones. Acta Medica Mediterranea. 2016;32:2075-2078. The authors wish to thank Mr. H. Argasi at the Research
Consultation Center (RCC) of Shiraz University of Medical
Sciences for his invaluable assistance in editing this manuscript. In addition, the authors wish to thank Mr. Akber Enveri for his
invaluable assistance in the Turkish translation. 5. Pierce J, Warrier N, Antonopoulos C, Siegel N, Husain D. Iris abscess a rare
presentation of intravenous drug abuse associated Candida endophthalmitis. Am J Ophthalmol Case Rep. 2016;18:27-29. Ethics 6. Doran O, Foley B. Acute complications following extracorporeal shock-wave
lithotripsy for renal and ureteric calculi. Emerg Med Australas. 2008;20:105-
111. Informed Consent: Received. Peer-review: Externally peer-reviewed. 7. Toshikuni N, Ujike K, Yanagawa T, Suga T, Shimizu T, Kusuda Y. Okamoto
M, Ogawa T, Yuasa S. Candida albicans endophthalmitis after extracorporeal
shock wave lithotripsy in a patient with liver cirrhosis. Intern Med. 2006;45:1327-1332. Authorship Contributions Authorship Contributions Surgical and Medical Practices: Mohammad Shirvani,
Concept: Mohammad Shirvani, Mehrnoosh Maalhagh,
Design: Mohammad Shirvani, Mehrnoosh Maalhagh, Data
Collection or Processing: Mohammad Shirvani, Mehrnoosh
Maalhagh,
Analysis
or
Interpretation:
Mohammad
Shirvani, Shahla Hosseini, Mehrnoosh Maalhagh, Sahar
Mohaghegh, Literature Search: Mohammad Shirvani, Shahla
Hosseini, Writing: Mohammad Shirvani, Sahar Mohaghegh. 8. Lavine JA, Mititelu M. Multimodal imaging of refractory Candida
chorioretinitis progressing to endogenous endophthalmitis. J Ophthalmic
Inflamm Infect. 2015;5:54. 9. Yuan Z, Liang X, Zhong J. The Disease Course of Bilateral Endogenous
Fungal Endophthalmitis. J Clin Exp Ophthalmol. 2017;8:639. 9. Yuan Z, Liang X, Zhong J. The Disease Course of Bilateral Endogenous
Fungal Endophthalmitis. J Clin Exp Ophthalmol. 2017;8:639. 10. Inn FX, Md Noh UK, Jasman MH. Endogenous endophthalmi 10. Inn FX, Md Noh UK, Jasman MH. Endogenous endophthalmitis as a septic
complication postureteroscope candiduria. Urol Ann. 2017;9:83-85. 10. Inn FX, Md Noh UK, Jasman MH. Endogenous endophthalmitis as a sept
complication postureteroscope candiduria. Urol Ann. 2017;9:83-85. Conflict of Interest: No conflict of interest was declared by
the authors. Conflict of Interest: No conflict of interest was declared by
the authors. complication postureteroscope candiduria. Urol Ann. 2017;9:83-8 11. Barbosa IAF, Wanzeler ACV, Gomes HAP, Lúcio KCV, Jorge EC, Schellini
SA. Bilateral endogenous fungal endophthalmitis secondary obstructive
pyelonephritis. Rev Bras Oftalmol. 2017;76:33-36. Financial Disclosure: The authors declared that this study
received no financial support. Financial Disclosure: The authors declared that this study
received no financial support. 296
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ASO Author Reflections: Advanced Imaging Allows Better Detection of Peritoneal Metastases
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Annals of surgical oncology
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cc-by
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PRESENT Peritoneal metastases (PM) are diagnosed in up to 30% of
patients with colorectal cancer, especially in patients with T4
disease.1 Cytoreduction and hyperthermic intraperitoneal
chemotherapy (HIPEC) improve the survival of patientswith
colorectal PM, resulting in an overall survival of 56 months
in selected patients with limited peritoneal disease.2 Unfor-
tunately, PM are often missed during elective primary tumor
resection. Second, during follow-up after initial curative
treatment, PM are mostly diagnosed in an advanced stage. Current diagnostic imaging modalities such as positron
emission tomography/computed tomography (PET/CT)
have a detection limit of 5 mm and the multiple small peri-
toneal lesions are frequently missed.3 The presence of
advanced intra-abdominal disease such as omental cake can
be easily visualized, but curative options in this stage are
limited. Since cytoreduction and HIPEC results in good
survival in patients with limited peritoneal disease, accom-
panied by a low morbidity due to the limited amount of
resections, extensive effort should be made to improve early
diagnosis of PM. Advanced imaging could improve the detection of PM
during elective colorectal cancer resection and diagnostic
laparoscopy. Narrow-band imaging (NBI), near-infrared
indocyanine green fluorescence imaging (NIR-ICG), and
spray-dye chromoendoscopy (SDCE) were prospectively
compared with white-light imaging for the detection of PM
in 28 patients.4 NBI substantially increased sensitivity from
80.0% with white light to 96.0% (p = 0.008), without loss
of specificity (73.1% vs. 74.8%, p = 0.804). NIR-ICG and
SDCE were not considered of value: NIR-ICG did not
result in fluorescent PM and SDCE did not visualize the
whole peritoneum. NBI is not only a promising and safe method but is also
practical, time efficient, and does not require extra costs. Its
main implication could be early detection of PM during
evaluation of the peritoneum at the time of primary tumor
resection. This allows early referral to specialized HIPEC
centers, which is key for oncologic outcome. Second, NBI
could help determine feasibility of a complete cytoreduc-
tion prior to HIPEC. Third, improved visualization of PM
may optimize cytoreduction. Ann Surg Oncol (2019) 26:165–166
https://doi.org/10.1245/s10434-018-7083-4 Ann Surg Oncol (2019) 26:165–166
https://doi.org/10.1245/s10434-018-7083-4 ASO AUTHOR REFLECTIONS ASO Author Reflections is a brief invited commentary on the article,
‘‘Narrow-Band Imaging Improves Detection of Colorectal Peritoneal
Metastases: A Clinical Study Comparing Advanced Imaging
Techniques,’’ Ann Surg Oncol. https://doi.org/10.1245/s10434-018-
7005-5. The Author(s) 2018
First Received: 14 November 2018;
Published Online: 26 November 2018
N. R. Sluiter, MD
e-mail: n.sluiter@vumc.nl FUTURE NBI is a practical technique that is available on most
laparoscopic systems, facilitating quick translation into
clinical practice. Fluorescence with antibody-coupled ICG
is a promising method but is currently not widely available
and requires further research prior to clinical implemen-
tation. Issues to be addressed include identification of
optimal antibody targets (vascular endothelial growth fac-
tor or carcinoembryonic antigen) and cost-effectiveness. Furthermore, investigation of the value of advanced
imaging in terms of oncologic outcomes is pivotal, war-
ranting prospective randomization between techniques. ASO Author Reflections is a brief invited commentary on the article,
‘‘Narrow-Band Imaging Improves Detection of Colorectal Peritoneal
Metastases: A Clinical Study Comparing Advanced Imaging
Techniques,’’ Ann Surg Oncol. https://doi.org/10.1245/s10434-018-
7005-5. 166 N. R. Sluiter, J. B. Tuynman link to the Creative Commons license, and indicate if changes were
made. link to the Creative Commons license, and indicate if changes were
made. Currently,
a
trial
evaluating
second-look
diagnostic
laparoscopy after T4 colorectal cancer resection aims for
detection of PM at a clinically occult stage, which is
expected to translate into survival benefit (ClinicalTrials.-
gov: NCT03413254). To further improve survival of
patients with colorectal PM, early diagnosis by advanced
(epi-)genetic analyses in peripheral blood as liquid biopsies
has been studied.5 Together with advanced intraoperative
tumor detection, this could allow for cytoreduction and
HIPEC in an early and curable stage of peritoneal disease,
and, consequently, improve oncologic outcomes. REFERENCES 1. Koppe MJ, Boerman OC, Oyen WJ, Bleichrodt RP. Peritoneal
carcinomatosis of colorectal origin: incidence and current treat-
ment strategies. Ann Surg. 2006;243(2):212–22. 2. Sluiter NR, Rovers KP, Salhi Y, et al. Metachronous peritoneal
metastases after adjuvant chemotherapy are associated with poor
outcome after cytoreduction and HIPEC. Ann Surg Oncol. 2018;25(8):2347–56. 3. Koh JL, Yan TD, Glenn D, Morris DL. Evaluation of preoperative
computed tomography in estimating peritoneal cancer index in
colorectal
peritoneal
carcinomatosis. Ann
Surg
Oncol. 2009;16(2):327–33. DISCLOSURES
Nina
Roelie
Sluiter
and
Jurriaan
Benjamin
Tuynman have no conflicts of interest to disclose. DISCLOSURES
Nina
Roelie
Sluiter
and
Jurriaan
Benjamin
Tuynman have no conflicts of interest to disclose. 4. Sluiter NR, Vlek SL, Wijsmuller AR, Brandsma HT, De Vet
HCW, Van Grieken NCT, et al. Narrow-band imaging improves
detection of colorectal peritoneal metastases: a clinical study com-
paring
advanced
imaging
techniques. Ann
Surg
Oncol. https://doi.org/10.1245/s10434-018-7005-5 OPEN ACCESS
This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.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 5. Wan JC, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come
of age: towards implementation of circulating tumour DNA. Nat
Rev Cancer. 2017;17(4):223–238.
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Pressure as a Limiting Factor for Life
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Life
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cc-by
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Concept Paper Concept Paper Received: 3 June 2016; Accepted: 9 August 2016; Published: 17 August 2016 Received: 3 June 2016; Accepted: 9 August 2016; Published: 17 August 2016 Received: 3 June 2016; Accepted: 9 August 2016; Published: 17 August 2016 ved: 3 June 2016; Accepted: 9 August 2016; Published Abstract: Facts concerning the stability and functioning of key biomolecular components suggest
that cellular life should no longer be viable above a few thousand atmospheres (200–300 MPa). However, organisms are seen to survive in the laboratory to much higher pressures, extending into
the GPa or even tens of GPa ranges. This is causing main questions to be posed concerning the
survival mechanisms of simple to complex organisms. Understanding the ultimate pressure survival
of organisms is critical for food sterilization and agricultural products conservation technologies. On Earth the deep biosphere is limited in its extent by geothermal gradients but if life forms exist in
cooler habitats elsewhere then survival to greater depths must be considered. The extent of pressure
resistance and survival appears to vary greatly with the timescale of the exposure. For example,
shock experiments on nanosecond timescales reveal greatly enhanced survival rates extending to
higher pressure. Some organisms could survive bolide impacts thus allowing successful transport
between planetary bodies. We summarize some of the main questions raised by recent results and
their implications for the survival of life under extreme compression conditions and its possible
extent in the laboratory and throughout the universe. Keywords: extreme conditions; high pressure treatment; food technology; protein stability; cell wall
biochemistry; bacterial survival; interplanetary transport; seeds; spores; Artemia cysts Rachael Hazael 1,2, Filip Meersman 3, Fumihisa Ono 4 and Paul F. McMillan 1,* 1
Christopher Ingold Laboratories, Department of Chemistry, University College London, 20 Gordon Street,
London WC1H 0AJ, UK; r.hazael@ucl.ac.uk 1
Christopher Ingold Laboratories, Department of Chemistry, University College London, 20 Gordon Street,
London WC1H 0AJ, UK; r.hazael@ucl.ac.uk Earth Sciences Department, University College London, Gower Street, London WC1E 6BT, UK
Biomolecular & Analytical Mass Spectrometry Group, Department of Chemistry, University of Antwerp,
Groenenborgerlaan 171, B-2020 Antwerpen, Belgium; f.meersman@ucl.ac.uk 2
Earth Sciences Department, University College London, Gower Street, London WC1E 6BT, UK
3
Biomolecular & Analytical Mass Spectrometry Group, Department of Chemistry, University of Antwerp,
Groenenborgerlaan 171, B-2020 Antwerpen, Belgium; f.meersman@ucl.ac.uk
4
Department of Applied Science, Okayama University of Science, 1-1 Ridaicho, Kitaku, Okayama 700-0005, 2
Earth Sciences Department, University College London, Gower Street, London WC1E 6BT, UK
3
Biomolecular & Analytical Mass Spectrometry Group, Department of Chemistry, University of Antwerp,
Groenenborgerlaan 171, B-2020 Antwerpen, Belgium; f.meersman@ucl.ac.uk g
,
p
,
g
;
4
Department of Applied Science, Okayama University of Science, 1-1 Ridaicho, Kitaku, Okayama 700-0005,
Japan; fumihisaono@yahoo.co.jp *
Correspondence: p.f.mcmillan@ucl.ac.uk; Tel.: +44-020-7679-4610 *
Correspondence: p.f.mcmillan@ucl.ac.uk; Tel.: +44-020-7679-4610 Academic Editor: William Bains Academic Editor: William Bains www.mdpi.com/journal/life life
Concept Paper life
Concept Paper 1. Introduction Life is currently only known to have evolved on Earth, although it has been hypothesized that it
could have emerged and might be present elsewhere in the universe [1]. In fact, our own expeditions
into space might be in the process of transporting live or dormant organisms outside our planetary
envelope, thus perhaps “seeding” life elsewhere. Until quite recently, life on Earth was considered to
occupy a narrow range of warm, damp habitats near the planetary surface [2–4]. However, both active
and dormant organisms are now known to proliferate in an ever-widening range of habitats in which
they are exposed to extreme physical and chemical environments [5]. These include high or low
temperatures, adverse chemical conditions, intense radiation and particle beam fluxes, and exposure to
extremely high mechanical stress and strain rate conditions. The survival of organisms under extreme
static or dynamic compression has implications for determining the possible extent of deep biospheres
on Earth and on other planetary bodies, as well as in the laboratory and inside industrial pressure
vessels used for preservation of foodstuffs, pharmaceuticals and agricultural products. Pressure
inactivation in the food industry is primarily enabled in open/flow systems operating at several
hundred MPa whereas the methods considered here involve closed systems operated at much higher Life 2016, 6, 34; doi:10.3390/life6030034 www.mdpi.com/journal/life Life 2016, 6, 34
Life 2015, 5, 34 2 of 8
2 of 8 pressures extending into the GPa range. These are where we examine pressure effects as ultimately
limiting the survival of organisms. New findings reveal the greatly increased survival rate of simple and
complex life forms during shock compression on a nanosecond timescale, supporting the hypothesis
of transport between planetary bodies via bolide impact. systems operated at much higher pressures extending into the GPa range. These are where we
examine pressure effects as ultimately limiting the survival of organisms. New findings reveal the
greatly increased survival rate of simple and complex life forms during shock compression on a
nanosecond timescale, supporting the hypothesis of transport between planetary bodies via bolide
impact. Unicellular prokaryotic organisms including bacteria and archaea are the simplest and most
adaptive organisms that survive and thrive in some of the harshest environments on Earth [6]. Extremophiles or “superbugs” colonize hot geysers and submarine oceanic vents, under Arctic
conditions, in acidic, alkaline and salt lakes, survive radiation exposure and high vacuum
conditions [7–11]. 1. Introduction These and other more complex organisms have been found within the ultrahigh
pressure environments of the deepest ocean trenches, and extending to several kilometers below
the oceanic and continental crust [12]. For example, at these depths the pressure can attain several
hundreds to thousands of atmospheres (1 kbar = 100 MPa ≈1000 atm). On Earth the hydrostatic
pressure gradient is approximately 10 MPa/km−1 in the oceans while those in the rocky crust increase
to approximately 15–28 MPa/km−1. The temperature rises rapidly with depth and this could provide
the main parameter limiting the extent of the deep biosphere [4,13]. Although it is difficult to quantify
the exact values of extreme pressures that might be encountered by biological organisms on other
planetary bodies it is clear that pressures in the 100–1000 MPa range and above could be experienced
at lower temperatures [14–16] (Figure 1). p
Unicellular prokaryotic organisms including bacteria and archaea are the simplest and most
adaptive organisms that survive and thrive in some of the harshest environments on Earth [6]. Extremophiles or “superbugs” colonize hot geysers and submarine oceanic vents, under Arctic
conditions, in acidic, alkaline and salt lakes, survive radiation exposure and high vacuum conditions
[7–11]. These and other more complex organisms have been found within the ultrahigh pressure
environments of the deepest ocean trenches, and extending to several kilometers below the oceanic
and continental crust [12]. For example, at these depths the pressure can attain several hundreds to
thousands of atmospheres (1 kbar = 100 MPa ≈ 1000 atm). On Earth the hydrostatic pressure gradient
is approximately 10 MPa/km−1 in the oceans while those in the rocky crust increase to approximately
15–28 MPa/km−1. The temperature rises rapidly with depth and this could provide the main
parameter limiting the extent of the deep biosphere [4,13]. Although it is difficult to quantify the exact
values of extreme pressures that might be encountered by biological organisms on other planetary
bodies it is clear that pressures in the 100–1000 MPa range and above could be experienced at lower
temperatures [14–16] (Figure 1). Figure 1. Cartoon illustrating the current extent of the survivability of organisms. This comprises both
the habitable zone of the terrestrial biosphere to the important work of laboratory experiments, which
extend current field sampling sites and expands the current physical limitations for life. Figure 1. Cartoon illustrating the current extent of the survivability of organisms. 1. Introduction This comprises
both the habitable zone of the terrestrial biosphere to the important work of laboratory experiments,
which extend current field sampling sites and expands the current physical limitations for life. Figure 1. Cartoon illustrating the current extent of the survivability of organisms. This comprises both
the habitable zone of the terrestrial biosphere to the important work of laboratory experiments, which
extend current field sampling sites and expands the current physical limitations for life. Figure 1. Cartoon illustrating the current extent of the survivability of organisms. This comprises
both the habitable zone of the terrestrial biosphere to the important work of laboratory experiments,
which extend current field sampling sites and expands the current physical limitations for life. 2. Pressure Stability Limits for Biological Macromolecules and Their Complexes
2. Pressure Stability Limits for Biological Macromolecules and Their Complexes It might be thought that a reductionist approach could be helpful to fix the absolute limits for
life survival under extreme compression, by determining a set of pressures above which the
constituent macromolecules can no longer exist in a functional state. However, laboratory results
reveal that this principle does not operate as simply as might be expected. The pioneering
experiments of Bridgman [17] first revealed protein denaturation at pressures in the 700–800 MPa
range. Irreversible protein denaturation due to pressure application is now well documented, and it
typically shows an onset above 200–500 MPa at room temperature, completed by ~800 MPa. Dissociation of protein complexes that are critically important for biological functioning occurs at
lower pressure, by around 200–300 MPa. Reduced functionality of membrane proteins or detachment
of these proteins from the membrane typically occurs as a function of increasing pressure. However,
t u tu al ha
e i
the
e
b a e a
ote t
e
b a e
otei
f o
total i du ed i a ti atio
It might be thought that a reductionist approach could be helpful to fix the absolute limits for life
survival under extreme compression, by determining a set of pressures above which the constituent
macromolecules can no longer exist in a functional state. However, laboratory results reveal that
this principle does not operate as simply as might be expected. The pioneering experiments of
Bridgman [17] first revealed protein denaturation at pressures in the 700–800 MPa range. Irreversible
protein denaturation due to pressure application is now well documented, and it typically shows
an onset above 200–500 MPa at room temperature, completed by ~800 MPa. Dissociation of protein
complexes that are critically important for biological functioning occurs at lower pressure, by around
200–300 MPa. Reduced functionality of membrane proteins or detachment of these proteins from the
membrane typically occurs as a function of increasing pressure. However, structural changes in the 3 of 8 Life 2016, 6, 34 membrane can protect membrane proteins from total induced inactivation [18]. In addition, laboratory
studies of the complex phospholipid structures that form the primary structures determining cell walls
indicate that these undergo phase transformations that would render them rigid and unviable for
biological functioning above the 200–300 MPa range. In deep-sea organisms an increase in unsaturated
lipids incorporated into the cellular membrane occurs. 3. Actual Results for Bacterial Survival into the GPa Range Food scientists have long known that although there are few survivors from an initial bacterial
sample by 700 MPa, some survivors persist beyond this commonly used “Pascalization” threshold [21]. A first report that bacteria might survive in a viable state to GPa pressures was presented by
Sharma et al. using in situ determinations of metabolic activity in a diamond anvil cell [22]. Survivability of certain bacteria has now been demonstrated to extend into the 2–3 GPa range by
culturing survivors recovered after high pressure treatments [23,24]. Here it was demonstrated that
survivors that had been previously exposed to high pressure conditions could survive to even higher
pressures. Those results contain important information about bacterial survival mechanisms, as well
as posing new questions and hypotheses concerning the actual upper pressure limits for cellular
life forms. First, it is obvious that the bacterial survival results indicate that the pressure stability of proteins or
lipid cell bilayers, do not constitute a primary barrier to cellular integrity or the continued functioning
of biological processes under extreme compression conditions, that well exceed the established stability
limits of the biomolecules exposed under in vitro laboratory conditions. While there are several
hypotheses for the modification strategies adopted by various organisms to survive extreme high
pressure treatment, the fundamental biophysical or biochemical protection mechanisms are not yet
understood [18]. They also lead to hypotheses that bacterial life might exist to extreme depth within
“cold” planets with low geothermal gradients, with ultimate limits extending well into the GPa pressure
range. The extent of such deep planetary viability of cellular organisms will extend to increasing greater
pressures as they are cycled between depth horizons. However, such cycling processes, will likely be
associated with energy transfers and a geothermal gradient, and it is not known how that might affect
“bugs” exposed to the extraterrestrial high-P, T environments. 2. Pressure Stability Limits for Biological Macromolecules and Their Complexes
2. Pressure Stability Limits for Biological Macromolecules and Their Complexes High pressures also lead to the appearance
of ordered ‘gel-like’ phases that can result in an increase in hydration of the lipid head group and
preserve the lamellar membrane structure [18]. Inclusion of other molecules including polysaccharides
into the membranes renders these resistant to low temperature treatment and leads to cryopreservation
strategies that might also contribute to high pressure survival of the cell envelope structure [19,20]. These key results suggest that exposure of organisms to beyond a limiting pressure range extending up
to a few hundred MPa, should define the extent of life survival conditions. However, that prediction is
not borne out by laboratory results, or by the documented existence of prokaryotes, as well as even
more complex organisms under deep Earth conditions, at pressures extending into the GPa range. 4. Water in Cells One key issue concerns the high pressure behavior of water that is an essential biological
component constituting approximately 70% of both prokaryotic and eukaryotic cells. Liquid water
provides a dynamic medium in which molecules can diffuse and interact, and the structure and
function of biomolecules are to a large extent determined by the properties of water [25]. It has
long been assumed that water has to be in its liquid state to facilitate life. Pure H2O crystallizes at
approximately 1 GPa to produce the dense ice-VI phase [26]. However, water inside cells contains
dissolved ions and it interacts strongly with macromolecules within the nanoconfined environment [27]. Key questions to be answered concern the physical and chemical properties of intracellular water
under extreme compression, to determine to what pressures the biological functions are maintained. Experimental studies of dynamic processes using techniques including optical spectroscopy, nuclear
magnetic resonance (NMR) and quasi-elastic neutron scattering (QENS) are under way to examine
live cells under high pressure conditions [22,28–31]. Life 2016, 6, 34 4 of 8 5. Timescales of Biological Processes It is now established that viable prokaryotic cells can survive pressurization into the 2–3 GPa
range under static compression conditions on a laboratory timescale, and that some fractions of the
initial population can be recovered to form colonies at ambient pressure [22–24]. It is not yet known if
the cells continue to function normally at high pressure, although in situ spectroscopic studies at lower
pressure (to 200 MPa) indicate a dramatic slowdown in metabolic rates [20,30,31]. Such subsurface
organisms have been termed “zombie cells”, where the slow rates of metabolism may mean that
these cells may not reproduce on timescales that are comparable to the surface world [32]. We can
hypothesize that functioning cellular life might be present to well beyond the current observed limit of
150 MPa on Earth [33], as well as to depths of many kilometers below the surface of “colder” planets. We can also hypothesize that one of the effects of extreme pressurization is to lead to a lower cell
density and a slower evolutionary path. It is also well known that cells of simple and complex organisms have established protective
mechanisms against long periods of desiccation and adverse physical conditions by entering
a cryptobiotic or dormant state [34,35]. These mechanisms could also play a part in maintaining
essential biomolecules and their functional components in a viable form, to pressures that might
extend well into the GPa range. That would permit survival of organisms exposed to extreme
compression conditions over cosmological periods of time that might be resuscitated when conditions
improve. Although the density of surrounding mineral phases increases with depth inside planetary
bodies, there always remain intergranular spaces or inclusions within individual minerals that could
accommodate such intact cellular organisms. 7. Compression on a Rapid Timescale
7. Compression on a Rapid Timescale Laboratory experiments using traditional pressurization techniques have noted some
dependence of the survival of organisms dependent on the timescale of the pressure exposure. Mostly
those results can be explained in terms of biological processes such as the accumulation and expulsion
of damaged cellular components [45]. However, dynamic shock compression techniques that allow
probing of events on nanosecond timescales reveal further mechanisms for survival of organisms to
even higher pressures. Increased survival rates are recorded for wild type and pressure-adapted
bacterial strains at 2 GPa, and some bacteria have been reported to survive following exposure to
shock pressures as high as 78 GPa, with a rise time on the order of ns, and a pulse duration of μs
[46,47]. Here, it becomes important to consider the timescale of the mechanical relaxation response
of the bacterial cell envelope. For compression timescales lower than approximately 1 ms the cell
walls exhibit a solid-like “glassy” response and this may protect the internal biochemical structures
from the full effects of the shock [48]. Here, an upper pressure limit would be fixed by the fracture
strength of the bacterial cell wall, that may extend even into the megabar range (~100 GPa) for the
short timescale events [45]. In contrast to the bacterial results, shock studies on seeds and spores into
the 1–5 GPa range result in lack or delay of germination and altered behavioral or growth patterns
[42,49–51]. The difference between unicellular and organisms with a complex cellular structure is not
known. The results lead to the hypothesis that at least some simple organisms may survive
interplanetary transport and delivery to a new host via impact events providing the shock
temperatures remain sufficiently low [52–54]. Finally, recent work has established the production of
amino acids via hypervelocity shock synthesis from precursor molecules contained within cometary
ices [55]. Those results lead to possibilities that creation of at least some of the building blocks of life
itself might have occurred during high pressure impact events
Laboratory experiments using traditional pressurization techniques have noted some dependence
of the survival of organisms dependent on the timescale of the pressure exposure. Mostly those results
can be explained in terms of biological processes such as the accumulation and expulsion of damaged
cellular components [45]. However, dynamic shock compression techniques that allow probing of
events on nanosecond timescales reveal further mechanisms for survival of organisms to even higher
pressures. 6. Complex Lifeforms Masayuki Saigusa (Biology) at
Okayama University, with help from undergraduate students in both their laboratories. The tardigrades
for the study were mainly collected by Mr. Taro Uozumi. Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs,
although slightly less energetically than other samples exposed for shorter times [40]. The resuscitated
organisms were shown to survive for at least 7 days following the exposure to high pressure
conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-
pressurization tardigrade, from which the still image is extracted, is shown in the supplementary
information and was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. Masayuki
Saigusa (Biology) at Okayama University, with help from undergraduate students in both their
laboratories. The tardigrades for the study were mainly collected by Mr. Taro Uozumi. Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs, although
slightly less energetically than other samples exposed for shorter times [40]. The resuscitated organisms
were shown to survive for at least 7 days following the exposure to high pressure conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-pressurization
tardigrade, from which the still image is extracted, is shown in the Supplementary information and
was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. Masayuki Saigusa (Biology) at
Okayama University, with help from undergraduate students in both their laboratories. The tardigrades
for the study were mainly collected by Mr. Taro Uozumi. 6. Complex Lifeforms Initial beliefs held that the oceanic depths were devoid of life. However, expeditions have now
fully demonstrated the presence of complex life existing within the oceanic abyss and also at depths
to 2 km in mineshafts drilled into the continental crust [36–39]. High pressure effects on organisms
ranging from Artemia cysts in a cryptobiotic state, to various spores and seed varieties, as well as the
remarkable tardigrade creatures known as “water bears”, have been investigated using large volume
techniques at 7.5 GPa, holding the samples at pressure for timescales extending from a few hours to
several days [40–42] (Figure 2). Remarkably, most of the pressurized organisms resume their normal
biological function once they are recovered to ambient conditions. It is obvious that the mechanically
resistant coatings of seeds and other multicellular envelope structures protect the interior structures
from compressive stress, but those effects remain to be fully quantified. It is known that seed coatings
can resist high fracture stresses and rupture at low moisture content [43]. Artemia eggs hatch upon
exposure to water and the nauplii show normal swimming behavior. Likewise, water bears exposed to
pressurization become motile without apparent effects from the extreme compression [44]. Some seeds
do show changes in their subsequent growing behavior, indicating that some pressurization effects
on the biochemical apparatus have been perceived. The results lead to hypotheses that complex
multicellular life forms might exist in a functionally active state at pressures into the GPa range, or in
hibernation with a surrounding protective envelope around the cellular components, to pressures
extending to tens of GPa, on Earth as well as elsewhere in the universe. 5 of 8
f Life 2016, 6, 34 Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs,
although slightly less energetically than other samples exposed for shorter times [40]. The resuscitated
organisms were shown to survive for at least 7 days following the exposure to high pressure
conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-
pressurization tardigrade, from which the still image is extracted, is shown in the supplementary
information and was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. Masayuki
Saigusa (Biology) at Okayama University, with help from undergraduate students in both their
laboratories. 6. Complex Lifeforms The tardigrades for the study were mainly collected by Mr. Taro Uozumi. 0.2 mm
Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs, although
slightly less energetically than other samples exposed for shorter times [40]. The resuscitated organisms
were shown to survive for at least 7 days following the exposure to high pressure conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-pressurization
tardigrade, from which the still image is extracted, is shown in the Supplementary information and
was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. Masayuki Saigusa (Biology) at
Okayama University, with help from undergraduate students in both their laboratories. The tardigrades
for the study were mainly collected by Mr. Taro Uozumi. 0.2 mm 0.2 mm Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs,
although slightly less energetically than other samples exposed for shorter times [40]. The resuscitated
organisms were shown to survive for at least 7 days following the exposure to high pressure
conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-
pressurization tardigrade, from which the still image is extracted, is shown in the supplementary
information and was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. Masayuki
Saigusa (Biology) at Okayama University, with help from undergraduate students in both their
laboratories. The tardigrades for the study were mainly collected by Mr. Taro Uozumi. Figure 2. One of the five tardigrades which could tolerate exposure to 7.5 GPa for 12 h in a dormant
(cryptobiotic) state, and then be revived by addition of water. The animal could move its legs, although
slightly less energetically than other samples exposed for shorter times [40]. The resuscitated organisms
were shown to survive for at least 7 days following the exposure to high pressure conditions [40]. A video documenting the remarkable recovery and swimming activity of this post-pressurization
tardigrade, from which the still image is extracted, is shown in the Supplementary information and
was obtained by Prof. Fumihisa Ono (Physics) in collaboration with Dr. 7. Compression on a Rapid Timescale
7. Compression on a Rapid Timescale Increased survival rates are recorded for wild type and pressure-adapted bacterial strains
at 2 GPa, and some bacteria have been reported to survive following exposure to shock pressures as
high as 78 GPa, with a rise time on the order of ns, and a pulse duration of µs [46,47]. Here, it becomes
important to consider the timescale of the mechanical relaxation response of the bacterial cell envelope. For compression timescales lower than approximately 1 ms the cell walls exhibit a solid-like “glassy”
response and this may protect the internal biochemical structures from the full effects of the shock [48]. Here, an upper pressure limit would be fixed by the fracture strength of the bacterial cell wall, that may
extend even into the megabar range (~100 GPa) for the short timescale events [45]. In contrast to the
bacterial results, shock studies on seeds and spores into the 1–5 GPa range result in lack or delay of
germination and altered behavioral or growth patterns [42,49–51]. The difference between unicellular
and organisms with a complex cellular structure is not known. The results lead to the hypothesis
that at least some simple organisms may survive interplanetary transport and delivery to a new host
via impact events providing the shock temperatures remain sufficiently low [52–54]. Finally, recent
work has established the production of amino acids via hypervelocity shock synthesis from precursor
molecules contained within cometary ices [55]. Those results lead to possibilities that creation of at
least some of the building blocks of life itself might have occurred during high pressure impact events. Life 2016, 6, 34 6 of 8 References 1. Melosh, H.J. The rocky road to panspermia. Nature 1988, 332, 687–688. [CrossRef] [PubMed] 2. Yayanos, A.A. Microbiology to 10,500 Meters in the Deep-Sea. Ann. Rev. Microbiol. 1995, 49, 777–805. [CrossRef] [PubMed] 2. Yayanos, A.A. Microbiology to 10,500 Meters in the Deep-Sea. Ann. Rev. Microbiol. 1995, 49, 777–805. [CrossRef] [PubMed] 3. Edwards, K.J.; Becker, K.; Colwell, F. The Deep, Dark Energy Biosphere: Intraterrestrial Life on Earth. Ann. Rev. Earth Planet. Sci. 2012, 40, 551–568. [CrossRef] 4. Gold, T. The deep, hot biosphere. Proc. Nat. Acad. Sci. USA 1992, 89, 6045–6049. [CrossRef] [PubMed]
5
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hild L J
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[PubMed] 5. Rothschild, L.J.; Mancinelli, R.L. Life in extreme environments. Nature 2001, 409, 1092–1101. [CrossRef]
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1998, 95, 6578–6583. [CrossRef] [PubMed] 7. Huber, J.A. Making methane down deep. Science 2015, 349, 376–377. [CrossRef] [PubMed] 8. Inagaki, F.; Hinrichs, K.-U.; Kubo, Y.; Bowles, M.W.; Heuer, V.B.; Hong, W.-L.; Hoshino, T.; Ijiri, A.; Imachi, H.;
Ito, M.; et al. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor. Science 2015, 349, 420–424. [CrossRef] [PubMed] 9. Paulino-Lima, I.G.; Pilling, S.; Janot-Pacheco, E.; de Brito, A.N.; Barbosa, J.A.R.G.; Leitão, A.C.; Lage, C.D.A.S. Laboratory simulation of interplanetary ultraviolet radiation (broad spectrum) and its effects on Deinococcus
radiodurans. Planet. Space Sci. 2010, 58, 1180–1187. [CrossRef] 10. Baker-Austin, C.; Dopson, M. Life in acid: pH homeostasis in acidophiles. Trend. Microbiol. 2007, 15, 165–171. [CrossRef] [PubMed] 1. Padan, E.; Bibi, E.; Ito, M.; Krulwich, T.A. Alkaline pH Homeostasis in Bacteria: New Insights. BBA Biome
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Onstott, T.C. Nematoda from the terrestrial deep subsurface of South Africa. Nature 2011, 474, 79–82. [CrossRef] [PubMed] 13. Schiffries, C.M.; Hazen, R.M. Why Deep Carbon? Rev. Miner. Geochem. 2013, 74, 1–6. 14. Kargel, J.S.; Kaye, J.Z.; Head, J.W.; Marion, G.M.; Sassen, R.; Crowley, J.K.; Ballesteros, O.P.; Grant, S.A.;
Hogenboom, D.L. Europa’s Crust and Ocean: Origin, Composition, and the Prospects for Life. 8. Conclusions Although the highest pressure to which organisms are known to be exposed on Earth does
not exceed 150 MPa, laboratory results indicate that at least a fraction of the population of bacteria
or complex life forms can survive exposure to pressures far above this threshold. The observed
effects depend strongly on the experimental conditions. Nevertheless, the data suggest that pressure
represents a limiting factor for life, albeit at values beyond those experienced on Earth. It raises
a number of questions, especially as life forms are found to survive pressures at which individual
cellular components are thought to be in a dysfunctional state. It also touches upon the very
fundamental question of how to define life and opens perspectives on the probability of life elsewhere
in the universe. Supplementary Materials: The referenced video is available online at http://www.mdpi.com/2075-1729/
6/3/34/s1. Acknowledgments: Our work on the topic of pressure as a limiting factor for biological survival has been
supported by grants from the Leverhulme Trust (UK) and the Deep Life program of the Deep Carbon Observatory
(Sloan Foundation, USA). Author Contributions: All authors contributed equally to the ideas represented in the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Icarus 2000,
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High Pressure Survival in Shewanella oneidensis MR-1 into the GigaPascal Pressure Range. Front. Microbiol. 2014, 5. [CrossRef] [PubMed] 25. Ball, P. Water as an Active Constituent in Cell Biology. Chem. Rev. 2008, 108, 74–108. [CrossRef] [Pu 26. Bove, L.E.; Klotz, S.; Strassle, Th.; Koza, M.; Teixeira, J.; Saitta, A.M. Translational and Rotational Diffusion in
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[PubMed] 30. References Picard, A.; Daniel, I.; Testemale, D.; Kieffer, I.; Bleuet, P.; Cardon, H.; Oger, P.M. Monitoring microbial
redox transformations of metal and metalloid elements inder high pressure using in situ X-ray absorption
spectroscopy. Geobiology 2011, 9, 196–204. [PubMed] 31. Foglia, F.; Hazael, R.; Simeoni, G.G.; Appavou, M.-S.; Moulin, M.; Haertlein, M.; Forsyth, V.T.; Seydel, T.;
Daniel, I.; Meersman, F.; et al. Water Dynamics in Shewanella oneidensis at Ambient and High Pressure using
Quasi-Elastic Neutron Scattering. Sci. Rep. 2016, 6, 18862. [CrossRef] [PubMed] 32. Colwell, F.S.; D’Hondt, S. Nature and Extent of the Deep Biosphere. Rev. Miner. Geochem. 2013, 75, 547–574. [CrossRef] 33. Zeng, X.; Birren, J.-L.; Fouquet, Y.; Cherkashov, G.; Jebbar, M.; Querellou, J.; Oger, P.; Cambon-Bonavita, M.-A.;
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012053. [CrossRef] 43. Gupta, R.K.; Das, S.K. Fracture resistance of sunflower seed and kernal to compressive loading. J. Food Eng. 2000, 46, 1–8. [CrossRef] 44. Ono, F.; Minami, K.; Saigusa, M.; Matsushima, Y.; Mori, Y.; Takarabe, K.; Saini, N.L.; Yamashita, M. Life of
Artemia under very high pressure. J. Phys. Chem. Solids 2010, 71, 1127–1130. [CrossRef] 5. Govers, S.K.; Aertsen, A. Impact of high hydrostatic pressure processing on individual cellular resuscita
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46. Hazael, R.; Fitzmaurice, B.C.; Foglia, F.; Appleby-Thomas, G.J.; McMillan, P.F. Bacterial survival following
shock compression in the GigaPascal range. Icarus 2016, submitted. 46. Hazael, R.; Fitzmaurice, B.C.; Foglia, F.; Appleby-Thomas, G.J.; McMillan, P.F. Bacterial survival following
shock compression in the GigaPascal range. Icarus 2016, submitted. 47. Burchell, M.J.; Mann, J.R.; Bunch, A.W. Survival of bacteria and spores under extreme shock pressures. Mon. Not. R. Astron. Soc. 2004, 1278, 1273–1278. [CrossRef] 48. Vadillo-Rodriguez, V.; Beveridge, T.J.; Dutcher, J.R. Surface Viscoelasticity of Individual Gram-Negative
Bacterial Cells Measured Using Atomic Force Microscopy. J. Bacteriol. 2008, 190. [CrossRef] [PubMed] 49. Mustey, A.; Leighs, J.A.; Appleby-Thomas, G.J.; Wood, D.C.; Hazael, R.; McMillan, P.F.; Hazell, P.F. The effect
of hydrostatic vs. shock pressure treatment of plant seeds. J. Phys. Conf. Ser. 2014, 500, 182025. [CrossRef] 49. Mustey, A.; Leighs, J.A.; Appleby-Thomas, G.J.; Wood, D.C.; Hazael, R.; McMillan, P.F.; Hazell, P.F. The effect
of hydrostatic vs shock pressure treatment of plant seeds J Phys Conf Ser 2014 500 182025 [CrossRef] 50. Burchell, M.J.; Mann, J.; Bunch, A.W.; Brand, P.F.B. Survivability of Bacteria in Hypervelocity Impact. Icarus
2001, 547, 545–547. [CrossRef] 51. Price, M.C.; Solscheid, C.; Burchell, M.J.; Josse, L.; Adamek, N.; Cole, M.J. Survival of yeast spores in
hypervelocity impact events up to velocities of 7.4 km·s−1. Icarus 2013, 222, 263–272. [CrossRef] 52. Beck, P.; Gillet, P.; El Goresy, A.; Mostefaoui, S. Timescales of shock processes in chondritic and martian
meteorites. Nature 2005, 435, 1071–1074. [CrossRef] [PubMed] 53. Horneck, G.; Stöffler, D.; Ott, S.; Hornemann, U.; Cockell, C.S.; Moeller, R.; Meyer, C.; de Vera, J.P.; Fritz, J.;
Schade, S.; et al. References Microbial Rock Inhabitants Survive Hypervelocity Impacts on Mars-Like Host Planets: First
Phase of Lithopanspermia Experimentally Tested. Astrobiology 2008, 8, 17–44. [CrossRef] [PubMed] 54. Fajardo-Cavazos, P.; Langenhorst, F.; Melosh, H.J.; Nicholson, W.L. Bacterial Spores in Granite Survive
Hypervelocity Launch by Spallation: Implications for Lithopanspermia. Astrobiology 2009, 9, 647–657. [CrossRef] [PubMed] 55. Martins, Z.; Price, M.C.; Goldman, N.; Sephton, M.A.; Burchell, M.J. Shock synthesis of amino acids from
impacting cometary and icy planet surface analogues. Nat. Geosci. 2013, 6, 1045–1049. [CrossRef] © 2016 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/). Cranfield University
CERES Research Repository https://dspace.lib.cranfield.ac.uk/ Staff publications (CDS) Cranfield Defence and Security Downloaded from CERES Research Repository, Cranfield University Pressure as a limiting factor for life Hazael, Rachael Hazael, Rachael
2016-08-17
Attribution 4.0 International Hazael, Rachael
2016-08-17
Attribution 4.0 International Hazael R, Meersman F, Ono F, McMillan P, Pressure as a limiting factor for life, Life, Vol. 6.
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Problem of Work on a High Energy Synchrotron Submitted to Large Radiation Dose
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IEEE transactions on nuclear science
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EUROPEAN ORGANIZATION FOR NUCLEAR RESEARCH EUROPEAN ORGANIZATION FOR NUCLEAR RESEARCH CERN/MPS/SM 75-1
26.2.1975 CERN/MPS/SM 75-1
26.2.1975 CERN/MPS/SM 75-1
26.2.1975 CERN/MPS/SM 75-1
26.2.1975 PROBLEM OF WORK ON A HIGH ENERGY SYNCHROTRON Paper to be submitted to 1975 Particle Accelerator Conference,
Washington, D.C. Paper to be submitted to 1975 Particle Accelerator Conference, 2. Effects of Radiation on the CPS Synchrotron Magnet Three main effects have to be considered: on magnet
laminated blocks, on main excitation coils, on pole face
windings.1,2,3,4,5.6,7,8. 2.1. Magnet laminated blocks. Each of the 100 CPS magnet
units consists of 10 laminated blocks made of 1.5 mm
steel sheets, glued together with araldite . Under radia-
diation and magnetic field, loose front sheets break
the araldite pole face windings already weakened by
radiation. After a series of breakdowns, we have estimated the
reliability f of the original magnet units as f = 1 - p,
where p is the breakdown probability. To do so we con
sider the radiation dose R (in rad) absorbed by the iron
at a specific point of the unit, always at the same lo
cation which is considered as the normalized reference
point on all units. This point (point A in Fig. 1) is
located at the entrance of the first block, 5 cm below If they are not handled too much, the original pole
face windings could withstand 109 rad. They are progres
sively exchanged by better ones, able to support 3.109
rad. SUBMITTED TO LARGE RADIATION DOSE R. Gouiran Paper to be submitted to 1975 Particle Accelerator Conference, Washington, D.C. PROBLEM OF WORK ON A HIGH ENERGY SYNCHROTRON
SUBMITTED TO LARGE RADIATION DOSE R. Gouiran
CERN, Geneva, Switzerland R. Gouiran
CERN, Geneva, Switzerland beam axis, which is the. nearest place to the proton
beam. This location is the most exposed and the weakest
region of the magnet unit. f ≈ - 0.9 log (R/8.5 108) f ≈ - 0.9 log (R/8.5 108) which means that there is no old unit in good condition
for doses above 8.5 108 rad, and that the reliability f
starts to decrease below 1 somewhere around 5.107 rad. Consequently the damaged steel blocks have to be
repaired, and the pole face winding sheets have to be
exchanged. 2.2. Main excitation coils. These aluminium water cooled
coils are insulated by fiber glass and mica ribbons
moulded in araldite. In some places the araldite is des
troyed by radiation and the insulation is warranted only
by the mica laminations, which shows the necessity to
always provide a mica barrier in any araldite insulation
submitted to radiation. The coils are no longer water
proof and special care has to be taken to avoid water
leaks. Some coils will have to be repaired or exchanged
after a dose of around 8.108 rad, but where they are lo
cated, these coils receive 4 times less radiation than
the magnet block and the pole face windings at the ref
erence point A. 2.3. Pole face windings. These windings are moulded in
an araldite sheet fitting the magnet pole profile and
fixed by stainless steel belts (Fig. 1). Due to the CPS
structure, 400 of such sheets, each 2.20 m long, provide
the necessary multipole field corrections in order to
adjust the magnetic field and to compensate iron satura
tion. Araldite and connecting cable insulation become
hard and brittle. These pole face winding sheets have to
be exchanged gradually, in particular where the magnet
steel blocks have to be repaired, because they do not
stand up very well to dismounting. 1. Introduction Running a high energy synchrotron towards higher
and higher proton intensity begets series of problems
related to equipment damages and repairs and to works in
radiation areas. Maintenance should therefore become an
integral part of the organization and some methods
should be developed for evaluating and predicting irra
diation of personnel and equipment. Firstly, organic
components have to be surveyed and eventually exchanged;
secondly, work done by the staff in radioactive areas
should be strictly controlled and minimized. To reach
these goals radiation and radioactivity should not only
be measured but also predicted years in advance. Radia
tion doses are computed, measured and predicted accor
ding to the proton loss pattern around the accelerator. A correlation scheme of doses versus accelerator opera
tion is then deduced to predict the dose on any sensi
tive part of the accelerator. In the same way a radio
active pattern is computed, measured and accurately pre
dicted in terms of dose rate. Work on the accelerator is
studied in detail in order to forecast its duration and
the corresponding dose absorbed by the staff. With these
methods, it is easy now not only to predict the fate of
the equipment and the doses to be received by the staff,
but also to organize the work accordingly and possibly
to program the accelerator operation (proton intensity
and proton uses like target operation, dumping, extrac
ting etc.) following these predictions. The reliability f (only between 0 and 1) is given 3. Radiation Measurements 3.1. Radiation dosimetry. After several years of study,
calibration and intercomparison between different dosi
meters, we decided to use only small glass dosimeters
(Schott glass PDG11, 1.5 and 5 mm thick, coloured by
radiation and measured via a Beckmann spectrophotometer)
Of course these dosimeters are not very accurate and
fade rapidly9 but they are very easy to handle and mea
sure, and all we ask for is a good reproducibility. All
radiation measurements and equipment surveying should be
made with the same dosimeters and the same methods, be
cause we need accuracy only for relations between cause
and effect, not for absolute values. Fig. 1. Cross section of magnet pole profile, with
vacuum chamber and pole face windings. The relation between the measured glass absorbence
A and the apparent dose received R (in rad) looks more
or less like Fig. 1. Cross section of magnet pole profile, with
vacuum chamber and pole face windings. 1. (1) tracting, injecting, dumping etc.). e k
λ is a constant for a given wavelength (kλ = where k
λ is a constant for a given wavelength (kλ =
0.377 at λ = 510 nm which is the maximum sensitivity
peak, and kλ = 0.245 at λ = 330 nm which is the minimum
of sensitivity just before the UV peak). where k
λ is a constant for a given wavelength (kλ =
0.377 at λ = 510 nm which is the maximum sensitivity
peak, and kλ = 0.245 at λ = 330 nm which is the minimum
of sensitivity just before the UV peak). So it is possible now to get a fairly accurate pre
diction of the radiation pattern around the accelerator
when we know: a) the sharing of accelerated protons between acceler
ator users (in %) The fading of the absorbence A^ at day t compared
to the absorbence A2 two days after irradiation, is
approximatively b) the efficiency (in proton losses) of each proton
use b) the efficiency (in proton losses) of each proton
use At - A2 1.036 [1 + 0.115 log 1/t]
(2) At - A2 1.036 [1 + 0.115 log 1/t] (2) c) the total accelerated protons c) the total accelerated protons The position of the dosimeter on the magnet unit
is carefully noted in order to normalize, via a correc
tion factor, its reading to the dose received at the
reference point (see Fig. 4. Radioactivity Measurements Though radioactivity does not jeopardize the syn
chrotron future, it is nevertheless more important than
radiation on human grounds because it is directly bound
to the dose received by the maintenance staff whose duty
is to give a good tool to physicists. It is of prime
importance to study the impact of the machine induced
activity on the accelerator maintenance in order to
guarantee that the synchrotron could be safely operated
in the future, and also in order to schedule proton uses
for minimizing radioactivity in the ring during main
tenance shut-down periods Another source of radiation is more diffuse and
difficult to correlate to any operation, but it remains
more or less proportional to the number of accelerated
protons. We have called it the "banal dose", and it
follows a very regular "wriggle" pattern: it is 2.5
times higher in a focusing section than in a defocusing
one. 4.1. Dose rate measurements. In order to normalize pre
dictions, we consider always dose rates measured at
40 cm from the vacuum tubes of the hundred straight sec
tions between magnets, after two days of machine stop
(because this survey is done as a routine by the Health
Physics Group on the Monday morning following the acce
lerator stop on the Saturday evening, every four weeks). As a general rule the machine is stopped before week
ends or official holidays in order to leave the maximum
possible length of cooling time before starting works
inside the ring tunnel. The radiation azimuthal pattern shows a series of
hot and quiet regions around the ring. At the end of
1974, the most exposed reference point had received
4.109 rad (near internal target 1), but the magnet
unit at this location has been exchanged three times ! The less exposed reference point had received 2.107 rad
in the meantime. The sum of the hundred doses at the
reference points of the 100 magnet units was 1.5 1010
rad at that time. This sum is expected to reach 6.1O10
rad in 1984. A correction factor is then used to know the dose
rate at contact, and which is 7 on average, although it
could reach 14 near a target. 4.2. Radioactivity predictions. 3. Radiation Measurements 1), where it is not possible
to install anything. Dosimeters are changed and mea
sured at regular intervals. d) the dose pattern around the ring, in rad per inter
acting proton in the radiation sources used in a
given operation. e) the "banal non correlated dose" which is always
statistically present during any acceleration. 3.2. Loss and radiation pattern. Magnet irradiation
comes from different origins. Protons lost in an in
ternal target; an extraction septum (magnetic or elec
trostatic) , or an injection deflector beget high irra
diation on the following magnet unit and the two or
three subsequent units. However, because of the beta-
tronic oscillations and the closed orbit deviations, we
find rather large beam losses on some specific hot
spots far away from the original radiation sources. It
is possible to correlate directly the doses received by
some magnet units to a given proton use in the acceler
ator and to plot a radiation pattern correlated to a
loss pattern. This correlation is made by statistical
comparison between dosimeter readings on one hand, and
by a careful analysis of the CPS beam loss monitors
around the ring on the other hand, for each specific
accelerator operation. This method is used at the CERN synchrotron and it
allows to predict the fate of a sensitive equipment
years in advance and consequently to programme exchange
or repairs in due time7. years in advance and consequently to programme exchange
or repairs in due time7. 5. Dose Received by Working Staff The knowledge of the dose rate repartition is not
sufficient for a good estimation of the probable dose
to be received by any worker. The dose received at loca
tion i is the dose rate Di given by (4) multiplied by
the number of hours spent for the job and also by three
reduction factors, which we have called respectively13:
a) the "delay factor" fd , b) the "presence factor", f
and c) the "equipment factor", fe 4.3. Radioactivity pattern. As for irradiation the azi
muthal pattern shows hot regions where 1 rem/h at 40 cm
after two days could be observed (near targets, extrac
tion or injection septa etc.) and quiet regions where
the dose rate is often below 5 mrem/h, for the same
conditions. a) a) the delay factor comes from the fact that jobs on
very active pieces is delayed as much as possible,
and is done d days after the start of the machine
shut-down where Di has been measured or estimated. As a general rule we take fd ≃ 2/log [1 + (200/d)]
which is a fair enough approximation. We note that
fd ≃ 1.5 already after 10 days. It is also possible to correlate directly the dose
rate in some straight sections to a given proton use in
the accelerator and to plot a radioactive pattern cor
related to a loss pattern for each specific accelerator
operation. We also consider the more diffuse radio
activity, difficult to correlate to any operation and
which remains more or less proportional to the number
of accelerated protons, and what we have also called
the "banal dose rate". As the radiation, it follows the
regular "wriggle" pattern, being 2.5 times higher in a
focusing section than in a defocusing one. b) the presence factor is due to the fact that for a
one hour job the worker does not stay quiet at
40 cm from the vacuum pipe during this one hour! According to the job he stays at various distances
and fp should take account of this fact. This
reduction factor is specific for a given well
known standard job and should be accurately mea
sured for any typical intervention. It is between
1 and 5, generally around 4. 4. Radioactivity Measurements b) to estimate an "old" activity as given by a mean
operation during the last two years preceding this
last monthly period (as long as the accelerated
proton intensity has not varied too much during
the last two years) and which is supposed to cool
down during the last month according to the
Sullivan-Overton formula (3). This method is used at the CERN synchrotron and it
allows to predict the dose rate at any location where
people have to work, months in advance, and consequently
to programme the maintenance and the accelerator opera
tion in order to minimize, and to maintain as low as
possible, the dose received by any staff member. This "two components" simplification does not
introduce errors larger than 10 to 15%. For more accu
racy the "old" radioactivity can be split into two
yearly slices if necessary. These two components (fresh
and old) contribute nearly fifty-fifty to the final
dose rate. 4. Radioactivity Measurements Several authors13,14,15
16,18,19 have tried to define the dose rate measured at
the contact of a thick and isolated iron piece after 30
days of continuous irradiation followed by 1 day of
cooling, in rem/h per (stars/cm3s). This value, known
as ω30,1 has been taken here equal to 8.107. 3.3. Radiation prediction. Assuming that the reference
point is radially 5 cm below beam axis and longitudi
nally between 1 to 3 m from a direct radiation source
(targets, septa etc.) it is possible to compute theore
tically the absorbed dose in the iron and to compare
with the experimental results10,11,12, per proton lost
in the radiation source. Though it varies from case to
case, it is not too far from 2.5 10-10 rad per inter
acting proton in the radiation source. Then for each
proton lost, a correlation pattern of irradiation is
given around the accelerator. The free cooling follows fairly well the Sullivan-
Overton formula17 which says that the dose rate D(t)
after t days cooling following T days of continuous
irradiation is D(t) = k log [1 + (T/t)]
(3) D(t) = k log [1 + (T/t)] (3) The exact proton losses are then determined by the
knowledge of the various efficiencies of the accele
rator operation for each proton use (targetting, ex where k is just a proportional factor. 2. ton per second for a target, and 1.7•10-11 for a septum, Finally the fundamental formula we use for radio
activity prediction in the straight section i is: As for radiation the exact proton losses are then
determined by the knowledge of the various efficiencies
of the accelerator operation for each proton use (tar
getting, extracting, injecting, dumping etc.). As for radiation the exact proton losses are then
determined by the knowledge of the various efficiencies
of the accelerator operation for each proton use (tar
getting, extracting, injecting, dumping etc.). (4) Di(T,t) = Ki•Ii(p/s)•log[l + (T/t)]
(4) Di(T,t) = Ki•Ii(p/s)•log[l + (T/t)]
(4) Where Ii(p/s) is the average quantity of proton per
second, interacting in the source of radiation creating
the activity at location i during T days, k
i is a con
stant for each location to be defined experimentally,
and t is the cooling time in days. 4. Radioactivity Measurements Where Ii(p/s) is the average quantity of proton per
second, interacting in the source of radiation creating
the activity at location i during T days, k
i is a con
stant for each location to be defined experimentally,
and t is the cooling time in days. So it is possible now to get a fairly accurate
prediction of the radioactive pattern around the acce
lerator when we know a) the sharing of accelerated protons between acce
lerator users (in %) and the time schedule of this
use This formula shows that the radioactive cooling
becomes very slow after 4 months, where a factor 4 has
been already obtained, compared to the two days stan
dard cooling time. b) the efficiencies (in proton losses) of each proton
use The CPS synchrotron operation is divided in monthly
periods and this feature allows an important simplifi
cation for radioactivity prediction, the so-called "two
components" theory13: it says that, in order to know the
radioactivity at a given time, it is sufficient c) the total accelerated protons and the correspon
ding time schedule c) the total accelerated protons and the correspon
ding time schedule d) d) the dose rate pattern around the ring, in rem/h
per interacting proton per second in the radiation
source used in a given operation d) the dose rate pattern around the ring, in rem/h
per interacting proton per second in the radiation
source used in a given operation a) to consider in detail the last monthly period
giving a "fresh" radioactivity and a) to consider in detail the last monthly period
giving a "fresh" radioactivity and e) e) the "banal non correlated dose rate" which is al
ways statistically present after each acceleration. e) the "banal non correlated dose rate" which is al
ways statistically present after each acceleration. b) to estimate an "old" activity as given by a mean
operation during the last two years preceding this
last monthly period (as long as the accelerated
proton intensity has not varied too much during
the last two years) and which is supposed to cool
down during the last month according to the
Sullivan-Overton formula (3). 6. An Example We just try to figure out a very simple but fic
tive situation. Suppose that 1.7•1019 proton have been
accelerated per year with 10% allocated to target 1
whose efficiency is 80%. At this place of equation
(4) is 6.7•10-12 rem/h per interacting proton. 8. R. Gouiran, Groupe de travail sur l'aimant CPS
"Rapport sur l'état de l'aimant et son évolution. Mesures à prendre pour assurer sa survie",
MPS/SM/Note 74-13. 9. R. Gouiran, "Fading of glass dosimeters - prelim
inary results", MPS/SR/Note 73-39. We apply the two component theory. For the "old"
activity, the term I(p/s) will be 1.7•1019 x 10-1.2/
570.24•3600 (for 670 days), that is to say 5.8•1011. So after one month, the "old" components will contri
bute for 6.7•10-12.5.8•1011.log [1 + (670/30)] ≈ 0,531
rem/h. 10. R. Gouiran, "Taux d'irradiation des unités de 1'ai
mant. Statistiques et prévisions", MPS/MU/Note/EP
72-15 Rev. 11. J. Ranft, J.J. Routti, "Fluka and Magka, Monte-Carlo
program for calculating nucleon-meson cascades in
cylindrical geometries", CERN Lab.II RA/71-4. 12. J. Ranft, J.J. Routti, "Hadron cascade calculations
of angular distribution of integrated secondary par
ticles fluxes from external targets and description
of program FLUKU", CERN Lab.II RA/72-8. The "fresh" components will be (one month is one
tenth of a accelerator year and corresponds to 24 days
of acceleration): 13. R. Gouiran, "La radioactivité de l'aimant du CPS et
son influence sur la maintenance de l'anneau. Statistiques et prévisions", CERN/MPS/SR 73-5. 14. J. Ranft, K. Goebel, "Estimation of induced radio
activity around high energy accelerators from hadro
nic cascade star densities obtained from Monte-Carlo
calculations". 300 GeV/RAD. Note/70-24. So Di at 40 cm after 2 days stop would be 1.14 rem/h
at this place. So Di at 40 cm after 2 days stop would be 1.14 rem/h
at this place. Suppose a man has to change the vacuum section
near this target, but 10 days after the shut-down
start. So fd = 1.5. We know by past experience that for
this job fp = 4. As nothing has been removed yet,
fe = 1. The work lasts half an hour, so this worker
would receive 1.14 x 0.5/1.5•4•l ≃ 100 mrem. We should
not forget to add, to this dose, the "general" dose of
§ 5. which is, for 10 days "walking" in the ring,
TAR 10.3.5•10-3 = 175 mrem already, for TAR = 5 rem/h. 7. Conclusions The survey of irradiation and radioactivity is pos
sible by means of some accurate methods. It is absolu
tely necessary in order to not only organize maintenance
and repairs but also to protect working staff against
undue radioactive doses. Such surveys should become an
integrated part of any particle accelerator management
and they should be able to influence accelerator opera
ting schedule. (5) D = Di•log [1 + (200/d)]/2•fe•fp
(5) Unfortunately we should also add to these doses
received for a specific well known job, a general dose
received during the stay in the ring, outside the spe
cific job time. This dose comes from multiple small
tasks, going and coming, unnecessary stays in radio
active areas (the "talk-dose"!) etc. We should like to thank all the persons who have
contributed to this work, in particular Messrs. G. Munday, C. Germain, G. Plass, M. Nielsen, E. Barbe,
M. Höfert, A. Burlet, M. Van de Voorde, P. Valloton, the
PS Operating Staff and Health Physics Group. We should like to thank all the persons who have
contributed to this work, in particular Messrs. G. Munday, C. Germain, G. Plass, M. Nielsen, E. Barbe,
M. Höfert, A. Burlet, M. Van de Voorde, P. Valloton, the
PS Operating Staff and Health Physics Group. It is proportional to the time spent in the acce
lerator tunnel and is: TAR(rem/h).3.5•10-3 rem per
working day. (TAR is the sum of the 100 dose rate mea
sured at the standard 100 points as described in § 4.3. Its value at present is around 5 rem/h). 5. Dose Received by Working Staff The sum of the hundred dose rates measured at
40 cm after two days on each of the 100 straight sec
tion is called TAR ("Total Activity Ring"), and it
depends strongly upon the last month operation. In the
last two years it was around 5 rem/h and we expect that
it could reach 10 rem/h in 1984. c) the equipment factor comes when some active equip
ment has been removed before the job. When vacuum
chamber and straight section equipment have been
removed, fe is about 2. When equipment is removed
but not the vacuum chamber, fe becomes 1.7. We determine for each straight section i the
coefficient Ki of equation 4, for a given operation
leading to I (proton per second) lost in any specific
operation. Though it varies from case to case, this
is not too far from 6.7•10-12 rem/h per interacting pro 3. So the dose received by a person working for one
hour on location i, d days after the dose rate Di has
been estimated according to equation (4) is: References 1. M.H. Van de Voorde, "Effects of radiation on mate
rials and components", CERN 70-5. 2. K. Goebel (editor), "Radiation problems encountered
in the design of multi-GeV for research facilities"
CERN 71-20. For example, a foreman just walking in the tunnel
for a 20 days shut-down, without getting close to the
magnet, will recive at present 0.350 rem, which is not
negligible when it has to be added to more specific
doses. (At present we try to limit the total dose re
ceived during a 6 weeks annual shut-down at 2 rem per
person). 3. M.H. Van de Voorde, "Improvements on the properties
of organic materials in accelerator magnets",
ISR/MA/70-17. 4. M.H. Van de Voorde, "The effect of nuclear radiation
on the electrical properties of epoxy resins", CERN
68-13. The method described above has been used and is
very accurate as well as useful for staff dose survey,
in particular for people working on the vacuum system
and on the magnets themselves. 5. M.H, Van de Voorde, "Action des radiations ioni
santes sur les résines époxides", CERN 70-10 (1970). 5. M.H, Van de Voorde, "Action des radiations ioni
santes sur les résines époxides", CERN 70-10 (1970). 6. R, Gouiran, "Irradiation of CERN PS magnet steel
blocks", Note MPS/MU/EP 72-14, RG-1d. p
,
(
)
6. R, Gouiran, "Irradiation of CERN PS magnet steel
blocks", Note MPS/MU/EP 72-14, RG-1d. 7. R. Gouiran, "CPS High intensity running, implication
on magnet irradiation and radioactivity", MPS/SR/
Note 73-43. 6. An Example So the total absorbed dose will then be 275 mrem for
this simple example. 15. M. Barbier, North Holland Publ., "Induced radio
activity". 16. J. Ranft, J.T. Routti, "Fluka and Magka", Monte-
Carlo program for calculating nuclear meson cascades
in cylindrical geometries", CERN Lab.II RA/71-4. 17. A.H. Sullivan, T.R. Overton, "Time variation of the
dose rate from radioactivity induced in a high
energy accelerator", Health Physics, II, 1101-1105
(1965). 18. K. Goebel, M. Nielsen, "Prediction of future radio
activity in the PS ring", HP-70-90. 19. M. Höfert, M. Nielsen, J.M. Hanon, "Prediction of
future radiation levels from induced radioactivity
around the proton synchrotron and in regions near
external targets ...", HP-73-124. 4.
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Crenças Epistemológicas e o Processo de Aprendizagem da Homeopatia
|
Revista Brasileira de Educação Médica
| 2,015
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cc-by
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I Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. PALAVRAS-CHAVE –– Homeopatia;
–– Ensino;
–– Aprendizagem;
–– Educação Médica. Crenças Epistemológicas e o Processo de
Aprendizagem da Homeopatia
Epistemological Beliefs and the Learning
Process of Homeopathy Elizabeth Pinto Valente de SouzaI
Mauricio Abreu Pinto PeixotoI Elizabeth Pinto Valente de SouzaI
Mauricio Abreu Pinto PeixotoI RESUMO Observou-se em médicos homeopatas uma prática clínica em desacordo com seu discurso sobre o
modelo homeopático, baseado em racionalidade própria. Construiu-se a hipótese de que as crenças
epistemológicas modulam a tradução do conhecimento teórico para sua aplicação clínica. Crenças
epistemológicas são convicções individuais relativas ao conhecimento e a sua aquisição, e atuam como
mediadores cognitivos. Objetivou-se identificar a relação existente entre as crenças epistemológicas de
médicos egressos do curso de formação de especialista em Homeopatia e a dificuldade encontrada na
incorporação do modelo médico próprio dessa especialidade, determinando práticas médicas distintas. Foram realizadas entrevistas semiestruturadas com 14 médicos homeopatas, e o material foi subme
tido à análise de conteúdo, relacionando as racionalidades presentes no discurso do entrevistado aos
tipos de crenças epistemológicas. Sugere-se relação entre as crenças epistemológicas e a prática médica
dos egressos a explicar a dificuldade observada na apreensão do modelo homeopático. A compreensão
dessa interdependência, tanto pelo docente como pelo aprendiz, pode propiciar uma conscientização
sobre o processo de ensino-aprendizagem e orientar o aproveitamento dos estudos e sua aplicação
prática em Homeopatia. INTRODUÇÃO Na formação de médicos homeopatas na Escola Kentiana do
Rio de Janeiro (EKRJ), observou-se uma cisão entre a compre
ensão teórica do modelo homeopático e sua aplicação clínica. Tais profissionais, formados há pelo menos dois anos (exigên
cia da Associação Médica Homeopática Brasileira) com até 5,
10, 15, 20 ou mais anos de exercício, compartilhavam pelo me
nos dois pontos: o interesse pela Homeopatia e a dificuldade
característica do processo de aprendizagem dessa especialida
de – a apreensão do modelo próprio da Homeopatia, distinto
do que vige na biomedicina. Apesar de produzirem discurso compatível com a racio
nalidade homeopática, prescreviam medicamentos homeopá
ticos subordinados a uma perspectiva da entidade nosológica,
própria da racionalidade biomédica. O estudo das CE vem abrindo possibilidades para a com
preensão dos processos cognitivos. As CE têm sido uma área de
crescente interesse para psicólogos e educadores, que sugerem
que elas podem se conectar com outras crenças disciplinares e,
além disso, ligar-se a outras construções em cognição e motiva
ção. Supõe-se que seu estudo possa renovar as abordagens cog
nitivas, tais como a teoria da mudança conceitual ou mental3. Se na biomedicina o foco está na entidade nosológica, na
Homeopatia a atenção dirige-se à representação individual e
pessoal do doente, determinando racionalidades distintas1. Assim, se na biomedicina a terapêutica está voltada para a en
tidade nosológica, mesmo que modulada por características
individuais, na Homeopatia ela é dirigida pelo conjunto das
expressões sintomáticas de um dado paciente. Roex e Degryse4 listam três argumentos pertinentes a este
estudo em defesa da importância das CE na prática médica. Em primeiro lugar, apontam o crescimento exponencial da
literatura médica, que representa para o profissional um de
safio na aquisição do conhecimento novo. E, neste sentido, a
consciência sobre os critérios de aprendizado é fator modula
dor de relevo. Em segundo lugar, o fato de que em medicina
dificilmente os problemas são simples e claros. Ao contrário,
geralmente comportam diversas possibilidades diagnósticas
e terapêuticas. E é nestes contextos pouco estruturados que
as CE afloram com maior relevância, modulando mais forte
mente as decisões clínicas. E, por último, o fato de que o co
nhecimento científico precisa ser considerado na perspectiva
do seu contexto de produção e aplicado segundo as condições
específicas de cada situação. Essa distinção se expressa também no significado do que
seja o medicamento, diferente nas duas racionalidades mé
dicas. ABSTRACT A clinical practice was observed among homeopathic doctors which was at odds with their defence
of the homeopathic model based on its rationale. It was hypothesized that epistemological beliefs mo
dulate the translation from theoretical knowledge to its clinical application. Epistemological beliefs
are individual convictions related to knowledge and its aquisition and which act as cognitive media
tors. The objective of the study was to identify the existing relationship between the epistemological
beliefs of medical doctors who had completed their specialization in Homeopathy and the difficulty
experienced in incorporating the homeopathic medical model, resulting in distinct medical practices. Semi-structured interviews were conducted with fourteen homeopathic doctors, and the material un
derwent content analysis, relating the lines of reasoning argued by each interviewee to the types of
epistemological beliefs. A relationship is suggested between the epistemological beliefs and the medical
practice of these doctors which explains the observed difficulty in grasping the homeopathic model. The understanding of this interdependence, by both teacher and student, could raise awareness of
the teaching-learning process and lead toward better results in both the learning and the practice of
Homeopathy. Recebido em: 24/09/2013
Reencaminhado em: 03/02/2014
Aprovado em: 10/03/2015 218 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto Os estudos sobre as CE foram desenvolvidos por William
Perry2 e publicados em 1970. Foram realizados na Universida
de de Harvard para investigar por que alguns alunos se mos
travam desorientados frente à multiplicidade de conceitos e a
uma concepção relativizante do conhecimento, enquanto ou
tros ficavam à vontade em meio a um universo científico que
considerava dimensões contextualizadas e idiossincráticas do
conhecimento. Observaram-se posições que variavam do ab
solutismo radical à progressiva relativização do saber. O es
tudo permitiu compreender como os alunos interpretam suas
experiências educacionais. Assim a questão que se apresenta
é: o aluno, ao ser capaz de revisar suas noções de conhecimen
to, também seria capaz de mudar a forma de obtê-lo? INTRODUÇÃO Se o que tratamos é uma entidade nosológica, então o
medicamento se relaciona com ela de modo direto e recíproco,
isto é, para determinada doença cabe determinado medica
mento (ou categoria de fármaco) e vice-versa. Por outro lado,
na Homeopatia, ao considerar um conjunto de variáveis pes
soais (que incluem a entidade nosológica), compreende-se que
não existem doenças, mas doentes. Se, então, o que é tratado
é o doente, quebra-se a relação direta e recíproca entre fárma
co e doença. Como consequência, para pessoas com a mesma
entidade nosológica é possível administrar medicamentos di
ferentes. Na perspectiva do processo educacional, a cisão citada
sugere que, independentemente da razão para isto, os alunos
tiveram dificuldade de mudar suas visões quanto à racionali
dade fundante de suas práticas. Este último argumento é particularmente relevante na
medida em que o objetivo da pesquisa realizada foi identificar
as crenças epistemológicas dos médicos formados pelo curso
de Homeopatia da EKRJ sobre a natureza do conhecimento e
do saber homeopático, identificando eventuais relações com
a racionalidade médica que dirige a prática médica eleita por
eles, considerando o modelo proposto na escola. Assim, a pergunta que norteou a pesquisa foi: por que os
profissionais pós-graduados em Homeopatia têm dificuldade
na aplicação de seu modelo teórico? Na pesquisa realizada, trabalhou-se com a hipótese de
que as crenças epistemológicas (CE) dos alunos influenciaram
na aprendizagem do modelo proposto na EKRJ e, consequen
temente, na prática adotada por eles. As CE são convicções in
dividuais relativas ao conhecimento e à sua aquisição e, como
tal, orientam e condicionam a ação. Sujeitos e métodos A Homeopatia era
relacionada a outros saberes, seja por opção eletiva,
seja por necessidade. Aqui se inclui o profissional que
em seu manejo clínico do caso atém-se ao raciocínio
homeopático, fazendo uso das noções de totalidade,
permitindo-se, assim, utilizar medicações tanto
homeopáticas como não homeopáticas. Certeza: Refere-se a mutabilidade do conhecimento
Fixo
O conhecimento é absoluto. O profissional demonstra
em seus conceitos teóricos e práticos ligados à
Homeopatia, ideias próximas ao modelo biomédico
que praticava antes de ter cursado a EKRJ. Essas
concepções são ligadas à ideia de doença como
entidade clínica, patologia, visão antropológica
mecanicista e divisão. Evolutivo
O conhecimento é mutável. O profissional apresenta
ideias de totalidade relativas ao modelo médico
homeopático ensinado na EKRJ. Processo do Saber: maneira pela qual alguém chega ao saber
Fonte: Refere-se à origem do conhecimento. Onde se situa a autoria
do conhecimento. Externa
O profissional referiu-se de forma restrita e com
regularidade somente aos textos, cursos e mestres
da especialidade, estando ausentes referências à sua
própria participação nas assertivas Quadro 1
Critérios de classificação dos entrevistados segundo
suas Crenças Epistemológicas
Natureza do Conhecimento: é o que cada um acredita ser o
conhecimento
Simplicidade: Refere-se à estrutura do conhecimento A racionalidade médica utilizada pelos sujeitos foi identi
ficada segundo o grau de aderência a dois conjuntos de cate
gorias: as concernentes à racionalidade médica homeopática1
e as relativas à racionalidade médica ocidental contemporâ
nea, a biomedicina5. A racionalidade médica homeopática se expressa em cinco
dimensões – cosmologia, doutrina médica, morfologia, dinâ
mica vital, sistemas diagnósticos e de intervenção terapêutica1. Nesta perspectiva, quando se estabelece a concepção homeo
pática do homem e se refere à doença como desequilíbrio da
energia vital, ressalta-se a totalidade alterada do indivíduo ex
pressa nos sintomas que a compõem. Por isto, define-se o con
ceito de cura homeopática como a diminuição da suscetibilida
de do sujeito a adoecer, pelo reequilíbrio de sua energia vital. Discreto
O conhecimento é concreto, composto de fatos
conhecidos. O profissional expressa o conhecimento de
modo isolado, separado, excludente (ou isso ou aquilo),
sem conexões com outros conhecimentos ou atuações
médicas e denotando dificuldade de diálogo entre essas
instâncias. Entende a Homeopatia como a única forma
terapêutica de escolha, independentemente do contexto
em que atua ou das características do paciente; ou ainda
quando separava de forma rígida os casos em que
aplicava os procedimentos homeopáticos daqueles em
que fazia uso da alopatia. Sujeitos e métodos A amostra incluiu médicos de ambos os sexos. A faixa etária
variou de 30 a 62 anos, tempo de formação em Medicina de
cinco a 38 anos e tempo de prática homeopática entre um e 26
anos. Todos os médicos exerciam sua prática no Rio de Janeiro. REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
219 REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
219 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto das: a natureza do conhecimento, que se subdivide em certeza
e simplicidade do conhecimento; e a natureza do saber, que
abarca a fonte do conhecimento e a justificação por meio dele. Os critérios de classificação dos entrevistados podem ser con
sultados no Quadro 1. Nesse quadro, identificamos os valores
atribuídos às unidades de sentido extraídas dos discursos dos
entrevistados. Cada uma das dimensões das crenças episte
mológicas foi relacionada ao tipo de racionalidade presente, à
possibilidade de interação com outros conhecimentos e espe
cialidades, à forma de construção do conhecimento e às justi
ficativas para validação do saber homeopático. Para a construção do corpus de análise, foram realizadas
14 entrevistas semiestruturadas, número definido pelo critério
de saturação das respostas dos entrevistados. O período de
coleta foi de quatro meses, tendo ocorrido entre maio e setem
bro de 2010. As entrevistas foram gravadas e tiveram duração
média de 20 a 35 minutos. Para orientação das entrevistas, construiu-se um roteiro
de 12 perguntas abertas que buscavam identificar as concep
ções do entrevistado sobre o fenômeno da doença, o signifi
cado da Homeopatia e sobre sua prática clínica. As respostas
permitiram identificar tanto a racionalidade fundante de sua
prática, como suas crenças epistemológicas. Quadro 1
Critérios de classificação dos entrevistados segundo
suas Crenças Epistemológicas
Natureza do Conhecimento: é o que cada um acredita ser o
conhecimento
Simplicidade: Refere-se à estrutura do conhecimento
Discreto
O conhecimento é concreto, composto de fatos
conhecidos. O profissional expressa o conhecimento de
modo isolado, separado, excludente (ou isso ou aquilo),
sem conexões com outros conhecimentos ou atuações
médicas e denotando dificuldade de diálogo entre essas
instâncias. Entende a Homeopatia como a única forma
terapêutica de escolha, independentemente do contexto
em que atua ou das características do paciente; ou ainda
quando separava de forma rígida os casos em que
aplicava os procedimentos homeopáticos daqueles em
que fazia uso da alopatia. Relativo
O conhecimento é contingente. Sujeitos e métodos Para a identificação da racionalidade biomédica, fez
-se uso dos conceitos de Camargo Jr.5 Nesta racionalidade, a
cosmovisão está implícita numa visão analítico-mecanicista
na qual os fenômenos são considerados por uma causalidade
linear. A morfologia vital e a anatomia humana são baseadas
na fisiologia e anatomia clássica, onde os sistemas incluem
aparelhos, órgãos, tecidos e células dentro do contexto do nor
mal e do patológico. A teoria das doenças sustenta as entida
des clínicas como fixas, traduzidas em lesões objetiváveis que
devem ser investigadas e combatidas. O sistema diagnóstico
engloba a anamnese, a semiologia, o exame físico e os exames
complementares. A terapêutica está relacionada à prevenção,
aos procedimentos cirúrgicos, se necessário, e aos medicamen
tos que devem combater os agentes causadores da doença. vo
O conhecimento é contingente. A Homeopatia era
relacionada a outros saberes, seja por opção eletiva,
seja por necessidade. Aqui se inclui o profissional que
em seu manejo clínico do caso atém-se ao raciocínio
homeopático, fazendo uso das noções de totalidade,
permitindo-se, assim, utilizar medicações tanto
homeopáticas como não homeopáticas. Certeza: Refere-se a mutabilidade do conhecimento
Fixo
O conhecimento é absoluto. O profissional demonstra
em seus conceitos teóricos e práticos ligados à
Homeopatia, ideias próximas ao modelo biomédico
que praticava antes de ter cursado a EKRJ. Essas
concepções são ligadas à ideia de doença como
entidade clínica, patologia, visão antropológica
mecanicista e divisão. Evolutivo
O conhecimento é mutável. O profissional apresenta
ideias de totalidade relativas ao modelo médico
homeopático ensinado na EKRJ. Processo do Saber: maneira pela qual alguém chega ao saber
Fonte: Refere-se à origem do conhecimento. Onde se situa a autoria
do conhecimento. Externa
O profissional referiu-se de forma restrita e com
regularidade somente aos textos, cursos e mestres
da especialidade, estando ausentes referências à sua
própria participação nas assertivas Já as crenças epistemológicas foram identificadas com
base na classificação proposta por Hofer e Pintrich3. Tais au
tores afirmam que as crenças epistemológicas apresentam
dimensões múltiplas, a saber, crenças sobre a natureza do
conhecimento e do processo do saber. Sujeitos e métodos Essas duas áreas são
propostas como centrais para as teorias epistemológicas indi
viduais, cabendo a cada uma delas outras duas, o que forma
quatro dimensões de teorias epistemológicas, assim agrupa REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
220 REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA 39 (2) : 218 – 225 ; 2015
220 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto Interna
Nasce do indivíduo, sendo ele o sujeito do próprio
conhecimento, que o constrói na interação com outros. Quando se observou que o recurso aos textos, cursos e
mestres da especialidade era utilizado principalmente
como matéria-prima para a sua reflexão. Justificação: Raciocínio pelo qual os indivíduos avaliam as
alegações de conhecimento (*). Dualística
Apenas uma das quatro opiniões citadas (*) foi
utilizada para justificação do saber homeopático. Na
pesquisa realizada, entendeu-se a prática homeopática
como de natureza imprevisível e complexa,
necessitando, por isso, de contínua avaliação e
construção, sendo, então, inadequadas as posições
dualísticas excludentes, do tipo “somente isto é certo”
e “aquilo é errado”. Múltipla
O valor de aceitação de múltiplas opiniões, entretanto,
foi considerado quando as comprovações, alegações
e evidências abarcaram mais de uma forma, estando
de acordo com a compreensão da prática homeopática
referida acima. ao previsto na Resolução nº 196/96 do Conselho Nacional de
Saúde, com o nº de protocolo 042/2010. Interna
Nasce do indivíduo, sendo ele o sujeito do próprio
conhecimento, que o constrói na interação com outros. Quando se observou que o recurso aos textos, cursos e
mestres da especialidade era utilizado principalmente
como matéria-prima para a sua reflexão. As unidades de sentido Justificação: Raciocínio pelo qual os indivíduos avaliam as
alegações de conhecimento (*). Sete entrevistados apresentaram práticas médicas homeopáti
cas coerentes com o discurso fundado na teoria homeopática. Nestes, as CE apresentaram, nas dimensões certeza, estru
tura e justificação, a presença dos tipos evolutivo, relativo e
múltiplo, respectivamente. Quatro apresentaram uma prática
mista, em que as racionalidades biomédica e homeopática se
superpunham. Aqui, para cada dimensão, exceto a justifica
ção, os entrevistados se distribuíram igualmente entre os ti
pos. Finalmente, nos três restantes, que apresentaram conduta
homeopática fundada numa racionalidade puramente biomé
dica, em relação às mesmas dimensões (certeza, estrutura e
justificação), os tipos fixo, discreto e dualístico foram predo
minantes. (*) Inclui a evidência, a consolidação e a justificação de suas crenças, bem como o uso
que fazem da autoridade e da sabedoria. Para a justificação do saber homeopático foram
consideradas quatro possibilidades. Na primeira, o entrevistado justifica a Homeopatia,
referindo-se aos seus princípios básicos: a lei da semelhança e a experimentação no ho
mem são. Na segunda, a justificativa se dá pela evidência dos seus resultados clínicos na
perspectiva do modelo biomédico tradicional, em que se observa a cura da(s) entidade(s)
clínica(s) e o desaparecimento de seus sinais e sintomas. Na terceira, a cura é entendida
pelo viés da Homeopatia, em que o indivíduo é considerado em sua totalidade, sendo os
efeitos, nesse conjunto, o quadro a considerar para afirmar os resultados positivos da
terapêutica. Assim, esse é um campo de observação coerente com os preceitos teóricos re
lativos à racionalidade homeopática. A quarta forma, finalmente, justifica-se a partir da
interface que a ciência homeopática tem com outros saberes, como filosofia, etimologia,
analogia, simbologia e o estudo das substâncias que constituem os medicamentos (mine
ralogia, botânica, zoologia). Isto é, buscam-se nessas áreas de conhecimento argumentos
para defender a propriedade da ciência homeopática. Apresentam-se, a seguir, exemplos das unidades de sen
tido onde se ressalta o contraste entre os que apresentaram
práticas médicas homeopáticas coerentes com o discurso e
aqueles onde isto não ocorreu, categorizados em: cosmologia,
doutrina médica, morfologia e dinâmica vital, diagnose. Nas práticas médicas coerentes com o discurso: Na cosmologia, verificou-se a referência do entrevistado à
visão holística do ser imerso no Universo: “A minha prática clínica é fundamentada nos princípios filo
sóficos, homeopáticos e numa bagagem anterior que a gente
traz de outras formações. As unidades de sentido Eu procuro usar, na minha prática
clínica, tudo o que eu aprendi a respeito do homem. Não só
voltado para a visão homeopática, mas tudo aquilo que corro
borava a visão holística do homem”. (E2) “A minha prática clínica é fundamentada nos princípios filo
sóficos, homeopáticos e numa bagagem anterior que a gente
traz de outras formações. Eu procuro usar, na minha prática
clínica, tudo o que eu aprendi a respeito do homem. Não só
voltado para a visão homeopática, mas tudo aquilo que corro
borava a visão holística do homem”. (E2) Para análise dos dados, optou-se pela análise de conteú
do6, considerada pertinente por integrar um conjunto de ins
trumentos metodológicos que se aplicam a discursos diversi
ficados. Mais especificamente, utilizou-se a análise temática
como técnica de análise. A análise temática foi transversal,
isto é, recortou o conjunto das entrevistas por meio do refe
rencial de categorias teóricas aplicadas sobre as respostas. No
presente estudo, as CE, suas dimensões e tipos representaram
os temas extraídos dos conjuntos dos discursos, considera
dos dados segmentáveis e comparáveis de acordo com seus
valores. Na dimensão doutrina médica, ele demonstrou estar em
consonância com o conceito homeopático – que considera a
doença como expressão do desequilíbrio da energia vital, ini
ciado na mente antes da exteriorização no físico – e contem
plou a forma singular do adoecimento segundo uma visão
global, em que o objeto de estudo é o “sujeito doente” e não
a doença: A apresentação da análise obedeceu ao seguinte formato:
identificação das unidades de sentido, sua classificação e cons
trução de uma defesa da hipótese original do estudo. As uni
dades de sentido neste estudo caracterizaram-se como trechos
do discurso que permitiam, pelo uso dos critérios citados, a
identificação quer da racionalidade, quer da CE expressa pelo
sujeito. “Estar doente não é só estar com uma pneumonia, é qualquer
coisa no teu estado do humor, no teu estado de ânimo; tudo é
doença, uma diferente postura em relação a alguma situação;
um exagero em ver uma situação, um exagero na sua maneira
de responder em determinadas situações da vida, isso tudo é
doença, eu acho”. (E2) Todos os sujeitos foram identificados com pseudônimos,
de forma a preservar o anonimato. As unidades de sentido (E4) “Frente a sua maneira de reagir nas diferentes coisas e situa
õ
t
id
d
lí i “Frente a sua maneira de reagir nas diferentes coisas e situa
ções que acontecem em sua vida ou com doenças clínicas; mas
ele pode ser uma pessoa que não tenha uma doença manifesta
e ela mostra uma reatividade que reflete o desequilíbrio”. (E4) Na diagnose, considerando o doente em sua totalidade
imerso no mundo e Universo, assim se pronunciou o entre
vistado: Na doutrina médica, evidencia-se a ausência do conceito
de totalidade, em que os planos aparecem compartimentaliza
dos, como no modelo biomédico: “O que aconteceu na sua vida naquele dia? O que estava
acontecendo? O que você estava fazendo? O que houve? Quem é você? A gente não pergunta, mas fica na nossa, é o
nosso pensamento interior, quem é essa pessoa? O que a trou
xe aqui? O que eu posso tratar? Não é o que ele está faltando,
é o que eu tenho a descobrir. O que é digno de tratar deste
paciente, então é isso que determina a prescrição. Indepen
dentemente do que ele tenha, ele pode ter uma pereba, uma
pneumonia, uma amigdalite”. (E3) “Doente é quando o teu físico não está bem, quando o teu
emocional não está bem, quando o teu espiritual não está
bem; é quando o teu psíquico não está bem, não está em equi
líbrio. Quando você não consegue, com todas as dificuldades,
com todas as adversidades, você não consegue se sentir bem,
feliz. [...] isso pode se manifestar tanto através do físico quan
to através do teu psíquico”. (E1) As unidades de sentido A pesquisa foi aprovada
pelo Comitê de Ética em Pesquisa EEAN/HESFA, atendendo REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
221 REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA 39 (2) : 218 – 225 ; 2015
221 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto Tabela 1
Relação entre as CE e a prática clínica
Prática
Estrutura
Fonte
Justificação
Certeza
Discreto
Relativo
Interna
Externa
Dualístico
Múltiplo
Fixo
Evolutivo
Homeopática (7)
0
7
7
7
0
7
0
7
Mista (4)
4
4
4
4
0
4
4
4
Biomédica* (3)
3
0
3
3
2
1
3
0
Total (14)
* Prática homeopática na qual se prescrevem medicamentos homeopáticos subordinados a uma perspectiva da racionalidade biomédica. Número entre parênteses = número de sujeitos entrevistados em cada categoria. * Prática homeopática na qual se prescrevem medicamentos homeopáticos subordinados a uma perspectiva da racionalidade biomédica. Número entre parênteses = número de sujeitos entrevistados em cada categoria. * Prática homeopática na qual se prescrevem medicamentos homeopáticos subordinados a uma perspectiva da racionalidade biomédica. Número entre parênteses = número de sujeitos entrevistados em cada categoria. pois o entrevistado sugeriu que não adianta dar o medicamen
to homeopático se o paciente não está bem consigo mesmo,
sendo que é exatamente esse o objetivo de cura para a Ho
meopatia, a medicação deve deixar o indivíduo bem consigo
mesmo: Na morfologia e na dinâmica vital, o entrevistado escla
receu, em consonância com o conceito homeopático, que con
sidera a doença como expressão do desequilíbrio da energia
vital, que se manifesta antes mesmo de que qualquer entidade
clínica apareça, indicando implicitamente a noção de totalida
de quando se referiu aos diferentes níveis de sua expressão: “Nos casos crônicos, eu diria que prescrevo mais com uma
medicação paliativa. Para pacientes que vêm reclamando da
quela artrose que não tem jeito, faço um rhus tox, uma arni
ca, vai mandar fazer uma fisioterapia, uma condroitina, uma
glucosamina, não adianta insistir em uma artrose que já está
lá toda calcificada, achando que vai melhorar com uma me
dicação que você vai demorar um tempão pra repertorizar”. (E8) “Frente a sua maneira de reagir nas diferentes coisas e situa
ções que acontecem em sua vida ou com doenças clínicas; mas
ele pode ser uma pessoa que não tenha uma doença manifesta
e ela mostra uma reatividade que reflete o desequilíbrio”. Na morfologia e dinâmica vital o entrevistado relatou: Na cosmologia, verificou-se que os conceitos de doença e
de cura estavam relacionados à entidade clínica. O entrevista
do utilizou a Homeopatia tendo a entidade clínica como obje
tivo; segundo ele, ela é indicada nas patologias que considera
reversíveis. Tais afirmações contrariam o ensinado na EKRJ,
em que o doente é tratado por sua individualidade, indepen
dentemente da patologia de que seja portador. O conceito de
doença não é coerente com o do modelo médico homeopático, “Doença é estar em desequilíbrio. É mais a parte da emoção,
ou seja, você não consegue resolver um problema, não está
bem com você. Com isso, você vai somatizar e produzir a
doença e aí você vai adoecer porque não conseguiu externar. Engoliu tanto sapo que acabou fazendo um tumor. Isso é o
desequilíbrio, fez uma contenção enorme e não saiu em pa REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
222 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto lavras, você não foi correr, não foi lutar um boxe e fez um
tumor. É uma contenção que você cria e ela passa a não exis
tir devido a vários fatores externos, daí você desenvolve uma
doença”. (E8) lavras, você não foi correr, não foi lutar um boxe e fez um
tumor. É uma contenção que você cria e ela passa a não exis
tir devido a vários fatores externos, daí você desenvolve uma
doença”. (E8) forte contraste, observam-se nos três entrevistados fundados
na racionalidade biomédica, em relação às mesmas dimensões
(certeza, estrutura e justificação), os tipos fixa, discreta e du
alística (esta um pouco menos). Finalmente, dentre os quatro
entrevistados caracterizados como de prática mista, o que se
observa é uma distribuição homogênea entre os vários tipos. Já a dimensão fonte do conhecimento não foi capaz de discri
minar as diferentes práticas. Quando o entrevistado se refere à doença como desequilí
brio, pareceu se apropriar de um conceito homeopático, mas,
no contexto em que o termo está inserido, a noção de dese
quilíbrio é relativa a uma causalidade linear característica do
modelo mecanicista biomédico. Não comparecem as noções
de processualidade, unidade, totalidade, que proporciona
riam uma visão mais dinâmica do adoecimento. Ele trabalhou
com a ausência da externalização de um problema emocional
como causa da doença. Essa é uma explicação linear comum e
psicossomática: tal emoção levando a tal doença. Na morfologia e dinâmica vital o entrevistado relatou: Não perce
bemos esforço na singularização e individualização do caso. Discussão As crenças, em termos genéricos, podem funcionar como im
portantes mediadores cognitivos. Elas são o motor interno,
determinam a motivação intrínseca, que depende dos desejos,
percepções e motivações do indivíduo7. Interferem, pela sua
transferência, no objeto em estudo, influenciando a interpreta
ção e a aquisição do novo conhecimento. Na diagnose e na terapêutica tem-se a entidade clínica
como objetivo: As CEs relacionadas a posições mais relativizantes e cons
trutivas quanto à obtenção do conhecimento são consideradas
facilitadoras; ao contrário, posturas mais rígidas e absolutas
relacionadas à posição objetivista são tidas como obstaculiza
doras2,8. “Primeiro identificando o quadro, como agora, que eu peguei
uma adenite, esse paciente que acabou de sair. Mas uma ade
nite submaxilar mandibular e de uma evolução que a criança
deitou e acordou no dia seguinte com um gânglio de 5 cm”
[...] (E13) Segundo Quian e Alvermann10 (apud Pecharromán e
Pozo9), parece existir uma íntima relação entre a consideração
do conhecimento como absoluto e a obtenção de conclusões
dogmáticas. Assim como nas crenças absolutistas, em que o
conhecimento é tido como fixo, baseado nas descrições de fe
nômenos e garantido pela autoridade, os alunos que abraçam
tais crenças manifestam grande resistência a se desprender de
concepções equivocadas e encontram dificuldade na mudança. REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
223 A relação entre as CE e a prática médica homeopática Uma vez identificadas as unidades de sentido, e os entrevista
dos classificados segundo a coerência entre seu discurso e a prá
tica médica homeopática, foram identificados três grupos: (i)
prática médica homeopática, (ii) prática médica homeopática/
biomédica e (iii) prática médica biomédica. No primeiro, havia
coerência entre o discurso teórico homeopático e a prescrição;
no último, a prescrição era baseada nos conceitos da biomedici
na; já no grupo intermediário ou misto, os conceitos homeopá
ticos e biomédicos estavam superpostos pelo menos em parte. Colbeck11 concluiu que as CE são construídas constantemen
te e podem ser modificadas pelo próprio indivíduo, assim como
pela interação social em seu ambiente educacional. Por meio
desse processo, melhores resultados podem ser alcançados. Para incorporar outra racionalidade, são necessárias cren
ças facilitadoras, que possam incorporar o “novo”, em que os
elementos de verificação não são necessariamente os mesmos
do modelo antes apreendido. Por exemplo, uma formação mé
dica pautada no modelo biomédico precisa da incorporação
de outro modelo de racionalidade para possibilitar o aprendi
zado da Homeopatia, considerada no presente estudo. Na etapa seguinte, relacionou-se o tipo de prática médica
homeopática em que está implícita uma racionalidade médica
própria com as crenças epistemológicas presentes no discurso
de cada um dos médicos entrevistados. A Tabela 1 compila os
achados encontrados na pesquisa e contrasta os resultados en
tre as crenças e a prática clínica. Assim, é cabível questionar-se sobre o processo pelo qual
a cisão observada se dá. Aventamos três linhas de argumen
tação que, a nosso ver, tendem a corroborar nossos achados. Na Tabela 1 observa-se clara separação, na perspectiva
das CE, entre os entrevistados que apresentaram diferentes
práticas clínicas. Naqueles cuja prática médica era sustentada
pela racionalidade homeopática, todos os sete apresentaram
nas dimensões certeza, estrutura e justificação a presença dos
tipos evolutivo, relativo e múltiplo, respectivamente. Já em Em primeiro lugar, a visão construtivista da aprendiza
gem postula que ela se dá por um processo ativo de constru
ção. Neste sentido, embora a informação possa ser transmitida
de um indivíduo a outro, o conhecimento é idiossincrásico. Dito de outra forma: o que o professor ensina não é necessaria REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
223 http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto mente aquilo que o aluno aprende. CONCLUSÕES Não foi objetivo desta pesquisa demonstrar que a linha de tra
balho desenvolvida e ensinada na instituição formadora de es
pecialistas (EKRJ) é a correta, nem estabelecer juízo de valores
sobre as diferentes linhas de trabalho, nem mesmo estabelecer
comparações entre elas. É claro, há limites nas conclusões, tais como em relação
ao tamanho da amostra e aos fatores externos que podem ter
tido um papel interveniente relevante nos achados. O estudo
se restringiu aos aspectos cognitivos e, em especial, às CE. No
entanto, apresentamos algumas razões de ordem teórica e em
pírica a fundamentar nossa hipótese de trabalho. Finalmente, o aporte à Teoria da Decisão permite dividir a
decisão clínica em duas categorias; as principalmente progra
madas, e as que não o são. No primeiro caso, encontramos os
casos em que o quadro é claro e explícito. Além disto, a indica
ção terapêutica já é de consenso e compartilhada amplamente
pela comunidade. Neste caso, a decisão é quase automática e
alicerçada em todo o conhecimento teórico. Há pouca refle
xão crítica sobre os procedimentos indicados. Já no caso das
outras, o quadro é complexo e sutil. Múltiplas variáveis en
tram em jogo. Aqui, então, embora baseado no conhecimento
vigente, o médico é obrigado a refletir e a relacionar sua baga
gem cultural com sua experiência clínica14. Esta pesquisa demonstrou que existiam coincidências
entre CE e o processo de aprendizagem no que se refere à
incorporação da racionalidade médica homeopática. A teo
ria da CE elucidou importantes aspectos do processo ensino
-aprendizagem experimentado pelos médicos entrevistados,
já que permitiu identificar as crenças epistemológicas de cada
médico e o modo como influenciaram na aquisição de uma
nova racionalidade, condicionando determinado tipo de prá
tica homeopática. E aqui fica claro como CE rígidas podem explicar a obser
vada cisão discurso/prática. Em primeiro lugar, elas dificul
tam a construção do conhecimento, já que, se ele é fixo e está
vel, fica mais difícil integrar o novo ao antigo, modificando-o. Favorece-se assim o que Ausubel15 denomina aprendizagem
mecânica. Aprende-se, desta forma, um conjunto de fatos des
conectados. Confirmou-se que existe relação entre as crenças episte
mológicas e a prática médica dos egressos. Observou-se que
aqueles que possuíam crenças epistemológicas construtivistas
tinham práticas médicas condizentes com a formação que ti
veram. Já os que mantinham posições epistemológicas obje
tivistas tinham práticas médicas não condizentes com o que
lhes foi ensinado. A relação entre as CE e a prática médica homeopática A construção implica um
processamento cognitivo, que visa à produção de sentido no
que é aprendido. Para isto, o aprendiz seleciona porções rele
vantes do material novo, organiza-o numa estrutura coerente
e o integra ao que já possui12. pendentemente da realidade clínica, o profissional tenderá a
considerá-los como típicos, tomando decisões principalmente
programadas. Neste caso, então, privilegiará o corpus da bio
medicina, já que mais antiga, solidificada e, principalmente,
pouco alterada em função de suas CE, que afirmam ser o co
nhecimento fixo e absoluto. Em segundo, referimo-nos à forma como o conhecimento
médico se estruturou na mente do profissional. Os conceitos
aprendidos podem ser estruturados de forma genérica ou es
pecífica. Quando genéricos, os conceitos são mais abrangentes
e gerais, podendo ser aplicados em qualquer situação clínica. Já os específicos se aplicam apenas a um dado problema. O
que surpreende neste caso é que Coderre et al.13 demonstra
ram que o desempenho diagnóstico se mostrou mais adequa
do quando seus conceitos eram mais específicos, alinhando-se
mais diretamente ao desafio clínico. REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
224 CONCLUSÕES Em seguida, neste contexto de fatos desconexos, favorece
-se ainda mais a estruturação do conhecimento em conceitos
mais gerais. É, portanto, razoável supor que seja mais fácil
para o profissional que professe CE absolutistas aprender a
teoria homeopática de modo mais superficial, estruturando-a
em conceitos mais genéricos. Com base nos resultados desta pesquisa, sugerimos que
a identificação das CE dos médicos, alunos do curso de espe
cialização, durante o período de sua formação em Homeopatia
pode ser um valioso recurso para a formação de profissionais
mais conscientes e alinhados com a racionalidade homeopática. A questão apresentada por Perry2 – se o aluno, ao ser ca
paz de revisar suas noções de conhecimento, também seria
capaz de mudar a forma de obtê-lo – parece-nos poder ser res
pondida de forma positiva. Assim, torna-se mais compreensível a cisão discurso/
prática aqui discutida, na medida em que a mera repetição de
conceitos não demanda mais que a manipulação de genera
lidades. No entanto, sua aplicação no diagnóstico de um pa
ciente específico mobiliza maior complexidade no raciocínio. Ao conhecer suas concepções epistemológicas, o aluno
poderá ter maior controle sobre elas, pois irá perceber a in
fluência das CE no processo de aprendizagem e na posterior
aplicação destas. A compreensão dessa interdependência, tan Finalmente, como resultado deste processo de aprendiza
gem mais superficial e pouco integrado, favorece-se a realiza
ção de diagnósticos mais estereotipados. Nestes, então, inde REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
224 Elizabeth Pinto Valente de Souza & Mauricio Abreu Pinto Peixoto http://dx.doi.org/10.1590/1981-52712015v39n2e02242013 9. Pecharromán I, Pozo JI. Cómo sé que es verdad?: Episte
mologías intuitivas de los estudiantes sobre el conocimien
to científico. Revista IENCI 2006;11(2). to pelo docente como pelo aprendiz, deverá propiciar uma
autorreflexão que instrumentalizará a gerência consciente de
tal processo de aprendizagem. Além disto, poderá orientar a
organização de futuras ações pedagógicas que possibilitem
melhor aproveitamento dos estudos e maior controle sobre o
aprendizado. 10. Quian G, Alvermann D. Role of Epistemological beliefs
and learned helplessness in secondary school students ‘le
arning science concepts from text. Journal of Educational
Psychology 1995; 87(2):282-292. Este artigo é resultado da pesquisa realizada para obten
ção do título de Mestre em Educação em Ciências e Saúde do
Núcleo de Tecnologia Educacional para a Saúde da Universi
dade Federal do Rio de Janeiro, em 2011. O presente estudo
teve apoio financeiro do CNPq. 11. CONCLUSÕES Colbeck D. “Understanding knowledge genesis by means
of multivariate factor analysis of epistemological belief
structures”. Information Research [on line]. 2007. 12(4)
[Capturado em 08 jun 2013]. Disponível em: http://Infor
mationR.net/ir/12-4/colis/colis16.html REFERÊNCIAS 12. Reynolds WM, Miller GE, orgs. Educational Psychology. Hoboken, New Jersey: John Wiley & Sons, Inc; 2003. p.49. 1. Campello MF, Luz HS. A Racionalidade Médica Homeopá
tica. In: Luz MT, Barros NF (Org.). Racionalidades Médicas
e Práticas Integrativas em Saúde. Rio de Janeiro: UERJ/
IMS/LAPPIS; 2012. p 73-102. 13. Coderre S, Jenkins D, McLaughlin K. Qualitative differen
ces in knowledge structure are associated with diagnos
tic performance in medical students. Advances in Health
Sciences. Education [on line].2008. 14(5) [Capturado em:
6 jun. 2013]; 677–684. Disponível em: http://link.springer. com/article/10.1007/s10459-008-9149-8 2. Perry WG. Forms of intellectual and ethical development
in the college years: A scheme, New York: Holt, Rinehart
and Winston; 1970 apud Hofer BK, Pintrich P. The develo
pment of epistemological theories: beliefs about knowled
ge and knowing and their relation to learning. Review of
Educational Research 1997;67(1):88-140. 14. Martinez C. Contribución de la teoría sobre la toma de
decisiones a la atención médica integral. Medisur. Re
vista Electrónica de las Ciencias Médicas en Cienfuegos
[on line]. 2009. 7(5) [Capturado em: 2 jun. 2013];308–
310. Disponível em: http://www.redalyc.org/articulo. oa?id=180014819003 3. Hofer BK, Pintrich P. The development of epistemological
theories: beliefs about knowledge and knowing and their
relation to learning. Review of Educational Research 1997;
67(1): 88-140. 15. Ausubel DP. Educational Psychology: A Cognitive View. 1o
ed. Holt, Rinehart and Winston; 1968. p. 109. 4. Roex A, Degryse J. Viewpoint: introducing the concept of
epistemological beliefs into medical education: the hot
-air-balloon metaphor. Academic Medicine [on line].2007. 82(6) [Capturado em: 27 abr. 2013]; 616–620. Disponível
em:
http://journals.lww.com/academicmedicine/Abs
tract/publishahead/Toward. 99378.aspx CONTRIBUIÇÃO DOS AUTORES Autora: Elaboração e revisão Co-autor: Orientação e revisão REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
225 CONFLITO DE INTERESSES 5. Camargo Jr KR. A medicina ocidental contemporânea. Rio
de Janeiro: UFRJ; 1993. p.7-19. (Série Estudos em saúde co
letiva, 065) Os autores declaram não haver conflito de interesses. Os autores declaram não haver conflito de interesses. ENDEREÇO PARA CORRESPONDÊNCIA
Elizabeth Pinto Valente de Souza
Rua Dom Francisco 163
Condomínio Santa Mônica Sul
Barra da Tijuca Rio de Janeiro
CEP 22793272
E-mail: betvalente@gmail.com 6. Bardin L. Análise de Conteúdo. Lisboa: Edições 70; 2010. Elizabeth Pinto Valente de Souza
Rua Dom Francisco 163
Condomínio Santa Mônica Sul
Barra da Tijuca Rio de Janeiro
CEP 22793272
E-mail: betvalente@gmail.com 7. Peixoto MAP, Brandão MAG, Santos G. Metacognição e
Tecnologia Educacional simbólica. Revista Brasileira de
Educação Médica 2007; 31(1):283-290. 8. Marchand H. Desenvolvimento intelectual e ético em es
tudantes do ensino superior – implicações pedagógicas. Revista de Ciências da Educação 2007; 9-18. REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
225 REVISTA BRASILEIRA DE EDUCAÇÃO MÉDICA
39 (2) : 218 – 225 ; 2015
225
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L’analisi del mutamento sociale nella sociologia di Luciano Cavalli
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Democrazia e società:
la sociologia
di Luciano Cavalli Democrazia e società:
la sociologia
di Luciano Cavalli VOL. 14, N° 27 • 2023
I S S N 2038 -3150 Firenze University Press
societàmutamentopolitica
r i v i s ta i ta l i a n a d i s o c i o l o g i a
Democrazia e società:
la sociologia di Luciano Cavalli Copyright © 2023 Authors. The authors retain all rights to the original work without any restric Open Access. This issue is distributed under the terms of the Creative Commons Attribution 4.0 International License
(CC-BY-4.0) which permits unrestricted use, distribution, and reproduction in any medium, provided you give ap-
propriate 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 (CC0 1.0) waiver applies to the data made
available in this issue, unless otherwise stated. REDAZIONE Lorenzo Viviani (direttore)
Lorenzo Grifone Baglioni
Pierluca Birindelli
Silvia Cervia
Carlo Colloca
Simona Gozzo
Marco Damiani
Stella Milani
Andrea Pirni
Ilaria Pitti
Stefano Poli
Luca Raffini
Andrea Valzania Stella Milani
Andrea Pirni
Ilaria Pitti
Stefano Poli
Luca Raffini
Andrea Valzania Stella Milani
Andrea Pirni
Ilaria Pitti
Stefano Poli
Luca Raffini
Andrea Valzania Lorenzo Viviani (direttore)
Lorenzo Grifone Baglioni
Pierluca Birindelli
Silvia Cervia
Carlo Colloca
Simona Gozzo
Marco Damiani societàmutamentopolitica
r i v i s ta i ta l i a n a d i s o c i o l o g i a Democrazia e società:
la sociologia di Luciano Cavalli Firenze University Press (Fondata da Gianfranco Bettin Lattes)
societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a COMITATO SCIENTIFICO Klaus Eder, Humboldt Universität, Berlin Stefano Monti Bragadin, Università di Genova Massimo Pendenza, Università di Salerno y
Jorge Arzate Salgado, Universidad Autónoma del Estado de México, Messico Copyright © 2023 Authors. The authors retain all rights to the original work without any restrictions. Democrazia e società: la sociologia di Luciano Cavalli Democrazia e società: la sociologia di Luciano Cavalli
A cura di Lorenzo Viviani A cura di Lorenzo Viviani Indice
5
Democrazia e società: la sociologia di
Luciano Cavalli. Per un’introduzione
Lorenzo Viviani
15
Accademia Toscana di Scienze e Lettere “La
Colombaria”
Sandro Rogari
17
Un debito intellettuale
Arnaldo Bagnasco
21
Il magistero di Luciano Cavalli come
sociologo critico
Giorgio Marsiglia
29
Le tre dimensioni di Luciano Cavalli. Maestro,
studioso, intellettuale pubblico
Roberto Segatori
41
Carisma e leadership nella sociologia della
democrazia
Lorenzo Viviani
61
I leader e le élite. Il contributo di Luciano
Cavalli alla sociologia politica
Carlo A. Marletti
73
I lati oscuri della democrazia
Gianfranco Bettin Lattes
85
La democrazia manipolata nella società
digitale
Paolo Giovannini
89
Carisma dei valori e carisma dei fini
Ambrogio Santambrogio
99
Carisma del sangue e familismo: un rapporto
controverso
Antonio Costabile
105
Storia e scienze sociali
Mario Caciagli
109
Religione e società: l’influenza di Weber sulla
sociologia italiana
Carlo Trigilia
113
123
129
139
L’in
149
153 Published by
Fi
U i Published by
Firenze University Press – University of Florence, Italy
Via Cittadella, 7 - 50144 Florence - Italy
http://www.fupress.com/smp Vol. 14, N. 27 • 2023 Vol. 14, N. 27 • 2023 Democrazia e società: la sociologia di Luciano Cavalli Citation: Lorenzo Viviani (2023) Democra-
zia e società: la sociologia di Luciano
Cavalli. Per un’introduzione. Soci-
etàMutamentoPolitica 14(27): 5-14. doi:
10.36253/smp-14329 Citation: Lorenzo Viviani (2023) Democra-
zia e società: la sociologia di Luciano
Cavalli. Per un’introduzione. Soci-
etàMutamentoPolitica 14(27): 5-14. doi:
10.36253/smp-14329 Lorenzo Viviani Copyright: © 2023 Lorenzo Viviani. This
is an open access, peer-reviewed arti-
cle published by Firenze University
Press (http://www.fupress.com/smp)
and distributed under the terms of the
Creative Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. Soltanto chi è sicuro di non cedere anche se il mon-
do, considerato dal suo punto di vista, è troppo stu-
pido o volgare per ciò che egli vuole offrirgli, soltan-
to chi è sicuro di poter dire di fronte a tutto questo:
«Non importa, andiamo avanti», soltanto quest’uo-
mo ha la «vocazione» per la politica Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Max Weber, La politica come professione (1919) Competing Interests: The Author(s)
declare(s) no conflict of interest. L’intento di questa nota vuol essere – consapevolmente – un non esausti-
vo quadro introduttivo del Beruf sociologico e intellettuale di Luciano Caval-
li. Non una agiografia di cui il Sociologo della Cesare Alfieri non avrebbe
alcun bisogno, data la rilevanza del suo pensiero e della sua opera, né una
sistematica rivisitazione di tutte le parti del suo pensiero. Due volumi di par-
ticolare importanza sono dedicati a questo. Il primo è Politica e società. Studi
in onore di Luciano Cavalli (1997) a cura di Gianfranco Bettin, in cui allievi,
collaboratori, colleghi della Cesare Alfieri e personalità del mondo accade-
mico e culturale italiano hanno sviluppato, ciascuno seguendo la specificità
dei propri studi, i temi connessi ai principali interessi di ricerca di Luciano
Cavalli. Il secondo è La sociologia di Luciano Cavalli (2008) a cura di Gian-
franco Bettin Lattes e Paolo Turi, in cui oltre a un approfondito ritratto intel-
lettuale di Luciano Cavalli viene ripresa la sua lezione sociologica a partire
dai due macro-ambiti della sociologia dei fenomeni politici e della teoria
sociologica e del mutamento sociale. Indice Indice
5
Democrazia e società: la sociologia di
Luciano Cavalli. Per un’introduzione
Lorenzo Viviani
15
Accademia Toscana di Scienze e Lettere “La
Colombaria”
Sandro Rogari
17
Un debito intellettuale
Arnaldo Bagnasco
21
Il magistero di Luciano Cavalli come
sociologo critico
Giorgio Marsiglia
29
Le tre dimensioni di Luciano Cavalli. Maestro,
studioso, intellettuale pubblico
Roberto Segatori
41
Carisma e leadership nella sociologia della
democrazia
Lorenzo Viviani
61
I leader e le élite. Il contributo di Luciano
Cavalli alla sociologia politica
Carlo A. Marletti
73
I lati oscuri della democrazia
Gianfranco Bettin Lattes
85
La democrazia manipolata nella società
digitale
Paolo Giovannini
89
Carisma dei valori e carisma dei fini
Ambrogio Santambrogio
99
Carisma del sangue e familismo: un rapporto
controverso
Antonio Costabile
105
Storia e scienze sociali
Mario Caciagli
109
Religione e società: l’influenza di Weber sulla
sociologia italiana
Carlo Trigilia
113
Cavalli, Weber e la formazione dei sociologi
sui testi classici
Rossana Trifiletti
123
L’analisi del mutamento sociale nella
sociologia di Luciano Cavalli
Marco Bontempi
129
Un’altra importante strada aperta da Luciano
Cavalli: lo studio del pensiero politico nei
classici della letteratura
Andrea Millefiorini
139
Il “realismo radicale” di Luciano Cavalli. Appunti per una riflessione
Franca Bonichi
L’intervista
149
Una lettura politica di Shakespeare. Intervista
a Luciano Cavalli
Renzo Ricchi
153
Appendice bio-bibliografica su autori e autrici 149
Una lettura politica di Shakespeare. Intervista
a Luciano Cavalli
Renzo Ricchi 153
Appendice bio-bibliografica su autori e autrici societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. Riflettere sulla sociologia di Luciano Cavalli offre
ai suoi allievi di ieri, di oggi e di domani, la possibilità
di un itinerario diverso da una pur rilevante storia del-
la sociologia, favorendo l’incontro con un Maestro che
guida nei temi e nei problemi del presente dialogando
costantemente, e invero in modo del tutto originale e
non di rado con una ricezione anticipata di autori e teo-
rie rispetto ai suoi tempi, con i classici della sociologia. Proprio lo studio dei classici costituisce non solo la pre-
messa necessaria, ma un’interazione costante rispetto al
programma di ricerca di Luciano Cavalli. Al tempo stes-
so dai classici della sociologia è ripresa la passione per i
“grandi temi”, pur con la ferma convinzione di rifiuta-
re astrattismi o un certo idealismo pseudo-sociologico
che cerchi di afferrare, quando non imporre, una teoria
generale della società. Cavalli propone una lettura della società e del-
la politica conoscendo approfonditamente la storia e il
metodo storico, così come la filosofia, l’antropologia e
la psicologia, finanche coltivando una profonda cultura
classica in ambito letterario. Nonostante la capacità di
interazione con altre discipline, ciò che emerge nel cor-
so di tutta la sua opera, dentro l’Accademia e nel più
ampio dibattito pubblico, è una precisa idea del senso
della sociologia e del compito del sociologo che rifiuta
ogni sincretismo interdisciplinare e che mai cede all’as-
senza di rigore del metodo sociologico, responsabili, in
ogni tempo, del derubricare la sociologia a opinionismo
di maniera. L’immaginazione sociologica di Cavalli, la
sua scelta di temi e problemi da affrontare, si muovono
costantemente e consapevolmente nell’ambito di una
chiara idea di sociologia, del significato di avalutatività e
del rapporto stringente fra sociologo e democrazia. Una
vocazione intellettuale che ne promuove senza ombra di
dubbio il pensiero e la ricerca al ruolo di Maestro. g
Nella introduzione a Il mutamento sociale. Set-
te ricerche sulla civiltà occidentale (1970), Cavalli rende
esplicita la sua prospettiva di dialogo con i classici nel
“far parlare” gli autori su cosa definisca il mondo occi-
dentale, quali siano le sue contraddizioni e i suoi conflit-
ti, quali dinamiche di cambiamento vengano delineate
anche nella comparazione con altri mondi. I saggi di cui si compo-
ne il numero fanno riferimento a studiosi appartenenti al
primo nucleo di collaboratori che hanno seguito Luciano
Cavalli da Genova alla Cesare Alfieri di Firenze, a coloro
che hanno preso parte alle prime edizioni del Dottorato
in Sociologia politica, a coloro che lo hanno conosciuto
all’interno della Sezione di Sociologia politica dell’Asso-
ciazione Italiana di Sociologia (AIS), e a coloro che han-
no avuto uno scambio intellettuale in fasi diverse del pro-
prio percorso accademico. lf l’Autore con cui si è interfacciato lungo tutto il suo pro-
gramma di ricerca, Weber. Quello fra Cavalli e Weber è
un dialogo ininterrotto e tutt’altro che ancorato a una
pur profonda, accurata e innovativa capacità esegetica
del primo rispetto al secondo. Cavalli dialoga con i temi
posti da Weber, ma da sociologo li porta nel proprio
tempo, non ipostatizzando la lezione weberiana ma ren-
dendola feconda. Inoltre, avvalendosi di una non comu-
ne e diretta conoscenza della sociologia e della sociologia
politica internazionale, in specie per la frequentazione
pionieristica della sociologia americana, Cavalli intro-
duce nel dibattito italiano sociologi oggi reputati classici,
dai Lynd a Mills, oltre a farsi portatore di un approccio
critico alla lezione di Parsons e a introdurre un socio-
logo di particolare originalità nello scenario europeo
quale Dahrendorf. Una conoscenza, quella della sociolo-
gia americana, che nei periodi di studio negli Stati Uni-
ti lo porterà a conoscere e a interagire con le principali
figure della sociologia politica d’Oltreoceano degli anni
Cinquanta e Sessanta, in specie Linz e Lipset, portando
nella sociologia politica italiana un respiro teorico ed
empirico innovativo. Nel dialogo con i classici Cavalli è
un puntuale e fine lettore dei testi originali, ma non si
pone l’obiettivo di fare mere glosse ai testi, né di limi-
tarsi a un esercizio filologico, quanto cerca costantemen-
te di svilupparne le trame di ricerca, arricchendole di
conoscenza sociologica e, in alcuni casi, anche ponendo
uno sguardo critico su aspetti di mancato sviluppo, o di
sottovalutazione di fenomeni. Pur coinvolgendo ampia parte delle autrici e degli autori che hanno col-
laborato alle due pubblicazioni sopra citate, l’idea di questo volume dal titolo
Democrazia e società: la sociologia di Luciano Cavalli nasce contestualmente
all’idea maturata da Gianfranco Bettin Lattes e da chi scrive, insieme a San-
dro Rogari, già Preside della Facoltà di Scienze politiche Cesare Alfieri dell’U-
niversità di Firenze e attuale Presidente dell’Accademia Toscana di Scienze
e Lettere “La Colombaria”, alla moglie di Luciano Cavalli, Anna Servettaz,
e ai figli Michele e Martino, di promuovere due giornate di studio dal tito-
lo Società e democrazia nel pensiero di Luciano Cavalli, convegno tenutosi a SOCIETÀMUTAMENTOPOLITICA 14(27): 5-14, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14329 6 Lorenzo Viviani Firenze presso la stessa Accademia “La Colombaria” nei
giorni 17 e 18 novembre 2022. Il presente volume che
appare come fascicolo monografico della rivista è stato
espressamente pensato a partire dalla primavera del 2022
come l’occasione per riflettere su temi sociologici “a par-
tire da” e non solo “su” Luciano Cavalli, a riprova di una
perdurante fecondità della sua opera per la futura ricerca
sociologica. Come si vedrà nella sezione finale di questa
nota introduttiva, i contributi fanno riferimento a diverse
generazioni di studiosi che hanno avuto una significativa
frequentazione con il Professore. I saggi di cui si compo-
ne il numero fanno riferimento a studiosi appartenenti al
primo nucleo di collaboratori che hanno seguito Luciano
Cavalli da Genova alla Cesare Alfieri di Firenze, a coloro
che hanno preso parte alle prime edizioni del Dottorato
in Sociologia politica, a coloro che lo hanno conosciuto
all’interno della Sezione di Sociologia politica dell’Asso-
ciazione Italiana di Sociologia (AIS), e a coloro che han-
no avuto uno scambio intellettuale in fasi diverse del pro-
prio percorso accademico. lf Firenze presso la stessa Accademia “La Colombaria” nei
giorni 17 e 18 novembre 2022. Il presente volume che
appare come fascicolo monografico della rivista è stato
espressamente pensato a partire dalla primavera del 2022
come l’occasione per riflettere su temi sociologici “a par-
tire da” e non solo “su” Luciano Cavalli, a riprova di una
perdurante fecondità della sua opera per la futura ricerca
sociologica. Come si vedrà nella sezione finale di questa
nota introduttiva, i contributi fanno riferimento a diverse
generazioni di studiosi che hanno avuto una significativa
frequentazione con il Professore. Il sociologo è in pieno un attore
del cambiamento e un “creatore sperimentale di nuove
forme sociali”, laddove si adoperi per realizzare un’ope-
ra di svelamento dei processi di dominazione, dovendo-
si infatti chiaramente contrapporre lo scienziato socia-
le ai “molti ras e molti don Rodrigo” della democrazia
manipolata (Cavalli 1964, p. 23). Un’emancipazione che
emerge non in contraddizione, ma in estrema continui-
tà con il “realismo radicale” di Cavalli, consapevole dei
rischi di un affidamento acritico all’idea di un illusorio
protagonismo delle masse (manipolate) in termini della
weberiana democrazia della strada, con un atteggiamen-
to critico non verso l’illuminismo kantiano della uscita
dalla condizione di minorità dell’individuo, ma ver-
so la manipolazione e i rischi insiti nel fare dei principi
dell’89 una chimera ideologica a sua volta responsabile
di degenerazioni anti-democratiche. In tutta la sua ope-
ra di ricezione e di sviluppo della sociologia weberiana
Cavalli ben conosce i limiti delle prospettive che dava-
no come acquisite l’emancipazione razionale dai simboli
e miti della religione della società moderna, analizzando
le forme di “re-incantamento” ad opera delle ideologie, le
“religioni laiche di redenzione”, e le manipolazioni che
operano anche, o forse soprattutto con la centralità dei
media, nelle democrazie contemporanee. Potremmo dire
che in ogni fase di effervescenza delle masse, specie lad-
dove la rottura rivoluzionaria si costruisca nel mito del
“popolo”, c’è sempre il rischio delle conseguenze inattese
che la storia mette in evidenza, così che non è raro pas-
sare dalla presa della Bastiglia, al Terrore di Robespierre
e infine approdare a Napoleone. La manipolazione incombe in ogni fase del muta-
mento sociale e politico, e il Cavalli realista radicale
h
di l d
i
h l
i
i valore emancipativo in nome della “gestione sociale del
potere” o della democrazia partecipativa, laddove dietro
quelle formule si celi la dominazione delle oligarchie di
partito della democrazia senza un leader (Cavalli 1973;
Cavalli 1981a). Sviluppando i limiti e le ambiguità della
stessa “illuminazione carismatica della ragione” messi in
evidenza da Weber, Cavalli si interroga sulla comples-
sa trama dei processi sociali e politici che connotano la
modernizzazione e le trasformazioni della democrazia,
affidando al ritorno del carisma laicizzato in politica, e
più in generale alla leadership, il ruolo di strumento di
contrapposizione sia alle degenerazioni della “democra-
zia della strada”, sia alla palude manipolativa del potere
delle oligarchie di partito. Un’emancipazione che
emerge non in contraddizione, ma in estrema continui-
tà con il “realismo radicale” di Cavalli, consapevole dei
rischi di un affidamento acritico all’idea di un illusorio
protagonismo delle masse (manipolate) in termini della
weberiana democrazia della strada, con un atteggiamen-
to critico non verso l’illuminismo kantiano della uscita
dalla condizione di minorità dell’individuo, ma ver-
so la manipolazione e i rischi insiti nel fare dei principi
dell’89 una chimera ideologica a sua volta responsabile
di degenerazioni anti-democratiche. In tutta la sua ope-
ra di ricezione e di sviluppo della sociologia weberiana
Cavalli ben conosce i limiti delle prospettive che dava-
no come acquisite l’emancipazione razionale dai simboli
e miti della religione della società moderna, analizzando
le forme di “re-incantamento” ad opera delle ideologie, le
“religioni laiche di redenzione”, e le manipolazioni che
operano anche, o forse soprattutto con la centralità dei
media, nelle democrazie contemporanee. Potremmo dire
che in ogni fase di effervescenza delle masse, specie lad-
dove la rottura rivoluzionaria si costruisca nel mito del
“popolo”, c’è sempre il rischio delle conseguenze inattese
che la storia mette in evidenza, così che non è raro pas-
sare dalla presa della Bastiglia, al Terrore di Robespierre
e infine approdare a Napoleone. l
b
f
d l scienze sociali di emanciparsi “dalla strumentalizzazione
dei poteri costituiti”, affidando al sociologo il compito di
cercare la soluzione al proprio problema senza chiedersi
se i suoi risultati fossero utili ai detentori del potere poli-
tico. Questa appare la stessa costante postura intellettua-
le di Luciano Cavalli nel corso della sua attività di ricer-
ca e di insegnamento, prima a Genova e successivamente
nella rifondazione della sociologia alla Cesare Alfieri di
Firenze, al cui interno inaugurò il corso di Sociolo-
gia nel 1966. Cavalli promuove un’idea della sociologia
che appare chiara fin dai tre libri pubblicati nel biennio
1964-1965, Il sociologo e la democrazia, La democrazia
manipolata e La città divisa. Sociologia del consenso e
del conflitto in ambiente urbano. La sociologia e la socio-
logia politica partecipano attivamente del processo di
emancipazione dell’individuo, e della società, dalle for-
me di oppressione indotte dai processi di manipolazio-
ne da parte del potere. Un itinera-
rio di ricerca che viene portato avanti in Sociologie del
nostro tempo (1973), titolo che già in sé contiene il sen-
so di una prospettiva sociologica, dal momento che gli
autori e le opere trattate contribuiscono a chiarificare
le dinamiche “dell’epoca in cui viviamo, del processo
in cui siamo immersi, dei fenomeni che tocchiamo con
mano”. In queste pagine vengono presentati gli autori
che accompagnano Cavalli in tutto il suo itinerario di
ricerca, da Marx a Durkheim, da Mosca a quello che è Nella introduzione al testo Masters of Sociological
Thought: Ideas in Historical and Social Context del 1971,
Lewis Coser prendeva spunto dal concetto di avalutati-
vità in Weber per identificare la necessità da parte delle 7 Democrazia e società: la sociologia di Luciano Cavalli Democrazia e società: la sociologia di Luciano Cavalli
scienze sociali di emanciparsi “dalla strumentalizzazione
dei poteri costituiti”, affidando al sociologo il compito di
cercare la soluzione al proprio problema senza chiedersi
se i suoi risultati fossero utili ai detentori del potere poli-
tico. Questa appare la stessa costante postura intellettua-
le di Luciano Cavalli nel corso della sua attività di ricer-
ca e di insegnamento, prima a Genova e successivamente
nella rifondazione della sociologia alla Cesare Alfieri di
Firenze, al cui interno inaugurò il corso di Sociolo-
gia nel 1966. Cavalli promuove un’idea della sociologia
che appare chiara fin dai tre libri pubblicati nel biennio
1964-1965, Il sociologo e la democrazia, La democrazia
manipolata e La città divisa. Sociologia del consenso e
del conflitto in ambiente urbano. La sociologia e la socio-
logia politica partecipano attivamente del processo di
emancipazione dell’individuo, e della società, dalle for-
me di oppressione indotte dai processi di manipolazio-
ne da parte del potere. Il sociologo è in pieno un attore
del cambiamento e un “creatore sperimentale di nuove
forme sociali”, laddove si adoperi per realizzare un’ope-
ra di svelamento dei processi di dominazione, dovendo-
si infatti chiaramente contrapporre lo scienziato socia-
le ai “molti ras e molti don Rodrigo” della democrazia
manipolata (Cavalli 1964, p. 23). In
continuità con Weber, ma anche con la sociologia criti-
ca di Mills e con la sua riflessione sull’immaginazione
sociologica, Cavalli ha ben chiare quanto siano rilevan-
ti le condizioni stesse dei processi di socializzazione dei
sociologi. Al pari di quanto accade per la manipolazione
delle masse, anche per i sociologi permangono processi
di socializzazione che scoraggiano vedute di insieme del
mondo attuale e dei relativi problemi sociali e politici. È questo per Cavalli il piano inclinato su cui rischia di
scivolare la sociologia, favorendo l’affermarsi di “omni-
bus research professor”, termine ripreso da Sorokin che
rimanda a una ricerca innocua per il potere, incentra-
ta sulle mere predisposizioni di survey, statisticamente
elaborate quanto conoscitivamente povere di valore e di
capacità di articolare concetti e quadri di insieme, per lo
più concentrate sulla sola “conoscenza verbale” (Caval-
li 1964, pp. 32-33). Da ciò deriva un malvezzo che ben
poco ha a che fare con la missione della sociologia, abiu-
rata in nome della subordinazione ai gruppi dominanti
che in qualsiasi epoca storica, e in qualsiasi ambito del
sapere, si adoperano per controllare, imbrigliare e di fat-
to neutralizzare la scienza. Si tratta di un contesto cul-
turale che favorisce l’emergere di sociologi “specialisti di
angusti orizzonti”, non dissimili dagli “specialisti senza
spirito” di Weber, che di fatti abdicano alla compren-
sione del proprio tempo. Luciano Cavalli non solo non
hai mai indugiato, ma ha preso ampiamente le distanze
dalla tentazione di entrare a far parte della schiera dei
chierici della scienza intesa in funzione ancillare rispetto
al potere politico, e lo stesso magistero educativo man-
tiene intatto lo spirito emancipativo della sociologia, mai
abbandonando il monito weberiano per cui “il profeta e
il demagogo non si addicono alla cattedra universitaria”
(Weber [1919] 2004, p. 30). pegno senza rinchiudersi in un astrattismo e in una neu-
tralità assiologica rispetto ai problemi del proprio tempo. Un tratto che richiama la stessa biografia intellettuale di
Weber e la sua ricezione che troppo spesso ha disgiunto
l’attore sociale dallo studioso, così come la dimensione
storica da quella analitica, perpetuando una forzatura
di derivazione parsonsiana (Magatti 2016). Su questo si
misura il significato di “sociologo impegnato” che Cavalli
ha in mente e che mette in atto nella sua attività di ricer-
ca. Un sociologo che ha una posizione e che è capace di
esprimere un giudizio di valore sui temi del proprio tem-
po non equivale a un attivista o un ideologo che piega i
risultati della propria ricerca al fine di proselitismo o di
programma politico. È lo stesso Cavalli che ci offre la chiave interpreta-
tiva del senso della sociologia e del rapporto tra socio-
logia e democrazia, richiamando quella mirabile sintesi
del Beruf del politico, ma parimenti dello scienziato, che
si pone come stella polare delle battaglie culturali e degli
ideali umani. Cavalli ha come riferimento esplicito la
postura intellettuale di Weber, e in particolare la figura
di “uomo eroicamente teso a fare il suo dovere nella sua
«vocazione»” (Cavalli 1964, p. 40). Voluto e consapevo-
le è il richiamo all’asceta intra-mondano che sostanzia il
Beruf di chi persegue la sua causa non opponendo etica
dell’intenzione e etica della responsabilità, ma facen-
dole coesistere e permettendo ad esse di completarsi in
uno sforzo unitario costante. Se nelle pagine relative alla
leadership carismatica e alla democrazia questo tema
emerge in Weber e in Cavalli come chiave interpretati-
va della conciliazione del carisma con la società razio-
nalizzata, è sempre in questo snodo di ricerca e di vita
che si sostanzia la specifica vocazione del sociologo in
relazione allo svelamento della democrazia manipolata. Uno svelamento che è impegno civile e scienza, passione
e rigore metodologico, azione rivolta all’autoconsapevo-
lezza dei processi e azione a sostegno di un’educazione
pubblica alla democrazia. L’impegno per la democrazia
di Cavalli non è mai democraticismo, non è assenza di
responsabilità, né manto ideologico che cela l’intento di
dominazione da parte di vecchie e nuove religioni laiche
di redenzione, ma è quella libertà che promuove il plu-
ralismo e al contempo l’uso consapevole della ragione
che consente l’ordine. È questa la prospettiva del Cavalli
realista radicale che affida alle élite e alla leadership un
compito centrale nel rendere possibile la democrazia,
assumendo quest’ultima come valore da difendere e da
promuovere. Nell’opera e nel magistero di Cavalli la democrazia è
al centro delle domande di ricerca, aspetto che permet-
te di connotarlo, anche rispetto al Sociologo di Erfurt,
come un sociologo della e per la democrazia. Partendo
dalla ineludibile relazione fra sociologia e studio della
politica, Cavalli offre così una definizione stessa del fare
“sociologia politica”, allontanandola dalle secche euristi-
che delle “sociologie speciali”, e recuperando la lezione
dei classici mostra chiaramente come non sia sociolo-
gicamente possibile scindere lo studio della politica da
quello della società e del mutamento sociale. Non appaia
quindi una meticolosa puntigliosità meramente accade-
mica l’insistere sul non aderire alla pur affermatasi pro-
spettiva di una sociologia della politica o una sociologia
dei fenomeni politici, ma la consapevolezza che la defini-
zione sociologia politica vada ben oltre la questione ter-
minologica recuperando appieno la lezione cavalliana. g
In tutta la sua opera Cavalli si pone come un socio-
logo critico, dove la critica non ha alcun fondamento
ideologico, non è iscritta in appartenenze o consorterie
più o meno note, ma è a fondamento di un contributo
originale e non di rado contro-corrente nella lettura dei
processi sociali. Non una sociologia del micro, del pic-
colissimo, né un’astrazione filosofica o para-filosofica,
come tali teorizzazioni che albergano sulla “volante Iso-
la di Laputa”, un vezzo in cui sovente ricade una teoria
sociologica non consapevole della sua epistemologia,
quando non direttamente acerba dei suoi strumenti
teorici ed empirici. Al tempo stesso Cavalli ha chiaro il
rischio, ieri come oggi, dell’incombere di altri fenomeni
degenerativi delle scienze sociali, ancor più se valutati
nell’ottica del ruolo della sociologia per la democrazia. Non solo il sociologismo, ossia la prospettiva che spie-
ga i fenomeni sociali unicamente in ragione di variabili
strutturali, negando la centralità dell’apporto dell’indi-
viduo nella storia, ma la stessa sociografia, come studio
empirico, più descrittivo che analitico, che elude non
tanto le grandi teorizzazioni, quanto gli interrogativi i
La manipolazione incombe in ogni fase del muta-
mento sociale e politico, e il Cavalli realista radicale
che studia la democrazia sa che la stessa partecipazione
dei cittadini alla politica può essere manipolata nel suo 8 Lorenzo Viviani stessi della sociologia e della libertà che ad essa è asso-
ciata come domande, metodo e risultati della ricerca. Una tensione intellettuale che fa di Caval-
li un sociologo per la democrazia, andando ben oltre la
sfumata constatazione dello stato di fatto della democra-
zia, come era stato per gli elitisti, ma anche oltre la teo-
ria politica weberiana che se assume la democrazia come Nel dialogo costante con Weber, Cavalli già a parti-
re dagli anni Sessanta assume la centralità di un fecondo
legame tra passione e studio, impegno e scienza, avendo
ben presente gli effetti di una Wertfreiheit malintesa, una
banalizzazione e al tempo stesso una distorsione del con-
cetto di avalutatività. L’avalutatività che Cavalli riprende
da Weber, e dalla sua stessa biografia umana e scientifica,
viene affrontata criticamente nelle sue aporie. La ricerca
sociologica serve per mettere alla prova, chiarificare e
avere consapevolezza dei valori che stanno a fondamento
delle domande di ricerca che persegue. Il contrario di un
disincanto razionalmente asettico rispetto allo studio del-
la società, della politica, della democrazia. Sono invece le
scelte di valore che animano la ricerca del sociologo, por-
tandolo a indagare i temi e i problemi che non suscitano
una mera curiosità, ma che animano la riflessione e l’im- 9 Democrazia e società: la sociologia di Luciano Cavalli 9 italiane, del Centro Interuniversitario di Sociologia Poli-
tica CIUSPO (con gli Atenei di Firenze, Perugia e Geno-
va), e delle collane di Working papers edite da il Mulino. In particolare, si deve all’impegno di Luciano Caval-
li un ruolo fondamentale nella nascita della Sezione di
Sociologia Politica dell’Associazione Italiana di Sociolo-
gia (AIS), di cui fu Coordinatore e che ha costituito un
motore di sviluppo della sociologia politica in Italia e a
livello internazionale, tanto in termini di ricerca che di
formazione di nuove generazioni di studiosi. Nata nella
metà degli anni Ottanta, la Sezione di Sociologia Politica
dell’AIS ha avuto un momento di sviluppo rilevante pro-
prio con l’impulso di Luciano Cavalli nella organizza-
zione del primo rilevante Convegno di San Miniato del
5-7 dicembre 1986, da cui origina il volume Leadership e
democrazia (1987), di particolare importanza per lo stu-
dio della politica in Italia e per il dialogo fra sociologia
politica e altre discipline. valore, tuttavia non ha nelle sue priorità lo sviluppo di
una teoria democratica. i C’è infine un ulteriore aspetto che completa il rap-
porto fra Cavalli e la vocazione alla democrazia della
sociologia, e che ne anima il senso dell’impegno accade-
mico e pubblico: l’educazione alla democrazia. Quest’ul-
timo tema ha recentemente assunto una nuova centra-
lità a partire dal dibattito sulla public sociology inaugu-
rato da Michael Burawoy nel suo Presidential Address
del 2004 all’American Sociological Association (ASA)
(Burawoy 2021), pur con un diverso significato di impe-
gno pubblico rispetto all’idea di Cavalli. Ancora più
recentemente si segnala come l’attenzione a tale tema
abbia portato alla scelta della stessa ASA di dedicare il
suo 118th Annual Meeting del 2023 al tema The educa-
tive power of sociology. Luciano Cavalli ha costantemen-
te orientato la sua attività di docente, così come il suo
più ampio impegno nella sfera pubblica, al fine di favo-
rire quella maturazione democratica che è condizione
imprescindibile di una cittadinanza attiva e, potremmo
aggiungere, adulta. fi Luciano Cavalli è stato quindi un vero e proprio
Maestro alla cui lezione e vocazione critica hanno attin-
to diverse generazioni di sociologi, pur intraprendendo
poi ciascuno di essi il proprio specifico ambito di ricer-
ca. Nella sua biografia intellettuale si legge inequivoca-
bilmente l’eco di quell’invito weberiano ad adempiere
nella vita personale e professionale alla “richiesta di ogni
giorno”, seguendo e perseguendo il demone che tiene i
fili della propria vita, ossia quella tensione fra sociologo
e democrazia che rimane lezione attualissima lasciata
dal Professore. gg
g
Consapevole del ruolo affidato agli intellettuali nel-
la democrazia, al pari della lezione classica di Dewey e
di quella sociologica di Mills, Cavalli recupera proprio
da quest’ultimo la necessità di dare ai giovani l’immagi-
nazione sociologica, sia stimolandone la fuoriuscita dal
senso comune dei condizionamenti storico-culturali, sia
formando giovani studenti e studiosi di scienze socia-
li capaci di assumere il ruolo di educatori di democra-
zia e di sperimentatori di nuove forme sociali. Evitando
weberianamente ogni volontà “apostolica” in termini di
proselitismo della cattedra, emerge il Cavalli fortemen-
te convinto del ruolo dell’università nella formazione
dei giovani, senza il timore di operare apertamente per
rendere le istituzioni universitarie il luogo della forma-
zione delle future élite in grado di rispondere ai proble-
mi del proprio tempo. Proprio questo è il senso più pro-
fondo del richiamo che Cavalli fa al ruolo del “ribelle”,
del dissenter, una figura che si sostanzia nella vocazione
all’uso della ragione nel contrastare il mainstream dei
condizionamenti sociali, politici e culturali, e non la
vacua adesione a un omologante clima di contestazione. L’impegno dello studioso e del Maestro non può quin-
di prescindere dalla centralità dell’università, ambito in
cui Cavalli assume con il suo arrivo a Firenze una serie
di ruoli di responsabilità come Direttore dell’Istituto di
Sociologia, Consigliere di Amministrazione dell’Ateneo,
Preside della Cesare Alfieri. Dall’Ateneo fiorentino par-
te anche quella azione feconda di rilancio in Italia della
sociologia politica, a cui Cavalli contribuisce come Fon-
datore del Dottorato in Sociologia politica che nel tempo
contribuirà a formare studiosi delle principali università Proprio in ragione della volontà di riflettere sul
nostro tempo recuperando le coordinate della lezione
di Luciano Cavalli, il volume di «SocietàMutamentoPo-
litica» dal titolo Democrazia e società: la sociologia di
Luciano Cavalli raccoglie saggi di diverse generazioni di
studiosi articolandosi idealmente in tre parti. La prima parte è dedicata al ricordo e al magistero
educativo e intellettuale. Arnaldo Bagnasco ripercorre il
“debito intellettuale” maturato nei confronti di Luciano
Cavalli negli anni trascorsi alla Cesare Alfieri di Firenze,
mettendo in risalto come tra gli autori oggetto della sua
riflessione vi fossero, fra gli altri, sociologi poco cono-
sciuti e dibattuti in Italia come Lynd e Mills, a riprova
di un approccio critico che portava Cavalli a interro-
garsi sul senso stesso della conoscenza sociologica, in
un modo originale rispetto anche al più recente dibat-
tito sulla sociologia pubblica. Pur nello sviluppo di un
autonomo itinerario di ricerca, Bagnasco riconosce l’in-
segnamento di Cavalli di cui si è avvalso nei termini di
un accrescimento intellettuale come parte di un ampio
bagaglio culturale maturato nella Cesare Alfieri. f i
Giorgio Marsiglia affronta il tema della sociologia
critica di Luciano Cavalli, di cui è stato uno dei primi 10 Lorenzo Viviani Carlo Marletti, dopo aver ricordato il clima intellet-
tuale al cui interno si è sviluppata la sua frequentazione
con Luciano Cavalli e altre figure, in particolare Filippo
Barbano, dedica il suo contributo alla sociologia politica
di Weber proposta da Luciano Cavalli. Viene così mes-
so in risalto lo specifico contributo di Cavalli al tema
del rapporto tra carisma e democrazia, confrontando la
prospettiva sociologico-politica dello stesso Weber con
altri autori, in specie Pareto. Una parte rilevante del sag-
gio viene poi dedicata alla attualizzazione della sociolo-
gia politica di Cavalli in relazione allo specifico campo
di ricerca dell’Autore, ossia la sociologia della comuni-
cazione. Continuando a dialogare con l’opera di Caval-
li, Marletti giunge a proporre una rivisitazione del tema
del carisma nelle società e nelle democrazie contempo-
ranee, alla luce dei risultati della ricerca nell’ambito del-
la comunicazione politica, ponendosi così pienamente
nell’ottica del volume, tesa a sviluppare nuove direzioni
di ricerca nel dialogo con il pensiero di Luciano Cavalli. allievi nel periodo genovese. Una consuetudine intellet-
tuale che porta Marsiglia a cogliere con estrema brillan-
tezza e conoscenza tutto il dipanarsi della lettura criti-
ca della società, e della stessa sociologia, alla luce dei
principali contributi di Cavalli. Ne emerge un ritratto
puntuale dell’idea di sociologia, del ruolo dei classici, e
anche del senso più fecondo della interdisciplinarità che
ha guidato la ricerca e il magistero educativo universita-
rio del Professore, specie ricordando il ruolo del Dotto-
rato di Sociologia politica. allievi nel periodo genovese. Una consuetudine intellet-
tuale che porta Marsiglia a cogliere con estrema brillan-
tezza e conoscenza tutto il dipanarsi della lettura criti-
ca della società, e della stessa sociologia, alla luce dei
principali contributi di Cavalli. Ne emerge un ritratto
puntuale dell’idea di sociologia, del ruolo dei classici, e
anche del senso più fecondo della interdisciplinarità che
ha guidato la ricerca e il magistero educativo universita-
rio del Professore, specie ricordando il ruolo del Dotto-
rato di Sociologia politica. Roberto Segatori chiude questa prima parte, invero
non schematicamente costruita ma ordinata secondo un
filo che consente di collegare l’itinerario intellettuale e
biografico di Luciano Cavalli. Il contributo di Segatori
si articola su tre pilastri, il Maestro conosciuto a distan-
za, lo studioso conosciuto da vicino e l’intellettuale
pubblico tra impegno e disincanto. Ne emerge un qua-
dro che mette in risalto la centralità del rigore scientifi-
co di Luciano Cavalli e al tempo stesso la sua interazio-
ne con i principali e più brillanti sociologi del proprio
tempo, da Franco Crespi a Luciano Pellicani. Fra i punti
toccati da Segatori emergono in particolare due aspetti. Il primo relativo al suo studio di Weber, teso a storiciz-
zare la riflessione e le categorie weberiane e a far emer-
gere il senso di una riflessione su cui sviluppare la sua
originale sociologia della democrazia e della leadership. Il secondo relativo alla postura dell’intellettuale che nel
realismo radicale riesce a coniugare il disincanto della
realtà studiata con la passione e l’impegno dell’intellet-
tuale pubblico. Gianfranco Bettin Lattes ci guida con conoscenza
e passione all’interno di uno dei nodi più rilevanti di
tutta la riflessione sociologica di Luciano Cavalli, quei
lati oscuri della democrazia che è compito del sociolo-
go studiare e svelare al fine di difendere e promuovere
la democrazia stessa. L’Autore, che ha collaborato con il
Professore fin dai suoi primi anni di ricerca, non si limi-
ta alla semplice riproposizione dei testi in cui più aper-
tamente Cavalli mette in evidenza il rischio di degene-
razioni o manipolazioni della democrazia, ma proprio
a partire da Cavalli offre nuovi spunti per la ricerca, in
particolare ponendo l’attenzione su quella “sottospecie
di democrazia manipolata che si può etichettare come
democrazia stereotipata”. Gli stereotipi, come conoscen-
za a basso contenuto di consapevolezza, si prestano per
l’Autore a processi di degenerazione della democrazia,
al pari del democraticismo su cui tanto Luciano Cavalli
ha messo in guardia dal potenziale di manipolazione da
smascherare proprio con la ricerca sociologica. È proprio
rifacendosi alla lezione cavalliana sulla rilevanza di una
cultura politica nazionale oltre il recinto e la manipo-
lazione partitocratica delle ideologie del Novecento, ma
anche alla constatazione dell’assenza di un repubblica-
nesimo civile, che Gianfranco Bettin Lattes offre nuove
direzioni di ricerca facendo di Luciano Cavalli un Mae-
stro di teoria e di metodo per le nuove generazioni. La seconda parte del volume è dedicata allo studio
della politica, della democrazia e della leadership da par-
te di Luciano Cavalli, con una particolare focalizzazio-
ne sul tema del carisma, che a tutt’oggi rappresenta uno
dei campi di ricerca su cui il magistero intellettuale di
Cavalli costituisce una pietra d’angolo imprescindibile
per gli studiosi a livello nazionale e internazionale. f Lorenzo Viviani affronta il tema della sociologia
della democrazia di Luciano Cavalli, ripercorrendone
lo studio e il dialogo con Weber proprio a partire dal-
la centralità del carisma. Ne emerge una ricezione di
Weber da parte di Cavalli che supera l’impianto con-
solidato di Parsons e anticipa in Italia gli sviluppi del
lavoro di autori come Tenbruck e Schluchter sull’ope-
ra del Sociologo di Erfurt, in una lettura che si pone a
fianco della più innovativa sociologia weberiana a livello
internazionale. Al tempo stesso il saggio si addentra in
quel rapporto tra carisma e democrazia con un leader
in chiave contemporanea che costituisce un terreno di
ricerca su cui Luciano Cavalli rimane un punto di riferi-
mento ineguagliato per la sociologia politica contempo-
ranea. Paolo Giovannini, uno dei primi allievi che ha
seguito il Professore da Genova alla Cesare Alfieri di
Firenze, di cui è stato Preside, si colloca appieno nella
attualità del pensiero del Professore, offrendo una let-
tura di un fenomeno di stringente attualità sociologica
partendo da uno dei temi più rilevanti della lezione di
Luciano Cavalli, la manipolazione. In particolare Gio- 11 11 Democrazia e società: la sociologia di Luciano Cavalli vannini si concentra sui “processi di digitalizzazione
utilizzabili (e utilizzati) a scopo manipolatorio”, met-
tendone in evidenza i rischi per la democrazia. Al pari
dell’approccio critico della sociologia di Luciano Caval-
li, Giovannini mette così in evidenza come nella tecno-
logia e nelle innovazioni da questa innescate si possano
celare meccanismi che se non compresi e smascherati
operano in senso contrario alla democrazia. Richiaman-
dosi appassionatamente alla figura del ribelle evocata da
Cavalli, Giovannini presenta, assieme al disincanto per
la dimensione globale delle nuove piattaforme digitali, i
social network e la dominazione invisibile ma pervasiva
dei padroni della rete, la possibilità di resistenza tramite
quella vocazione critica e controcorrente che appare con-
sustanziale all’impegno del sociologo. La terza parte del volume si confronta con temi, pro-
blemi e linee di sviluppo del pensiero di Luciano Caval-
li che chiamano in causa l’uso dei classici del pensiero
sociologico, il metodo di analisi e la capacità di porsi in
dialogo con altre discipline. Mario Caciagli discute un tema centrale nel “fare
sociologia” di Luciano Cavalli, quella capacità di uso del-
la storia coltivata dallo stesso Weber e che nelle sue opere
emerge come “strumento indispensabile per l’approccio
interdisciplinare al fenomeno studiato”. Caciagli richiama
l’intreccio costante fra fondamento storico (e storicizzato)
dell’analisi dei fenomeni politici, che nella sociologia di
Cavalli non indugiano mai in un astrattismo autoreferen-
ziale. L’attenzione di Caciagli si concentra sul “caso” Hit-
ler, come magistralmente emerge in Carisma e tirannide
nel secolo XX. Il caso Hitler (1982), definito apertamente
un “capolavoro”. Caciagli, scienziato politico ma al tem-
po stesso studioso raffinato con profonda consuetudine
con l’analisi storica e con altrettanta sensibilità sociolo-
gica, mette in risalto come per Cavalli la lezione storica
non sia finalizzata alla musealizzazione di fatti accaduti,
quanto svolga quella antica funzione di “magistra”, met-
tendo in guardia dalle potenzialità del “carisma demonia-
co” che emerge nel caso Hitler. p g
g
Ambrogio Santambrogio tratta uno dei temi centrali
della sociologia di Luciano Cavalli, il ruolo del carisma
che si afferma in fasi di crisi e che opera all’interno del-
la politica come strumento di rigenerazione di valori e
ordine. In linea con l’insegnamento di Maestri come
Luciano Cavalli e Franco Crespi, che a lungo dialogò
con il primo anche se con prospettive di ricerca diverse,
Santambrogio prende apertamente posizione contro una
sociologia della iper-specializzazione dallo sguardo corto
e poco profondo, la cui “moda” attuale porta ad abban-
donare la teoria sociale e politica, o a farne un orpello da
aggiungere a un empirismo vacuo. Recuperando l’ambi-
zione sociologica di cimentarsi con grandi temi, il che
ovviamente non significa con teorie astratte e autorefe-
renziali, viene così proposta una lettura del processo di
razionalizzazione e del ruolo che il carisma può avere
in una società complessa e individualizzata come quella
contemporanea. Ponendosi ancora una volta in dialogo
con Luciano Cavalli, l’Autore propone una revisione cri-
tica del carisma nella democrazia, definendolo “un cari-
sma dei fini e non più costitutivo di valori”, come tale
al centro della promozione dello sviluppo dell’autonomia
degli individui come unico valore effettivo. f g
Carlo Trigilia focalizza la sua riflessione a parti-
re dal volume di Luciano Cavalli Max Weber: religio-
ne e società (1968), muovendosi su tre piani connessi
fra loro. Il primo fa riferimento al rilevante contributo
in termini di storia del pensiero che a partire da questa
opera Luciano Cavalli offre a una più corretta ricezione
di Weber da parte della sociologia italiana di quel perio-
do. Il secondo rimanda al rapporto, fecondo ma non di
sovrapposizione né di sincretismo, fra storia e sociolo-
gia nell’opera di Cavalli, così da evitare generalizzazioni
proprie della – allora – prevalente impostazione sociolo-
gica neo-positivista. Il terzo, infine, entra più direttamen-
te nell’influenza che la ricezione di Weber da parte di
Luciano Cavalli ha avuto nel contribuire a superare una
perdurante e ipostatizzata “tesi Weber” sul rapporto tra
religione e capitalismo, riportando al centro la rilevan-
za della plurifattorialità di impronta weberiana nel con-
tribuire alla interpretazione sociologica del capitalismo. Trigilia mette in costante relazione economia e politica,
così come la sociologia economica con le trasformazioni
della democrazia. È in questo ambito che emerge il dia-
logo fra Trigilia e Cavalli all’interno della Cesare Alfieri
in fasi diverse del lavoro di ricerca di entrambi gli stu-
diosi. Un dialogo che Trigilia continua nel suo saggio, a
partire dalla dialettica fra due prospettive di democrazia,
una “kelseniana”, più legata agli sviluppi della sociologia
economica, e una sociologico-politica weberiana, di cui
Cavalli rimane interprete autorevole. Il confronto tra la f
Antonio Costabile affronta il tema delle possibili
relazioni tra carisma del sangue e familismo, con alcuni
specifici riferimenti alla storia italiana. In continuità con
la ricezione di Weber offerta da Luciano Cavalli, l’Au-
tore si inserisce nella riflessione sociologico-politica sul
carisma ponendosi l’obiettivo di verificare se possa esi-
stere un collegamento tra carisma del sangue, familismo
sociale e familismo politico. Lo sguardo di Costabile è
rivolto alle nuove sfide poste dalla fase attuale di nuova
tensione fra razionalizzazione e carisma, con l’emergere
di nuove forme di comunità (dalla dimensione sempre
più ristretta) che assumono una valenza politica nell’am-
bito della crisi di legittimazione politica che contraddi-
stingue le attuali democrazie. 12 Lorenzo Viviani Vengono così affrontate la natura e le forme del conflitto
politico in Italia, per evidenziare come Cavalli avrebbe
poi approfondito in successive pubblicazioni, i nodi cri-
tici, senza spiegazioni strutturaliste, ma assumendo la
rilevanza di variabili culturali, politiche e sociali. i “democrazia negoziale”, basata su quella che Trigilia defi-
nisce un’efficace condivisione del potere politico, e una
“democrazia maggioritaria e plebiscitaria”, con al centro
il ruolo della leadership e della personalizzazione, conti-
nuano a porre alla sociologia una sfida di ricerca teorica
ed empirica di assoluta attualità. Andrea Millefiorini richiama il legame fra il suo
Maestro, Luciano Pellicani, e Luciano Cavalli, a fonda-
mento dell’incontro dell’Autore con l’opera di Lucia-
no Cavalli. Millefiorini dedica il suo contributo ai temi
dell’ultimo volume pubblicato da Luciano Cavalli, Giulio
Cesare, Coriolano e il Teatro della Repubblica. Una lettu-
ra politica di Shakespeare (2006), mettendo in evidenza
la capacità di dialogo fra il sociologo politico e le fonti
letterarie, in primis quella di Shakespeare. Come osser-
va Millefiorini, Luciano Cavalli mostra come sia possibi-
le ricavare e analizzare il pensiero politico tramite una
lettura sociologica delle opere shakespeariane, mettendo
in risalto temi centrali per la ricerca socio-politica, dal
significato del Teatro della Repubblica ai temi del con-
flitto e dell’ordine nella “città divisa”. Ne emerge un’o-
pera in cui la formazione umanistica di Cavalli riesce a
fondersi con la sua lezione sociologica e ciò che ne sca-
turisce è un esempio paradigmatico di come studiare la
leadership attraverso fonti letterarie. f Rossana Trifiletti compie un accurato, approfon-
dito e appassionato itinerario sociologico nella lettura
dei classici ad opera di Luciano Cavalli, a partire da un
seminario nel 1968 dedicato a Max Weber: religione e
società. L’Autrice, pur nella propria ricerca incontrando
temi e autori altri rispetto a quelli di Cavalli, si colloca
a pieno nella sua lezione sull’uso e la ricezione dei clas-
sici, così come sull’uso della storia e della comparazione. Cavalli viene fatto dialogare con filoni di ricerca diversi,
e pur tuttavia tutti espressione di una critica al positivi-
smo e all’empirismo astratto perché astorico, proprio di
una certa sociologia, non solo americana, verso cui lo
stesso Cavalli ha avanzato argomentate critiche. In par-
ticolare Rossana Trifiletti ci conduce con conoscenza e
competenza nella originalità della lettura weberiana di
Cavalli, ponendolo a fianco di Tenbruck e Schluchter,
due dei principali studiosi del Weber che hanno contri-
buito a sottrarlo dalle secche della sola ricezione par-
sonsiana. Vi è infine il riconoscimento di un’apertura a
contributi altri della sociologia, finanche a un suo possi-
bile apprezzamento di riflessione sociologica più recente,
come la public sociology, senza però mai forzare l’autono-
mia e lo spirito critico di Cavalli in postume torsioni del
suo pensiero. Franca Bonichi affronta quello che nel suo saggio
viene definito il filo conduttore della postura intellettua-
le di Luciano Cavalli nel suo itinerario di ricerca socio-
logico-politico, ossia la prospettiva di realismo radicale,
in particolare attraverso un approfondito esame de Il
leader e il dittatore. Uomini e istituzioni di governo nel
“realismo radicale” (2003). l reso possibili le due giornate di studio tenutesi in ricor-
do del Professor Luciano Cavalli, e a tutti gli allievi, gli
amici e i colleghi del Professore che hanno preso parte al
Convegno, oltre a coloro che hanno reso possibile la rea-
lizzazione di questo volume. Desidero infine esprimere
un particolare ringraziamento a Gianfranco Bettin, Fon-
datore e a lungo Direttore di questa rivista, non solo ha
reso possibili il convegno dedicato al Professor Luciano
Cavalli e questo numero speciale di «SocietàMutamento-
Politica», ma ha continuato la lezione di Luciano Cavalli
facendo della Scuola fiorentina un luogo di ricerca e di
formazione di nuove generazioni di sociologi. Nel suc-
cedergli come Direttore della rivista porto un debito di
gratitudine e insieme l’onere e l’onore di continuare la
lezione sociologica e accademica appresa. pp
g
All’interno delle riflessioni sviluppate in occasione
del convegno tenutosi presso la Accademia “La Colom-
baria”, si segnala la brillante e argomentata ricerca di
Paolo Turi dedicata all’uso delle biografie nell’opera di
Luciano Cavalli. Turi ha offerto una documentata e pun-
tuale analisi di una metodologia di ricerca che va ben
oltre la capacità di Cavalli di far uso di fonti diverse per
la ricostruzione delle biografie personali e politiche dei
casi di studio. Pur se Luciano Cavalli non ha lasciato
una esplicita codificazione della “sua” metodologia su
tale tema, attraverso il lavoro di Turi emerge il valore
di un sociologo che nel solco weberiano non disgiunge
teoria ed empiria. A Paolo Turi, il cui ritratto biografico-
intellettuale di Luciano Cavalli consente di approfondire
l’uomo e lo studioso Luciano Cavalli, in specie alle nuo-
ve generazioni di studiosi (Turi 2008), ancora una volta
va il merito di aver messo in luce un aspetto fondamen-
tale per la conoscenza dell’opera di Cavalli, senza il qua-
le non si potrebbe comprendere appieno la profondità
dei risultati stessi della sua teoria sociologica. * * * Le immagini presenti in questo fascicolo appartengo-
no all’archivio personale della famiglia Cavalli e sono sta-
te gentilmente messe a disposizione di SocietàMutamento-
Politica per la pubblicazione del volume. A conclusione di questa introduzione sia consentito
a chi scrive mostrare un debito di riconoscenza rivolto
al Professor Luciano Cavalli per le lunghe conversazioni
avute negli anni e per quella che considero una lezione
sociologica che ha costantemente alimentato la ricerca di
quella immaginazione sociologica che solo un Maestro
sa stimolare. L’Autrice si muove con una
non comune conoscenza del pensiero di Cavalli sul rap-
porto tra élite e masse, e poi su quello fra leader e mas-
se, osservando come proprio l’impianto critico del pro-
prio approccio sociologico portasse Cavalli a svelare le
manipolazioni e le aporie ideologicamente orientate che
si celavano nell’acritica esaltazione della partecipazio-
ne di massa alla politica. Dopo aver messo in relazione
la sociologia di Cavalli con gli sviluppi, invero spesso
anticipati, delle più recenti ricerche dell’elitismo demo-
cratico, l’Autrice dedica una parte del suo saggio alla
critica che Cavalli rivolge al costruttivismo inteso nella
sua declinazione di “democraticismo umanitario”. Viene
così colta la critica che Cavalli rivolge a quelle prospet-
tive teoriche, e non di meno alle ideologie politiche, che
espungono il conflitto dall’orizzonte della politica e del-
la società, riaffermando invece che proprio il conflitto,
weberianamente inteso, fra individui, gruppi e Stati, è al
centro della storia. i p
Marco Bontempi ci guida dentro uno dei temi cen-
trali della ricerca sociologica di Luciano Cavalli, quel
mutamento sociale che emerge dal dialogo con la sele-
zione di autori classici a partire da tre specifiche pubbli-
cazioni, Max Weber: religione e società (1968), Il muta-
mento sociale (1970) e Sociologia della storia italiana
(1871-1974). L’Autore analizza le modalità di dialogo fra
Cavalli e i diversi autori affrontati, spiega i nodi cruciali
che ne hanno originato la scelta e si sofferma sull’origi-
nalità della trattazione mai scolastica, e neppure mera-
mente manualistica, dei casi trattati, riconducendoli al
filo conduttore dell’interesse sociologico mosso da que-
sti autori nell’impianto generale del pensiero di Luciano
Cavalli. Viene così spiegata anche la scelta di inserire
fra gli autori trattati i Lynd e Mills, selezione origina-
le e inusuale per quel tempo, richiamando il legame fra
una prospettiva critica della sociologia e l’attrazione da
parte di Cavalli per la figura del dissenter, “lo studioso
controcorrente, acribico appassionato e creativo”. È infi-
ne nella terza delle tre opere prese in considerazione che
per Bontempi emerge la capacità di Cavalli di calare nel-
la realtà storica del proprio tempo la teoria sociologica. Il numero monografico si conclude con un’intervista
di Renzo Ricchi a Luciano Cavalli a partire dal volume
Giulio Cesare, Coriolano e il Teatro della Repubblica. Una 13 Democrazia e società: la sociologia di Luciano Cavalli lettura politica di Shakespeare (2006), in una precedente
versione apparsa sulla rivista Nuova Antologia nel 2007. La possibilità di confrontarsi direttamente
con il Professore su temi di ricerca legati alla leadership,
al carisma e alla democrazia hanno costituito un onore
per chi, come chi scrive, ha cercato di sviluppare questi
campi di ricerca nella sua attività di sociologo politico. La profonda conoscenza, la disponibilità al dialogo, la
possibilità di ricevere insegnamenti, indicazioni, corre-
zioni, spunti di ricerca, testi da approfondire, costitui-
scono un patrimonio prezioso, dal punto di vista umano
e accademico, di cui chi scrive è estremamente ricono-
scente. In particolare, durante le conversazioni avute con
il Professore sono stati affrontati alcuni temi di partico-
lare rilevanza, quali la necessità di riprendere il filo di
una sociologia della democrazia, così come il dibattuto
tema del rapporto tra leadership e partiti, sia nei classici,
sia nella sociologia politica contemporanea. Tutto que-
sto nel corso di conversazioni sociologiche con la con-
sapevolezza di poter dialogare con uno dei Maestri della
sociologia italiana. A questo non rituale ringraziamento
se ne aggiungono altri, in primo luogo alla moglie del
Professor Luciano Cavalli, dott.ssa Anna Servettaz, e ai
figli. Un pensiero di gratitudine va al Professor Sandro
Rogari e all’Accademia La Colombaria per aver voluto e 1 Il testo qui riportato è il saluto del Professor Sandro Rogari, già Preside della Facoltà di Scienze
Politiche Cesare Alfieri dell’Università di Firenze e attuale Presidente dell’Accademia La Colom-
baria al Convegno dal titolo Società e democrazia nel pensiero di Luciano Cavalli, tenutosi a Firen-
ze nei giorni 17 e 18 novembre 2022. Il Convegno è stato organizzato con la partecipazione della
Sezione di Sociologia politica dell’AIS e della Scuola di Scienze Politiche “Cesare Alfieri” dell’Uni-
versità di Firenze. L’Accademia toscana di scienze e lettere “La Colombaria” è un’istituzione cultu-
rale, originariamente ospitata nella “torre colombaria” del Palazzo Pazzi a Firenze, di cui il Profes-
sor Luciano Cavalli è stato a lungo socio e poi socio onorario. RIFERIMENTI BIBLIOGRAFICI Bettin G. (a cura di) (1997a), Politica e società. Studi in
onore di Luciano Cavalli, CEDAM, Padova. Bettin G. (1997b), «Un sociologo per la democrazia», in
G. Bettin (a cura di) (1997a), Politica e società. Stu-
di in onore di Luciano Cavalli, CEDAM, Padova, pp. 11-31. Bettin G., Turi P. (a cura di) (2008), La sociologia di
Luciano Cavalli, Firenze University Press, Firenze. Burawoy M. (2021), Public Sociology. Between Utopia and
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(1965), La città divisa. Sociologia del consenso e del
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(1968), Max Weber: religione e società, il Mulino,
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(1970), Il mutamento sociale. Sette ricerche sulla civ-
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(1974), Sociologia della storia italiana 1861-1974,
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(1981), Il capo carismatico: per una sociologia weberi-
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ana della leadership, il Mulino, Bologna. Magatti M. (2016), «Il paradosso dell’eredità weberiana»,
in Studi di Sociologia, 54 (1): 85-91. p
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(1982), Carisma e tirannide nel secolo XX. Il caso Hit-
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(1982), Carisma e tirannide nel secolo XX. Il caso Hit-
ler, il Mulino, Bologna. Turi P. (2008), «Sociologia e politica nell’itinerario intel-
lettuale di Luciano Cavalli», in Bettin Lattes G., Turi
P. (a cura di), La sociologia di Luciano Cavalli, Fire-
nze University Press, Firenze, pp. 23-189. g
-
(2003), Il leader e il dittatore. Uomini e istituzioni di
governo nel “realismo radicale”, Ideazione, Roma. -
(2003), Il leader e il dittatore. Uomini e istituzioni di
governo nel “realismo radicale”, Ideazione, Roma. Weber M. (2004 [1919]), La scienza come professione. La
politica come professione, Einaudi, Torino. -
(2006), Giulio Cesare, Coriolano e il Teatro della
Repubblica. Una lettura politica di Shakespeare, Rub-
bettino, Soveria Mannelli.h -
(2006), Giulio Cesare, Coriolano e il Teatro della
Repubblica. Una lettura politica di Shakespeare, Rub-
bettino, Soveria Mannelli.h Coser L. A. (1971), Masters of Sociological Th ought: Ide-
as in Historical and Social Context, Harcourt Brace
Jovanovich, San Diego. RIFERIMENTI BIBLIOGRAFICI societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Citation: Sandro Rogari (2023) Accade-
mia Toscana di Scienze e Lettere “La
Colombaria”. SocietàMutamentoPo-
litica 14(27): 15-16. doi: 10.36253/smp-
14330 Sandro Rogari1 Copyright: © 2023 Sandro Rogari. This is
an open access, peer-reviewed article
published by Firenze University Press
(http://www.fupress.com/smp) and dis-
tributed under the terms of the Crea-
tive Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. Sono particolarmente lieto, a nome di tutta l’Accademia, di portare il
saluto dei Soci e mio personale a questo seminario che Gianfranco Bettin e
Lorenzo Viviani hanno promosso nella ricorrenza di un anno dalla scompar-
sa del loro comune maestro professor Luciano Cavalli. È prassi dell’Accademia ricordare con un seminario di studi e di ricordo
l’eminente socio scomparso. Tuttavia, in questo caso il mio saluto e la mia
partecipazione di esordio a questo seminario non hanno il carattere della
formalità. Luciano Cavalli è stato un grande sociologo e fondatore, o meglio
rifondatore, della scuola sociologica della Facoltà di Scienze Politiche “Cesare
Alfieri” da quando fu chiamato dall’Università di Genova nel 1966. Il pro-
fessor Cavalli era un maestro come lo si intende nel senso più nobile del ter-
mine dell’Accademia e fondatore di quella scuola di sociologia ad eminente
carattere politico che ha concorso a rendere grande la “Cesare Alfieri” fino
alla fine del passato secolo. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. i
p
È stato inoltre docente di innumerevoli generazioni di studenti e io stes-
so mi sono preparato all’esame di Sociologia con il professor Cavalli studian-
do sui testi che ora trovate esposti nelle nostre bacheche grazie alla dispo-
nibilità della famiglia e in particolare della signora Anna che è qui con noi
assieme ai figli e che concorre a rendere questa giornata di studi così parte-
cipata e coinvolgente. Io devo essere molto grato a Gianfranco Bettin, nostro
socio, e a Lorenzo Viviani per l’impegno profuso nella organizzazione di
questo incontro di studio. Ma debbo aggiungere che molti dei vostri volti mi
richiamano alla memoria i tempi della nostra comune storia in Facoltà, dei
tanti Consigli, dei momenti lieti o difficili condivisi, comunque sempre di
una comunità che allora era molto unita e solidale pur nelle differenziazioni
disciplinari e di competenze. Competing Interests: The Author(s)
declare(s) no conflict of interest. Citation: Arnaldo Bagnasco (2023) Un
debito intellettuale. SocietàMutamen-
toPolitica 14(27): 17-20. doi: 10.36253/
smp-14331 Arnaldo Bagnasco Copyright: © 2023 Arnaldo Bagnasco. This is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. Non volevo mancare alla giornata di studio in onore di Luciano Cavalli
che si tenne a Firenze nel marzo del 2005. Mi ero laureato con lui all’Università
di Genova, e dopo alcuni anni mi aveva chiamato al Cesare Alfieri come inca-
ricato di sociologia economica, una specializzazione in cui nel frattempo avevo
cominciato a farmi le ossa a Torino. Rimasi a Firenze fino al 1979. Il mio lavo-
ro si era messo per una strada che si allontanava dai temi e dagli approcci più
caratteristici di Cavalli; al momento del convegno non lo incontravo da mol-
to tempo. Dovevo scegliere un argomento che in qualche modo potesse esse-
re in sintonia con suoi interessi scientifici e che, insieme, ricordasse l’influenza
che lui ha avuto nella mia formazione di sociologo. La scelta poteva dunque
mostrarsi un po’ complicata, un rientro difficile, per così dire; invece, fu faci-
le e immediata, mi vennero subito alla mente tema e taglio di una relazione
appropriata, che presentai con il titolo Un’interpretazione neo-weberiana della
città di oggi. Mi rifacevo alla famosa idea di Weber che in intermezzi storici
particolari, quando poteri superiori sono confusi o indeboliti, le città tornano
ad acquistare capacità politica autonoma. Qualcosa di simile si era verificato di
recente in Europa e anche in Italia, di fronte alle difficoltà dello stato nazionale
e alla presenza in crescita dell’Unione Europea. Da quanto mi disse quel gior-
no, credo che Cavalli avesse apprezzato la relazione.l Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. Quella dunque era stata per me anche l’occasione di riflettere sul mio
debito intellettuale nei suoi confronti. Sono lieto di poter tornare a farlo oggi,
e lo considero doveroso. La facilità della mia scelta per la relazione al con-
vegno era la conferma che quel debito, nonostante percorsi scientifici che si
erano allontanati, esisteva ed era importante. Come dicevo, ho incontrato Cavalli come studente all’Università di
Genova. Ricordare il professor Cavalli è un po’ come è stato ricordare Antonio
Zanfarino, nel gennaio del 2020, alla vigilia della pandemia. Per me stesso SOCIETÀMUTAMENTOPOLITICA 14(27): 15-16, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14330 16 Sandro Rogari Sandro Rogari e per molti di noi significa ricordare anni cruciali della
nostra esistenza dove queste figure di maestri sono state
centrali per la nostra formazione ed hanno accompagna-
to la nostra crescita accademica. Non mi resta che dare la parola al Presidente della
Scuola di Scienze Politiche “Cesare Alfieri”, erede della
vecchia facoltà, professor Carlo Sorrentino. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a SOCIETÀMUTAMENTOPOLITICA 14(27): 17-20, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14331 Negli anni al Cesare Alfieri ho portato a compimen-
to la pubblicazione di un volume sulla problematica ter-
ritoriale dello sviluppo italiano, nel quale presentavo un
modello della società italiana come articolazione di tre
formazioni sociali, particolari per economia, politica,
società. Tre Italie: il Nord-Ovest della grande industria,
il Meridione del sottosviluppo, il centro-nordest dello
sviluppo di piccola impresa (1977). A Firenze conobbi
anche Carlo Trigilia e insieme progettammo una vasta
ricerca che potesse approfondire i caratteri di economia
e società della piccola impresa, comparando casi riguar-
danti Toscana e Veneto.i Per la tesi, mi propose una ricerca sul potere di
comunità, una prospettiva allora sviluppata negli Stati
Uniti, ma poco seguita in Italia. E questo può essere da
me riconosciuto come un secondo elemento di debito
intellettuale nei suoi confronti, era una via promettente
da seguire. Molta parte del mio lavoro negli anni suc-
cessivi, dedicata ai problemi dello sviluppo, sarà proprio
sugli aspetti dell’organizzazione della società nello spazio. g
p
g
p
Nei suoi primi anni di sociologo, Cavalli si era
impegnato in ricerche empiriche su Genova, che nell’in-
sieme interpretava come “città divisa”. L’espressione ser-
vì anche come titolo di un volume del 1965 che le rac-
coglieva. Testo nel quale evidenziava come a Genova
ceto medio e operai vivessero generalmente in quartieri
diversi, non si mescolassero nella vita sociale e negli sva-
ghi, e avessero forti pregiudizi reciproci» (Cavalli 1965). I quartieri operai erano visti da lui come periferie oppo-
ste ai ceti borghesi delle zone centrali e a oriente della
città. Di conseguenza, proseguiva, «Il libero dibattito e
la dialettica delle idee […] vengono meno in questo cli-
ma di nette contrapposizioni ideali» (Cavalli 1965: 258). Muro contro muro era una metafora che valeva anche
per l’Italia di quegli anni, e Cavalli sapeva bene che
vederla radicata in città e quartieri, che osservava con
ricerche di dettaglio, era necessario per correttamen-
te vederla operante nel sistema più vasto della socie-
tà nazionale. Anche questa è una movenza weberiana:
per Weber la città è uno dei tanti modi in cui la socie-
tà prende forma, il tema della città appartiene allo stu-
dio complessivo della società, non è un tema derivato; e
la città divisa di Cavalli è appunto per lui un momento
della sua riflessione generale sulla difficile via alla demo-
crazia in Italia. È qui che mi ha introdotto in sociologia per la strada di Max Weber. È stato un avvio che ha segnato tutto il mio successivo percorso professio-
nale, del quale gli sono profondamente grato. Esplorazioni nei campi diversi
delle sociologie di ieri e di oggi, finiscono per me con selezioni e sistema-
zioni che alla fine mi accorgo di provare a ricondurre, consapevolmente, o
direi anche quasi d’istinto al metodo di Weber, per lo meno a raffrontarle
con questo. q
Quella di Weber, si mantiene come una delle radici più salde e produttive
della sociologia, alla quale Cavalli si rifaceva negli anni di ripartenza della
disciplina in Italia dopo la guerra, e alla quale tornerà sovente in seguito. La
sua generazione si era assunta il compito di ritrovare la ricca tradizione clas- SOCIETÀMUTAMENTOPOLITICA 14(27): 17-20, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14331 18 Arnaldo Bagnasco sica della sociologia, di stabilirne il valore nella società
in cerca di sue strade dopo la guerra e l’epoca dei tota-
litarismi, stabilendo anche rapporti con le diverse scuole
nazionali attive in quel momento. Cavalli era impegnato
con convinzione nel compito della sua generazione. do che al momento purtroppo non aveva a disposizio-
ne qualche risorsa e posizione da propormi. La trovai a
Torino, e fu in quell’ambiente che presi la strada della
sociologia economica, che continuai a coltivare anche
quando Cavalli mi propose un posto di incaricato, che
di comune accordo fu appunto di sociologia economica,
al Cesare Alfieri dove lui si era stabilmente trasferito. Il programma d’esame a giurisprudenza era dedicato
appunto a Weber, e a un giovane in formazione, la socio-
logia si presentava subito come una cosa seria, merite-
vole d’impegno e sulla quale poter investire in un’epo-
ca di trasformazioni sociali non facili da interpretare e
da vivere. Cavalli ne mostrava il significato e l’utilità sia
nelle ricerche sul campo, sia nell’esplorazione delle risor-
se teoriche in grado di orientarle. Devo dire di quella strada intrapresa, perché se si
allontanava da temi e interessi diretti e tipici di Caval-
li, per me, a ben vedere, significava anche ritrovare per
altre vie e coltivare in una specifica direzione la radice
di Weber. Robert Lynd tornò poi nella comunità un decennio
dopo, e fra le due ricerche c’era stata la grande depres-
sione dei primi anni Trenta, dalla quale si stava uscendo. Nella seconda ricerca, pubblicata con il titolo Middle-
town in transition, vennero messi in luce con maggiore
consapevolezza le dinamiche di classe e i fenomeni di
potere. Ho fatto solo un breve accenno sull’ascendenza di
Weber per la nuova sociologia e la political-economy
comparata. Aggiungo solo, perché questo riguarda anche
il debito di cui parlo oggi, che il contributo italiano a
questa prospettiva analitica ha anche il merito di aver
inaugurato una sua specifica applicazione a livello regio-
nale, vale a dire a un livello più specifico dell’organizza-
zione sociale nello spazio. Ricordato tutto questo, Cavalli chiude il capitolo su
Lynd con riferimento a Knowledge for what?, il libro che
può essere considerato «prosecuzione e coronamento
– così dice – delle grandi ricerche» (Ivi: 448). Il testo in
effetti condensa e ripropone in modo esplicito la critica
radicale della società e della sociologia americana di quel
tempo. È una critica del capitalismo e del suo effetto disu-
manizzante, una critica della sociologia dominante che
non riesce a riconoscerlo. Le scienze sociali devono esse-
re lo strumento per fare il mondo più umano contro una
cultura dominante non umana. A giudizio di Cavalli, il
quasi-marxista Lynd, come lo chiama, «affronta un pro-
blema serio in modo ragionevole e difendibile […] cercava
in effetti le condizioni di una società democratica, piani-
ficata e unita, e riteneva che fossero da reperire anche nel
dominio psicologico e morale, in certe esigenze (o predi-
sposizioni) umane, da coltivare e irrobustire» (Ibidem). p
Nel 1970, Cavalli ha pubblicato con il Mulino un
volume intitolato Il mutamento sociale, che aveva come
sottotitolo Sette ricerche sulla civiltà occidentale. Come
scrive l’Autore, il libro «è un risultato di una lunga ricer-
ca: una delle tante ricerche che non sono mai finite, per-
ché il loro vero oggetto è la vicenda storica globale che
stiamo vivendo, il loro vero fine conoscitivo è il senso
di questa ricerca» (Cavalli 1970: VII). Un’affermazione,
questa, che espone l’interesse centrale di tutta la socio-
logia di Cavalli. L’Autore, evidentemente, in quella affer-
mazione indica insieme il tema unificante della sua par-
ticolare ricerca, e come sia possibile ritrovarlo per aspetti
in qualche modo per lui significativi in alcuni Autori
che seleziona. È dunque indispensabile considerare l’or-
ganizzazione sociale nello spazio, e soprattutto lo è in
Italia, dove è molto differenziata. Dal punto di vista teorico, questo significava anche
entrare nell’ambito di una corrente di studi riconosciuta
come nuova sociologia economica, e a prospettive inter-
disciplinari di political-economy comparata, dedicata alle
diverse forme nazionali di capitalismo e alla loro trasfor-
mazione. Arrivo allora al punto per quanto sto riflet-
tendo oggi. Nell’insieme di questa evoluzione, infatti,
diventava visibile, in particolare per noi sociologi, l’in-
fluenza persistente di Weber. Le radici classiche dell’evo-
luzione che dicevo sono infatti molte, ma Weber occu-
pa una posizione particolare e riconosciuta. Particolare
soprattutto per noi sociologi, nell’approdo della political-
economy comparata. l Sintetizzerei una rapida esposizione dell’influenza di
Weber in un elenco di punti. Il primo, naturalmente, è la centralità dell’interes-
se per il capitalismo, in particolare per il capitalismo
moderno in sue varie forme, ma che risale alla sua gene-
si, e si interroga sul possibile sviluppo. Più in generale è
la sua prospettiva di comparazione di formazioni sociali
storiche diverse; e poi la visione plurifattoriale dei feno-
meni in analisi e l’attenzione ai sistemi istituzionali che
compongono particolari assetti di formazione sociale. L’interesse per l’economia è importante in Weber, come
anche oggi ovviamente nella political-economy com-
parata, ma mai isolato da quello per la società nel suo
insieme; questo significa anche rifiuto del determinismo
economico e dell’economicismo presente fra i classici Ritorno ora a quanto prima dicevo sulla mia strada
che si allontanava dai temi e dagli approcci più caratte-
ristici di Cavalli. Mi aveva incoraggiato dopo la laurea
a proseguire il mio impegno in sociologia, aggiungen- 19 Un debito intellettuale in Marx. Ad un livello diverso di riflessione è ripreso
dalla political-economy comparata il metodo weberia-
no che non mira a una teoria generale della società, e
procede con la costruzione di tipi-ideali, modelli inter-
pretativi da usare nella ricerca come strumenti, da non
confondere con la realtà; ma anche ritorna la sua tipica
idea di avalutatività, che non esclude una particolare
visione di valori e interessi dell’osservatore, ma richiede
che l’osservatore, come analista, si attenga a un rigoro-
so confronto con i fatti nell’esame dei fattori causali di
un fenomeno. Lo scopo non è trovare scientificamen-
te un’impossibile soluzione politica e pratica ai proble-
mi nella società, ma chiarificare le scelte di un attore
intenzionale, del quale Weber è preoccupato di vedere
e conservare i margini di autonomia: La political-eco-
nomy comparata eredita l’attenzione alla combinazione
di assetti istituzionali, con attenzione a stato, mercato,
società, e alla regolazione istituzionale dei loro rappor-
ti. La considerazione alla società richiede, in particolare,
attenzione a diversità di interessi e valori, e rinvia alle
classi, ovvero alle basi sociali della regolazione, su cui
Weber ha fornito importanti elementi concettuali anco-
ra influenti. Nel suo insieme, la metodologia di Weber
si presenta come molto aperta e adattabile, invita alla
costruzione di molti strumenti da utilizzare nella ricer-
ca; il carattere non deterministico della visione weberia-
na significa anche impossibilità scientifica di prevedere
la fine della storia, e questo atteggiamento è conservato
contro il fascino acritico di facili combinazioni o all’op-
posto di visioni apocalittiche. Durkheim, Gaetano Mosca, Weber – e successivi –
Robert Lynd, Parsons, Wright Mills. Cavalli sa bene che
la lista potrebbe aprirsi ad altri, e che quelli che ha scel-
to si prestano anche ad altre letture. Questa comunque
attiene alla “sua” ricerca, ma aggiunge Cavalli, mi «con-
forta nella scelta anche il fatto che i miei autori sono tra
quelli più spesso indicati dai colleghi contemporanei,
come i “grandi” della sociologia» (Ivi: VIII). A prima vista, può destare una certa sorpresa trova-
re in questo elenco Robert Lynd, che in genere non vie-
ne ricordato oggi quanto gli altri, non sembra altrettan-
to influente. Di questo è consapevole anche Cavalli, ma
le sue ragioni si capiscono appena si continua a leggere. La principale ragione, oltre all’importanza da lui data
alle ricerche localizzate, sta nel fatto che Lynd, ma anche
Wright Mills, sono scelti perché critici radicali dell’Ame-
rica e della sociologia americana del tempo. Lynd, insie-
me alla moglie Helen, come sappiamo ha condotto all’i-
nizio degli anni Venti una ricerca su aspetti fondamentali
della cultura, delle relazioni sociali, delle condizioni eco-
nomiche degli abitanti di una città di medie dimensioni
chiamata da loro Middletown, pubblicata con questo tito-
lo solo più tardi, dopo un lungo lavoro di sistemazione,
nel 1929. La ricerca suscitò notevole interesse, perché
faceva emergere una società americana molto diversa da
quella raccontata dal mito dell’american dream. RIFERIMENTI BIBLIOGRAFICI Cavalli termina il capitolo su Lynd osservando che
«vi sono passi in Knowledge for What? dove fa capire che
secondo lui un cambiamento radicale poteva attuarsi, in
America, solo in circostanze storiche speciali, quali era-
no forse state quelle degli anni tra Depressione e Ripre-
sa» (Ivi: 450). È un’intuizione importante, e non è un
caso che nelle critiche condizioni, generali e del nostro
mondo occidentale in particolare, sia riemerso negli ulti-
mi decenni il tema dei diversi generi di sociologia. Non
si tratta di differenti tipi di teorie, o di applicazioni del-
la sociologia a campi diversi, ma appunto del problema
sul significato del fare sociologia nella società posto da
Lynd, riconosciuto come capostipite di questa moven-
za tematica che aveva annunciato con la sua domanda:
sociologia per che cosa? Bagnasco A. (1977), Tre Italie. La problematica territoriale
dello sviluppo italiano, il Mulino, Bologna.f pp
g
Cavalli L. (1965), La città divisa, Giuffrè editore, Milano. -
(1970), Il mutamento sociale, il Mulino, Bologna. pp
g
Cavalli L. (1965), La città divisa, Giuffrè editore, Milano. -
(1970), Il mutamento sociale, il Mulino, Bologna. g
p
Il tema è stato rilanciato da Raymond Boudon, in
occasione della lezione inaugurale dell’European Aca-
demy of Sociology, nel 2001, che ha proposto al riguardo
quattro tipi di sociologia. È poi stato ripreso nell’indi-
rizzo presidenziale di Michael Burawoy all’inaugurazio-
ne dell’American Sociological Association del 2004, con
una proposta alternativa di quattro tipi, necessari gli uni
agli altri. Queste importanti aperture hanno suscitato un
ampio dibattito, ripreso anche in Italia. Non è necessario
però ora entrare più in dettaglio nelle tipologie, e non è
mia intenzione provare a far rientrare Cavalli in qualche
tipo. Non so se lui abbia seguito la questione, se lo ha
fatto, penso che non si sia certo riconosciuto, per esem-
pio, nel tipo di sociologia scientifica che Boudon consi-
dera la «sociologia che veramente conta», e non si sareb-
be sentito in sintonia con la “sociologia pubblica” in
continua interazione discorsiva con movimenti e asso-
ciazioni su cui insiste Burawoy; credo che non si sarebbe
sentito da nessuna parte. Importa invece per me rilevare
come lui avesse un “suo” modo di riprendere la doman-
da cruciale di Lynd, di averne fatto una sua costante pre-
occupazione intellettuale, e che questa sia ora tornata in
primo piano. Sociologia per che cosa? Sono Autori classici – Marx e Engels, Anche Cavalli si chiedeva a cosa servisse la sua
sociologia. È una domanda alla quale risponde con l’in- 20 Arnaldo Bagnasco Arnaldo Bagnasco tento di chiarificazione e pedagogico, come lui stesso
riconosce, al pari di Lynd. È una visione che corrispon-
de al suo stile di pensiero, che può assumere altre for-
me, ma qui è rilevante che il significato pratico e politi-
co della sociologia è per lui un problema ineludibile, sul
quale interrogarsi, da lui avvertito con insistenza e con-
tinuità nella sua vita di studioso. e capacità critica nella società italiana di allora. Termi-
no l’intervento dicendo che, per quanto mi riguarda, ho
conservato l’interrogativo come stimolo costante per la
ricerca di un senso per il mio lavoro. Citation: Giorgio Marsiglia (2023) Il magi-
stero di Luciano Cavalli come sociolo-
go critico. SocietàMutamentoPolitica
14(27): 21-27. doi: 10.36253/smp-14332 Giorgio Marsiglia Giorgio Marsiglia Giorgio Marsiglia Copyright: © 2023 Giorgio Marsiglia. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Parlare di Luciano Cavalli come sociologo critico può sembrare ecces-
sivo solo a chi non ha condiviso con lui, come me, il percorso intellettuale
che lo ha portato a scegliere e poi praticare l’insegnamento e la ricerca della
sociologia per oltre quaranta anni. g
p
q
In effetti, il primo aspetto che vorrei mettere in rilievo a proposito del
magistero di Luciano Cavalli riguarda proprio i miei esordi come studen-
te universitario nel corso di laurea in Scienze Politiche della Facoltà di Giu-
risprudenza dell’Università di Genova. Avevo scelto quel corso di laurea
perché conteneva come materia complementare l’insegnamento di sociolo-
gia, tema che mi aveva molto interessato nell’ultimo anno di Liceo, studian-
do Comte e la sua teoria della sociologia come scienza positiva della società. Nel mio primo anno di Università avevo già sentito parlare dell’arrivo di un
nuovo docente di sociologia che non solo introduceva gli studenti alla cono-
scenza dei padri fondatori di una vera e propria sociologia critica, ma utiliz-
zava metodi e approcci del tutto innovativi rispetto al tradizionalismo, basato
sull’insegnamento cattedratico e dogmatico e sullo studio acritico dei libri di
testo, che dominava, allora, nel corso di laurea in Scienze Politiche, come in
tutta la facoltà di Giurisprudenza. Avevo, peraltro, scelto di dedicare il mio
tempo di matricola proveniente da un liceo classico di provincia alla frequen-
tazione ed allo studio di quelli che mi apparivano come i grossi scogli da
superare in avvio della mia carriera universitaria, ossia gli esami di Istituzioni
di Diritto Privato, Economia Politica e Statistica, tutti estranei alla formazio-
ne umanistica fin lì ricevuta al liceo. Decisi pertanto di rimandare la mia fre-
quenza del corso di sociologia al secondo anno. RIFERIMENTI BIBLIOGRAFICI È un interrogativo che i
suoi allievi hanno trovato già nella didattica e nei lavo-
ri dei primi tempi a Genova, nel periodo critico del-
la ripresa del paese, e poi in libri come quello di cui ho
appena parlato; o in La democrazia manipolata, pensato
come la costruzione portata al limite di un ideal-tipo in
senso weberiano, con il quale suscitare consapevolezza societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Nel frattempo, se ben ricordo,
all’inizio di settembre del ‘64, vidi esposto nella vetrina di una libreria situa-
ta nei pressi dell’Università il libro di Luciano Cavalli che recava il seducen-
te titolo Il Sociologo e la democrazia (Cavalli 1964a), titolo che corrispondeva
esattamente agli interessi che allora stavo maturando e che mi avevano por-
tato alla scelta del corso di laurea in Scienze Politiche. Acquistai il libro e fui
subito conquistato dal discorso che Luciano Cavalli vi sviluppava. Dalla lettu-
ra di quelle pagine, che non sentivo distaccate ed accademiche, bensì piene di
partecipazione ed anche urgenza per una sorta di missione assegnata al socio-
logo, ho avvertito per la prima volta, da giovane studente ancora sprovveduto
sulla sociologia e le sue tematiche, ma già sensibile al rapporto tra sociologia e
democrazia, l’emergere di una personale attenzione, un coinvolgimento, anzi, Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 21-27, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14332 22 Giorgio Marsiglia Giorgio Marsiglia che è stato fra i motivi delle mie successive decisioni ad
approfondire lo studio della sociologia. to attraverso i seminari diventati parte quasi integrante
delle sue lezioni dedicate a temi di rilevanza locale, come
il rapporto tra cultura e politica a Genova, mediante una
ricerca esplorativa che coinvolgeva studenti e laureandi;
fu sempre in questi seminari che egli ci espose anche il
tema del potere di comunità, a partire da una lettura cri-
tica del libro di Floyd Hunter Community Power Struc-
ture (Hunter 1969). Fu anche attraverso questi seminari
che mi apparve chiaro il senso della iniziale esperienza
di ricerca di Luciano Cavalli come sociologo non acca-
demico, con gli studi da lui condotti sugli abituri a
Genova, sul quartiere operaio, e sui giovani del quartie-
re operaio, ricerche condotte per conto dell’Ufficio Studi
del Comune di Genova, da lui diretto. Anche in queste
ricerche, infatti, era già presente una impostazione cri-
tica ed una attenzione particolare sul ruolo del sociolo-
go e degli intellettuali in genere nell’assicurare un reale
coinvolgimento delle autorità istituzionali nell’affrontare
problemi sociali urgenti, al di là delle mediazioni partiti-
che e ideologiche. Anche la lettura del libro Gli immigra-
ti meridionali e la società ligure (Cavalli 1964b), basato
su una ricerca condotta sotto la guida di Cavalli dell’I-
stituto di Scienze Sociali da lui fondato e diretto fra il
‘61 e ‘63, manifestava un approccio critico non solo ver-
so i metodi di ricerca puramente quantitativi da cui egli
stava prendendo progressivamente le distanze, ma dalla
spiegazione riduttiva dei processi migratori, allora impe-
rante, e dalla trascuratezza del decisivo ruolo mediatore
dei partiti e dei gruppi di pressione nel processo di inte-
grazione degli immigrati. In realtà, quando cominciai a seguire il corso tenuto
da Luciano Cavalli, ebbi una prima momentanea delu-
sione, in quanto il docente aveva deciso, per quell’anno
accademico, di dedicare le sue lezioni esclusivamente
alla sociologia di Max Weber, studioso di cui a mala-
pena io conoscevo il nome, ma l’iniziale delusione fu
subito bilanciata dal metodo che Cavalli adottava, riser-
vando l’ultima parte di ciascuna lezione agli interventi
degli studenti, a volte spontanei, ma più spesso solleci-
tati dal docente stesso che interpellava singolarmente i
pochi presenti, circa una dozzina. Inoltre, a fine lezione
Cavalli era disponibile a dare suggerimenti di letture ed
approfondimenti. Decisi quindi, dopo quella prima espe-
rienza, di dedicare la mia tesi di laurea alla sociologia,
continuando a seguire il corso tenuto da Cavalli anche
l’anno successivo. Fu allora che il professore accettò
volentieri la mia richiesta di laurearmi con lui e mi sug-
gerì di dedicare la tesi a Charles Wright Mills, che io
conoscevo indirettamente in quanto l’anno precedente,
in alcuni seminari paralleli che Cavalli teneva ci aveva
parlato di questo autore, illustrando il contenuto di L’éli-
te del potere, allora già tradotto in italiano (Wright Mills
1959a [1956]), e di L’immaginazione sociologica, appena
uscito in versione italiana (Wright Mills 1962 [1959]). A questo libro Luciano Cavalli aveva anche dedicato
una recensione (Cavalli 1959), che subito mi affrettai a
leggere, procurandomi al contempo i due libri di Mills
già citati, oltre che Le cause della terza guerra mondia-
le, anch’esso uscito in traduzione italiana qualche anno
prima (Wright Mills 1959b [1958]). L’indicazione di
Mills, eminente sociologo critico, non era priva, come
ebbi modo presto di constatare, di una personale adesio-
ne che Cavalli aveva maturato alla concezione millsiana
del compito “liberatorio” della sociologia, della ripro-
posta dei valori illuministici di Ragione e Libertà che
essa conteneva, nonché del superamento dell’approccio
neopositivista e struttural-funzionalista che dominava
nella sociologia americana e dal quale progressivamen-
te Cavalli aveva preso le distanze, dopo una prima ade-
sione ai suoi paradigmi nella seconda metà degli anni
‘50. In effetti, il suo prolungato soggiorno americano,
che lo aveva portato a contatti personali sia con Charles
Wright Mills, che con Robert Lynd, altro esponente di
un approccio non convenzionale e sostanzialmente criti-
co al quale Cavalli dedicherà poi una parte del suo libro
Il mutamento sociale (Cavalli 1970a) e la traduzione del-
le due seminali ricerche su Middletown (Cavalli 1970b). Continuando negli anni successivi la mia frequentazio-
ne dei suoi corsi, ebbi modo di verificare le capacità di
insegnamento e le aperture critiche di Cavalli, soprattut- 2. Con la pubblicazione di Democrazia manipolata
(Cavalli 1964c) la vocazione critica di Luciano Caval-
li si dispiegava completamente; insieme alla riproposta
di un ruolo strategico della sociologia nella creazione
delle condizioni di una democrazia autentica, ancor-
ché fondata sulla partecipazione di una minoranza alle
decisioni di una élite responsabile e illuminata, Cavalli
si soffermava con spietata lucidità sugli ostacoli che in
una democrazia manipolata si frappongono allo svilup-
po pieno delle facoltà individuali comunque direzionate. In questo volume risultava molto evidente, ed anche per
me molto convincente, la critica alla Chiesa Cattolica, e
al Partito Comunista come fautori di un conformismo
delle masse manipolate anche con il ricorso ai mezzi di
comunicazione di massa oltreché agli strumenti preva-
lenti di socializzazione primaria e secondaria. L’autore
non abbandonava comunque la sua fiducia nelle funzioni
critiche garantite da una sociologia propriamente conce-
pita. Mi furono ancor più evidenti, in questo libro, inol-
tre, gli influssi da un lato di Cahrles Wright Mills, a cui
si deve il concetto sociologico di manipolazione, dall’al- 23 Il magistero di Luciano Cavalli come sociologo critico tro di Max Weber e della sua Realpolitik della Ragione
e degli élitisti classici, diffidenti nella possibilità di una
democrazia fondata su una partecipazione universa-
le delle masse alla politica. Già nell’introduzione al testo di Goode e Hatt Metodolo-
gia della ricerca sociale (Goode e Hatt 1962), significati-
vamente intitolata La ricerca sociale e i “problemi italia-
ni” (Cavalli 1962) e da lui tradotto insieme con la moglie
Anna, Cavalli criticava decisamente la concentrazione
della ricerca sociologica sugli aspetti quantitativi e sulla
survey, proponendo una metodologia multidisciplinare e
il ricorso alla tecnica del case study, che presuppone un
allargamento alle metodologie qualitative dell’intervista
libera e dello studio di contesto ed una concentrazione
sul problema sociale da comprendere piuttosto che sulla
trattazione quantitativa di rapporti causali. In effetti le
ricerche degli anni ‘70 hanno ampiamente fatto ricorso
ad un approccio qualitativo e multifattoriale che trova
la sua ispirazione nelle intuizioni di Max Weber. Questo
atteggiamento favorevole ad una sociologia, per l’appun-
to, multifattoriale e comprensiva resterà un punto fermo
in tutta la produzione sociologica successiva di Cavalli. g
pp
Nel frattempo, la mia tesi su Mills si avviava al com-
pletamento ed era seguita da Luciano Cavalli con rari
ma sempre puntuali suggerimenti di letture ed appro-
fondimenti; il docente non mancò peraltro, di utiliz-
zare il mio lavoro in elaborazione su Mills, facendomi
intervenire all’interno del suo corso per illustrare alcu-
ni libri non ancora tradotti del sociologo americano, in
particolare White Collar (Wright Mills 1953), fonda-
mentale studio sulla classe media americana. Natural-
mente il magistero di Cavalli come sociologo critico si
è prolungato negli anni seppur rivisto ed integrato in
buona parte, in funzione dello sviluppo degli interes-
si dell’autore per i temi del cambiamento sociale e della
leadership democratica. Di questi sviluppi parlerò più
avanti, ma voglio qui sottolineare che la mia collabora-
zione con Cavalli per lo meno lungo tutti gli anni ‘70, è
sempre continuata, pur nella diversificazione degli inte-
ressi, all’insegna della condivisione sulla funzione critica
e illuministica della sociologia. Con il mio trasferimento
al Cesare Alfieri, infatti, ho avuto modo di sperimentare
una applicazione del metodo critico di Cavalli nel corso
di Sociologia del 1969-‘70. Infine, con La città divisa
(Cavalli 1965) si confermava il periodo di Cavalli come
sociologo direttamente critico ancora una volta preoccu-
pato della individuazione e rimozione delle cause e delle
manifestazioni di divisione nelle città contemporanee. In
questo libro, che ebbe poi una ripubblicazione ampliata
e una nuova introduzione da parte dell’autore (Cavalli
1978), Cavalli riprendeva le sue ricerche degli anni ‘50
inserendole in un nuovo contesto teorico e problemati-
co nel quale si combinavano la proposta di temi quali il
potere di comunità e la devianza urbana e la preoccupa-
zione per il progressivo erodersi delle basi del consenso,
di fronte, ancora una volta, al dispiegarsi dei processi di
manipolazione che impediscono ai singoli individui di
prendere coscienza delle proprie possibilità di realizza-
zione individuale e di gruppo. critiche molto apprezzate dal docente. Anche le ricerche
a cui ho partecipato, in seguito, sotto la guida di Luciano
Cavalli nel corso degli anni ‘70 non hanno mai trascura-
to questa attenzione all’aspetto anche pedagogico di una
sociologia critica di ascendenza indubbiamente millsia-
na. Devo poi riconoscere che affrontando, molto più tar-
di, lo studio della sociologia di Pierre Bourdieu, autore
per alcuni versi piuttosto lontano dalle posizioni teoriche
di Cavalli, ho riscontrato, con soddisfazione, che anche
nella concezione del sociologo francese relativa alle pos-
sibilità liberatorie della sociologia ritrovavo posizioni
analoghe a quelle espresse dal mio maestro, lungo tutti
gli anni ‘60 ed anche oltre, anche se Cavalli non avrebbe
condiviso la fiducia illimitata nelle possibilità di emanci-
pazione dal dominio che Bourdieu attribuisce alla socio-
logia come scienza della società. 3. Un secondo aspetto del magistero di Cavalli
come sociologo critico riguarda la già ricordata presa di
distanza e la messa in discussione dell’approccio meto-
dologico neopositivista e quantitativista della ricerca
sociologica al quale, come ho detto, Cavalli aveva ade-
rito nella seconda metà degli anni ‘50, anche in conse-
guenza del suo soggiorno americano. D’altra parte, la
nascente sociologia italiana del dopoguerra, in cerca di
istituzionalizzazione si stava sviluppando secondo i cri-
teri metodologici ed epistemologici prevalenti negli Stati
Uniti, anche in risposta alle ripulse crociane e alle dif-
fidenze, se non ostilità, del Partito Comunista Italiano. 4. C’è poi un terzo importante aspetto per cui il
magistero di Cavalli si è rivelato non solo a me come
eminentemente critico. Il suo rapporto con i sociologi
classici. Già in Max Weber: religione e società (Caval- In particolare, di Marx coglie e valo-
rizza, senza trascurarne la critica, l’attenzione ai proces-
si di alienazione presenti nelle società capitalistiche e ai
meccanismi di crisi del capitalismo, presenti nei Mano-
scritti economico-filosofici del 1844 (Marx 1949 [1844]) e
l’analisi delle dinamiche di crisi inevitabile del capitali-
smo, proposte nel primo libro de Il Capitale (Marx 1964
[1818-83]), ma anche le analisi storico-politiche contenu-
te ne Il 1848 in Germania e in Francia (Marx e Engels
1948 [1848]), in Il XVIII Brumaio di Luigi Bonaparte
(Marx 1947 [1851]) e negli scritti sulla Comune di Parigi,
La guerra civile in Francia (Marx 1902 [1871]), nei quali
veniva valorizzata l’idea di una democrazia partecipativa
di tipo assolutamente radicale. Cavalli prendeva ovvia-
mente le distanze da una impostazione filosofica che
vedeva Marx privilegiare il primato della dimensione
economica su tutti gli altri aspetti della vita sociale, ma
non trascurava di mettere in evidenza il valore euristi-
co dell’affermazione marxiana della predominanza del-
le idee della classe dominante sulle forme ideologiche e
culturali che si diffondevano nelle società capitalistiche. Anche la divisione in classi, presente nella società capi-
talistica, che Marx riconduceva alla divisione economi-
ca tra borghesia e proletariato e la sottovalutazione delle
altre classi (contadini, piccoli proprietari, classe media e
sottoproletariato urbano) veniva criticata ma al contem-
po assunta come utile strumento per l’analisi di concreti
sviluppi che la divisione di classe assumeva nelle società
contemporanee. Invece, di Durkheim, anch’esso ripro-
posto con criteri filologicamente accurati, veniva privile-
giato ed in particolare veniva evidenziato criticamente il
contributo dato dal sociologo francese alla comprensio-
ne dei meccanismi di minaccia al legame sociale, con-
tenuti soprattutto in Il Suicidio (Durkheim 1897) nelle
f
d l
i idi
i ti
i l
t d l
i idi anomico proprio di società in crisi ed in rapido cambia-
mento. Presentando il pensiero di Durkheim su Le forme
elementari della vita religiosa (Durkheim 1912), mentre
esponeva accuratamente contenuti e limiti dell’approccio
durkheimiano allo studio delle religioni, Cavalli enu-
cleava con grande perspicacia l’importanza del rituale
nella vita sociale e soprattutto la rilevanza dei proces-
si collettivi creativi nella produzione di nuovi valori e
nuove norme sociali. Cavalli aveva affidato ai tre
assistenti che lo avevano seguito da Genova, trai quali
io stesso, la responsabilità di gruppi di studio paralle-
li dedicati agli studenti più desiderosi di affrontare un
lavoro critico su alcune istituzioni della società italia-
na; aveva dato la più ampia autonomia ad un lavoro che
vedeva, comunque La democrazia manipolata (Cavalli
1964c) e La città divisa (Cavalli 1965) come testi di rife-
rimento fondamentali, ma prevedeva molteplici appro-
fondimenti, condotti dagli studenti con grande cura e
seguiti dai tre responsabili con intensa partecipazione. Il lavoro dei gruppi si era concluso con ampie relazioni 4. C’è poi un terzo importante aspetto per cui il
magistero di Cavalli si è rivelato non solo a me come
eminentemente critico. Il suo rapporto con i sociologi
classici. Già in Max Weber: religione e società (Caval- 24 Giorgio Marsiglia Giorgio Marsiglia li 1968) Cavalli accompagnava l’esposizione puntua-
le e filologicamente accurata dei contributi di Weber
allo studio sociologico delle religioni mondiali con una
discussione dei punti cruciali del pensiero del sociologo
tedesco ed una valutazione critica dell’applicabilità delle
sue proposte teoriche al contesto delle società contempo-
ranee, ed in particolare dell’Italia. Ma è con Il Mutamen-
to Sociale. Sette ricerche sulla civiltà occidentale (Caval-
li 1970a) che Cavalli, come suggerisce già il sottotitolo,
esponeva il pensiero degli autori considerati – Marx ed
Engels, Durkheim, Mosca, Weber, Lynd, Parsons, Mills
– interrogandoli, per così dire, sulle risposte che ciascu-
no di loro dà al mutamento sociale proprio delle società
occidentali e ne espone criticamente le risposte, discu-
tendone potenzialità e limiti, non riducendo la sua trat-
tazione ad una mera esposizione del pensiero dei singoli
autori e tanto meno ad una ricerca della “giusta” lettura
dei loro contributi. Nella contemporanea introduzio-
ne alla nuova traduzione italiana di Il Suicidio e L’edu-
cazione morale, pubblicati in unico volume dalla UTET,
Cavalli ribadiva l’utilità del pensiero di Durkheim nella
spiegazione dell’ordine sociale e nella comprensione del
rapporto tra individuo e società (Cavalli 1969), sottoli-
neando in modo critico come il sociologo francese, ne
L’educazione morale, attribuisse all’adesione degli indivi-
dui alle norme sociali, sentite come una sorta di apriori
kantiano, e garantite dai meccanismi di socializzazione,
la condizione dell’ordine sociale e la persistenza del lega-
me sociale. Quanto a Parsons, Cavalli non si limitava a
esporre criticamente i volumi fondamentali del socio-
logo americano, ovvero La struttura dell’azione sociale
(Parsons 1937) e Il sistema sociale (Parsons 1951), ma
discuteva in particolare il contributo successivo dato
da Parsons nel corso degli anni ‘60, alla comprensio-
ne delle dinamiche del mutamento sociale proprie delle
società occidentali. Quanto a Gaetano Mosca, che Caval-
li già presentava come autore classico della sociologia
politica, veniva messo in evidenza il contributo dell’au-
tore alla comprensione dei meccanismi di formazione
di una classe politica responsabile e successivamente di
un secondo strato della classe politica identificabile con
la burocrazia pubblica specialistica, mentre veniva dato
risalto alla negazione da parte di Mosca di un ruolo
autonomo della massa nella formazione delle decisioni
politiche che la riguardano. A questo approccio élitista
Cavalli dava il merito di essere molto utile per compren-
dere le dinamiche del potere nelle società democratiche. Non a caso, presentando poi i contributi di Mills, dei
quali ho in parte già parlato, Cavalli metteva in eviden-
za l’approccio genericamente élitista già utilizzato dal
sociologo americano nella analisi della élite del potere e
dei rapporti di potere nella società statunitense. Natu-
ralmente Cavalli riprendeva anche in tono di apprez-
zamento le critiche di Mills a Parsons e al predominio
dei metodi quantitativi nella sociologia contemporanea,
non solo americana, e riproponeva l’idea millsiana di
una fondamentale funzione illuministica della sociologia
orientata dai valori di Ragione e Libertà. Infine, la parte
dedicata a Robert S. Lynd, rilancia l’idea di una funzio-
ne liberatoria della sociologia che il sociologo americano li 1968) Cavalli accompagnava l’esposizione puntua-
le e filologicamente accurata dei contributi di Weber
allo studio sociologico delle religioni mondiali con una
discussione dei punti cruciali del pensiero del sociologo
tedesco ed una valutazione critica dell’applicabilità delle
sue proposte teoriche al contesto delle società contempo-
ranee, ed in particolare dell’Italia. Ma è con Il Mutamen-
to Sociale. Sette ricerche sulla civiltà occidentale (Caval-
li 1970a) che Cavalli, come suggerisce già il sottotitolo,
esponeva il pensiero degli autori considerati – Marx ed
Engels, Durkheim, Mosca, Weber, Lynd, Parsons, Mills
– interrogandoli, per così dire, sulle risposte che ciascu-
no di loro dà al mutamento sociale proprio delle società
occidentali e ne espone criticamente le risposte, discu-
tendone potenzialità e limiti, non riducendo la sua trat-
tazione ad una mera esposizione del pensiero dei singoli
autori e tanto meno ad una ricerca della “giusta” lettura
dei loro contributi. In particolare, di Marx coglie e valo-
rizza, senza trascurarne la critica, l’attenzione ai proces-
si di alienazione presenti nelle società capitalistiche e ai
meccanismi di crisi del capitalismo, presenti nei Mano-
scritti economico-filosofici del 1844 (Marx 1949 [1844]) e
l’analisi delle dinamiche di crisi inevitabile del capitali-
smo, proposte nel primo libro de Il Capitale (Marx 1964
[1818-83]), ma anche le analisi storico-politiche contenu-
te ne Il 1848 in Germania e in Francia (Marx e Engels
1948 [1848]), in Il XVIII Brumaio di Luigi Bonaparte
(Marx 1947 [1851]) e negli scritti sulla Comune di Parigi,
La guerra civile in Francia (Marx 1902 [1871]), nei quali
veniva valorizzata l’idea di una democrazia partecipativa
di tipo assolutamente radicale. Cavalli prendeva ovvia-
mente le distanze da una impostazione filosofica che
vedeva Marx privilegiare il primato della dimensione
economica su tutti gli altri aspetti della vita sociale, ma
non trascurava di mettere in evidenza il valore euristi-
co dell’affermazione marxiana della predominanza del-
le idee della classe dominante sulle forme ideologiche e
culturali che si diffondevano nelle società capitalistiche. Anche la divisione in classi, presente nella società capi-
talistica, che Marx riconduceva alla divisione economi-
ca tra borghesia e proletariato e la sottovalutazione delle
altre classi (contadini, piccoli proprietari, classe media e
sottoproletariato urbano) veniva criticata ma al contem-
po assunta come utile strumento per l’analisi di concreti
sviluppi che la divisione di classe assumeva nelle società
contemporanee. Da Dahrendorf poi,
sempre durante la prima metà degli anni ‘70 Cavalli
trarrà ispirazioni non irrilevanti non solo per l’interpre-
tazione del conflitto nelle società moderne ma anche per
la sua concezione dell’individuo dotato di ragione come
elemento da considerare nella dinamica consenso-con-
flitto al di là delle appartenenze di gruppo o di classe. l
pp
g
pp
Ma è con Max Weber che Cavalli stabiliva un dia-
logo privilegiato; senza trascurare gli aspetti più pro-
priamente formali delle proposte teoriche di Weber, e
senza tralasciare le prospettive offerte dalla sua socio-
logia “comprendente”, Cavalli proseguiva il suo dialogo
con Weber, dapprima ricostruendo e discutendo la sua
idea di Carisma e di Capo Carismatico con Il Capo Cari-
smatico. Per una sociologia weberiana della leadership
(Cavalli 1981) e, successivamente, con Carisma e tiranni-
de nel secolo XX. Il caso Hitler (Cavalli 1982) cercando
di verificare la teoria weberiana del Capo Carismatico,
applicandola al caso storico di Hitler. La figura intellet-
tuale di Weber ed il suo contributo al ruolo del Carisma
personale nel risolvere la crisi delle democrazie moder-
ne, soffocate dal regime dei partiti, resterà un riferimen-
to sempre presente nella produzione successiva di Caval-
li, anche quando si applicherà specificamente al tema
della crisi e al problema della disgregazione sociale nella
società italiana. 7. In definitiva, facendo ricorso ancora una volta a
strumenti interpretativi assunti selettivamente dall’a-
mato Weber, ma anche dall’élitismo democratico e da
sociologi della tradizione riformista, Cavalli riformulava
anche in scritti successivi dedicati alla crisi della politi-
ca italiana, la sua adesione per quanto ormai disincanta-
ta all’idea di una potenziale funzione illuministica della
sociologia. Il progressivo frammentarsi della disciplina
in una miriade di specializzazioni, rinchiuse in sé, favo-
rita anche dalle parziali riforme universitarie, trovava
Cavalli ormai pessimista nei fatti, benché ancora legato
ad una utopia della funzione positiva della sociologia. i In particolare, vorrei infine sottolineare la valen-
za critica del superamento, da parte di Cavalli, della
distinzione accademica tra sociologia politica e scienze
della politica. Egli rifiutava praticamente prima ancora
che teoricamente, il confinamento da parte della scienza
politica, della sociologia politica ad uno studio prevalen-
temente quantitativo dei risultati elettorali e dei sondag-
gi di opinione con esclusione di approfondimenti teorici
e di analisi dei movimenti politici rivendicati, appunto,
dalla scienza della politica. Invece, di Durkheim, anch’esso ripro-
posto con criteri filologicamente accurati, veniva privile-
giato ed in particolare veniva evidenziato criticamente il
contributo dato dal sociologo francese alla comprensio-
ne dei meccanismi di minaccia al legame sociale, con-
i
i
Il S i idi
(D
kh i
1897)
ll 25 Il magistero di Luciano Cavalli come sociologo critico scienza politica, anch’essa in cerca di una propria auto-
nomia, nonché della filosofia politica. In Sociologia della
storia italiana 1861-1974 (Cavalli 1974) questo approccio
interdisciplinare aveva una prima completa applicazio-
ne. Il libro, che alla sua uscita non riscosse il consenso
che meritava, né da parte della comunità sociologica, né
da parte degli storici, probabilmente perché usciva in un
contesto politico ideologicamente orientato, come quello
dell’Italia degli anni ‘70. Conteneva, in effetti, una plu-
ralità di approcci che combinavano l’analisi delle stati-
stiche sociali, la ricostruzione dei processi storici e dei
momenti di crisi e di cambiamento della società italiana
e insieme la proposta di una “promessa” ripetutamente
formulata dalla classe politica sotto forme differenti ma
mai consapevolmente accolta e fatta propria dalle mas-
se manipolate. Analizzando poi, con gli stessi strumen-
ti teorici la crisi italiana ne L’Italia promessa. Riflessio-
ni sulla crisi nazionale (Cavalli 1976), Cavalli dava una
ulteriore prova della sua propensione al ricorso a stru-
menti interdisciplinari per spiegare l’evoluzione della
crisi italiana ed i suoi problemi sociali e l’inadeguatezza
della classe politica a rispondere efficacemente alle esi-
genze manifestate dagli individui e dai gruppi e ai pro-
blemi sociali che si erano via via aggravati. town (Lynd e Lynd 1929) e Middletown in transition
(Lynd e Lynd 1937), dallo stesso Cavalli presentate cri-
ticamente nella traduzione italiana (Cavalli 1970b), ma
soprattutto in Knowledge for what? (1939). town (Lynd e Lynd 1929) e Middletown in transition
(Lynd e Lynd 1937), dallo stesso Cavalli presentate cri-
ticamente nella traduzione italiana (Cavalli 1970b), ma
soprattutto in Knowledge for what? (1939). 5. Il discorso critico di Cavalli proseguiva poi con i
due volumi risultanti dai suoi corsi di sociologia tenu-
ti nei primi anni ‘70: l’antologia Ordine e mutamento
sociale (Cavalli 1971) e successivamente Sociologie del
nostro tempo (Cavalli 1973) dove l’esposizione selettiva
e critica degli autori considerati in precedenza veniva
allargata ad altri autori quali lo storico Arnold Toynbee
ed il sociologo Ralf Dahrendorf. Cavalli, già a partire dalla
fine degli anni ‘60, ma ancor più, con l’istituzione di un
dottorato in sociologia politica nel 1983 si muove libera-
mente tra le due discipline utilizzandone selettivamente
i contributi più funzionali alle sue analisi politiche del-
la società italiana. Così nei suoi scritti già a partire dagli 6. Un ulteriore contributo del magistero di Cavalli
ad una sociologia critica è costituito dal suo superamen-
to delle barriere disciplinari che si erano sviluppate in
Italia, e non solo, nel corso del processo di istituzionaliz-
zazione o re-istituzionalizzazione della sociologia come
scienza autonoma. Partendo probabilmente dai suoi
studi iniziali come storico, ma a mio giudizio, con una
crescente convinzione dell’errata concezione della socio-
logia come scienza distinta dalle altre scienze umane,
Cavalli ha sempre praticato una sociologia che si arric-
chiva dei contributi non solo della storia e della storia
del pensiero filosofico e politico, ma anche della psico-
logia, della psicanalisi, dell’antropologia culturale e della 26 Giorgio Marsiglia Giorgio Marsiglia anni ‘70, egli, pur non rinunciando all’utilizzo di dati
elettorali e risultati dei sondaggi politici nella definizio-
ne delle strategie della classe politica nella competizione
per il successo elettorale, ripropone la sua visione di una
politica che in conseguenza delle trasformazioni sociali,
ed in particolare dello sviluppo dei mass media, si allon-
tana sempre di più dal suo ideale weberiano ed élitista
insieme, della realizzazione di una democrazia parla-
mentare guidata da leaders qualificati e responsabili. Ricordo, inoltre, che nella predisposizione dei program-
mi del dottorato e nell’assegnazione delle tesi di dotto-
rato, Cavalli utilizza concretamente gli apporti di polito-
logi, di teorici politici, di storici della politica oltreché di
sociologi politici e di antropologi. Questa impostazione
interdisciplinare del dottorato, sempre portata avanti
con forme didattiche innovative che prevedevano inter-
venti dei dottorandi e loro contributi su punti specifici,
ha prodotto risultati di grande rilievo con le tesi di dot-
torato che spaziavano su temi sia di teoria politica che di
ricerca in sociologia politica ma anche su contributi dei
sociologi classici all’analisi politica. Mondiale di Sociologia, tenutosi a Stresa nel 1962 e poi
raccolto nel volume Sociologi e centri di potere in Italia
(Aa.Vv. 1962) pubblicato da Laterza, che anticipava la sua
pratica di una sociologia critica. RIFERIMENTI BIBLIOGRAFICI Aa.Vv. (1962), Sociologi e centri di potere in Italia, Later-
za, Bari. Cavalli L. (1959), «Recensione, Wright Mills C., The Soci-
ological Imagination», in Notiziario di Sociologia, II,
3: 11-15. – (1962), «La ricerca sociale e i “problemi italiani”», in
Goode W.J. e Hatt P.K., Metodologia della ricerca
sociale, il Mulino, Bologna: VII-XX. – (1964a), Il Sociologo e la democrazia, Silva Editore,
Milano. 8. In conclusione, vorrei sottolineare che il magiste-
ro di Luciano Cavalli come sociologo critico si è dispie-
gato nel corso del suo lunghissimo itinerario intellet-
tuale già a partire dagli anni ‘50, pur nella evoluzione
del suo pensiero fino alle pubblicazioni più recenti. Il
numero non indifferente di sociologi che egli ha forma-
to senza per questo costituirsi come caposcuola, ha in
modi diversi generato una pratica della sociologia che
risente comunque del suo magistero critico. È pur vero
che Cavalli non si è mai presentato, a differenza di altri
illustri sociologi, come Franco Ferrarotti, quale propu-
gnatore di una sociologia critica. La sua posizione appar-
tata e sostanzialmente scevra di apparizioni mediatiche
ha forse comportato un ritardo nel riconoscimento del
suo contributo essenziale all’affermazione della sociolo-
gia e alla sua istituzionalizzazione nel corso degli anni
‘60 e ‘70. Va anche ricordato che Cavalli non era uscito
dal laboratorio sociologico cresciuto intorno all’espe-
rienza innovativa di Adriano Olivetti, ma conosceva ed
apprezzava personalmente i sociologi che ne erano stati
protagonisti, dallo stesso Franco Ferrarotti, a Luciano
Gallino e ad Antonio Carbonaro; e forse non è un caso
che il suo principale contributo alla sociologia critica,
cioè La democrazia manipolata (Cavalli 1964c), sia sta-
to pubblicato proprio dalle Edizioni di Comunità, volu-
te da Olivetti stesso. D’altra parte, egli è stato anche in
contatto con il gruppo sociologico milanese che si racco-
glieva intorno ad Angelo Pagani, e con altre realtà della
ricerca sociologica italiana. Va anche ricordato, in sede
conclusiva, il suo contributo al seminario del Congresso – (1964b), Gli immigrati meridionali e la società ligure,
Franco Angeli, Milano. g
– (1964c), La democrazia manipolata, Edizioni di Comu-
nità, Milano. – (1965), La città divisa, Giuffrè, Milano. f
– (1968), Max Weber: religione e società, il Mulino, Bolo-
gna. – (1969), «Introduzione», in Durkheim É., Il suicidio. L’educazione morale, UTET, Torino. – (1970a), Il mutamento sociale. Sette ricerche sulla civiltà
occidentale, il Mulino, Bologna. – (1970b), «Introduzione», in Lynd R.S. Al di là del mio personale riconoscimento di Lucia-
no Cavalli come maestro, va dunque tenuto presente che
il suo magistero di sociologo critico ha segnato momenti
importanti ed ha influenzato più generazioni della socio-
logia italiana. RIFERIMENTI BIBLIOGRAFICI e Lynd H.M.,
Middletown, I, Edizioni di Comunità, Milano: IX-
XXV. – (1971), Ordine e mutamento sociale, il Mulino, Bologna. – (1973), Sociologie del nostro tempo, il Mulino, Bologna. – (1974), Sociologia della storia italiana 1861-1974, il
Mulino, Bologna.l g
– (1976), L’Italia promessa. Riflessioni sulla crisi nazionale,
il Mulino, Bologna. g
– (1978), La città divisa, Giuffré, Milano [2a edizione
ampliata]. – (1981), Il Capo Carismatico. Per una sociologia weberi-
ana della leadership, il Mulino, Bologna. – (1982), Carisma e tirannide nel secolo XX. Il caso Hitler,
il Mulino, Bologna. g
Durkheim É. (1897), Le suicide, Félix Alcan, Parigi. – (1912), Les formes élémentaires de la vie religieuse,
Presses Universitaire de France, Parigi. 27 27 Il magistero di Luciano Cavalli come sociologo critico Citation: Roberto Segatori (2023) Le tre
dimensioni di Luciano Cavalli. Mae-
stro, studioso, intellettuale pubblico. SocietàMutamentoPolitica 14(27): 29-40. doi: 10.36253/smp-14333 Citation: Roberto Segatori (2023) Le tre
dimensioni di Luciano Cavalli. Mae-
stro, studioso, intellettuale pubblico. SocietàMutamentoPolitica 14(27): 29-40. doi: 10.36253/smp-14333 Roberto Segatori Copyright: © 2023 Roberto Segatori. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Il maestro (conosciuto a distanza). Per chi si è laureato con una tesi in
sociologia nel 1970, come me, l’incontro pur indiretto con Luciano Cavalli è
stato senz’altro importante. Coloro che volevano investire nello studio del-
la sociologia (e non avevano ancora una padronanza adeguata delle lingue
straniere), potevano contare sulla traduzione dei classici promossa da pochi
decenni da alcune meritorie case editrici. Così – giusto per restare ai casi
più noti – i principali lavori di Émile Durkheim e di Max Weber erano sta-
ti pubblicati dalle edizioni di Comunità fin dagli anni Sessanta: La divisione
del lavoro sociale (Durkheim 1962 [1893]), Le forme elementari della vita reli-
giosa (Durkheim 1963 [1912]) e Le regole del metodo sociologico (Durkheim
1963 [1895]), e i due volumi di Economia e società (Weber 1961 [1922]). Di
Durkheim, la Utet aveva poi reso in italiano Il suicidio. L’educazione morale,
non a caso con un’Introduzione di Luciano Cavalli (Durkheim 1969 [1897]). Einaudi aveva fatto tradurre e aveva pubblicato, ancora di Weber, i due sag-
gi confluiti ne Il lavoro intellettuale come professione (Weber 1948 [1919]) e
Il metodo delle scienze storico-sociali (Weber 1958 [1922]). La stessa casa edi-
trice aveva messo a disposizione dei lettori italiani le maggiori opere della
Scuola di Francoforte: Eros e civiltà di Herbert Marcuse (1964 [1955]), Dialet-
tica dell’illuminismo di Max Horkheimer e Theodor W. Adorno (1966 [1947]),
L’uomo a una dimensione di Marcuse ([1964] 1967), Eclissi della ragione di
Horkheimer (1967 [1947]), Dialettica negativa di Adorno (1970 [1966]). In
pochi anni i lavori tradotti in italiano della “Teoria critica della società” si
erano aggiunti, sul versante del disvelamento radicale delle dinamiche del
potere sociale, ad alcuni testi fondamentali di Karl Marx, Friedrich Engels
e Vladimir Il’ič Ul’janov (Lenin) pubblicati dagli Editori Riuniti. Il magistero di Luciano Cavalli come sociologo critico – (1969), Il Suicidio - L’educazione morale, UTET, Torino. Goode W.J. e Hatt P.K. (1962), Metodologia della ricerca
sociale, il Mulino, Bologna. Hunter F. (1969), Community Power Structure, University
of North Carolina Press, Chapel Hill. Lynd R.S. (1939), Knowledge for what?, Princeton Univer-
sity Press, Princeton. Lynd R.S. e Lynd H.M. (1929), Middletown, Constable,
London. – (1937), Middletown in Transition, Harcourt Brace, New
York. Marx K. (1964 [1818-83]), Il Capitale, Editori Riuniti,
Roma.ii – (1949 [1844]), Manoscritti economico-filosofici del 1844,
Einaudi, Torino. – (1947 [1851]), Il XVIII Brumaio di Luigi Bonaparte,
Edizioni in Lingue Estere, Mosca. g
– (1902 [1871]), La guerra civile in Francia, Mongini,
Roma. Marx K. e Engels F. (1948 [1848]), Il 1848 in Germania e
Francia, Rinascita, Roma. Parsons T. (1937), The Structure of Social Action,
McGraw-Hill, New York.h – (1951), The Social System, Free Press, New York. Wright Mills C. (1959a [1956]), L’élite del potere, Fel-
trinelli, Milano. – (1959b [1958]), Le cause della terza guerra mondiale,
Feltrinelli, Milano. – (1962 [1959]), L’immaginazione sociologica, il Saggia-
tore, Milano. – (1953), White Collar, Oxford University Press, New
York. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. Continuando ad esplicitare ulteriormente le sue
intenzioni, Cavalli sottolineava come tutta la trattazione
si ispirasse all’idea di mettere a confronto e a far dialo-
gare (anche in modo polemico) tra loro i diversi autori. Rileggendo il testo, si può infatti constatare che, il perno
delle prime quattro parti – dedicate agli studiosi europei
– è costituito dal pensiero di Marx ed Engels, rispetto ai
quali vengono “fatti reagire” Durkheim, Pareto e Weber;
le tre parti successive – centrate su autori statunitensi –
sono destinate all’illustrazione della differenziazione/
contrapposizione di Robert Lynd e Charles Wright Mills
rispetto alla “Grande Teoria” di Talcott Parsons. Tutto semplice, dunque? No, perché il salto dal prin-
cipale manuale universitario in uso nel periodo – l’allu-
sivo e gracilino Introduzione alla sociologia di Alex Inke-
les (1967 [1964]) edito da il Mulino – allo studio diretto
e approfondito degli autori classici non era, ad essere
sinceri, immediatamente facile. Questa impostazione serviva a Cavalli a dimostrare
che all’approccio mono-fattoriale di Marx ed Engels, pur
importante e approfondito in ben 146 pagine, fossero
da affiancare (e per certi versi) preferire gli sguardi più
aperti e meno condizionati di Durkheim, di Weber e in
parte di Mosca; nonché, alla stessa maniera, che il “siste-
ma fin troppo integrato” di Parsons fosse opportuna-
mente chiosato e relativizzato dagli studi e dalle ricerche
empiriche di Lynd e Wright Mills. Di fronte a questo gap di comprensione approfondi-
ta, diventava necessario (ieri, ma sospetto che il bisogno
sia anche di oggi) ricorrere a studiosi che si dedicassero
ad opere di presentazione-intermediazione del pensiero
teorico degli stessi classici. Se ne accorge presto il Muli-
no, che pubblica Tipologia e storia della teoria sociologica
di Don Martindale (1968 [1961]); se ne fa carico Later-
za, che pubblica Storia della sociologia di Friedrich Jonas
(1970 [1968]). Due anni più tardi Mondadori inizierà a
pubblicare Le tappe del pensiero sociologico di Raymond
Aron (1972 [1965]), un testo che conoscerà numerose
edizioni. Peraltro, tali strumenti si confermavano tutti
opera di autori non italiani e con testi (almeno nel caso
dei primi due) abbastanza macchinosi. Confesso che all’epoca (avevo 23 anni) non dedicai
molta attenzione all’Introduzione del Mutamento socia-
le (Cavalli 1970). Magari mi chiesi come mai, accanto ad
autori indiscutibilmente “classici”, Cavalli si fosse impe-
gnato a presentare analiticamente anche il pensiero di
studiosi in fama di outsider come Lynd e Wright Mills. Del filo-
ne funzionalista del ‘900 (sia in chiave critica sia in chiave conservatrice o
struttural-funzionalista), Il Mulino aveva provveduto a pubblicare Teoria e
struttura sociale di Robert K. Merton (1959 [1949]) e La struttura dell’azio-
ne sociale di Talcott Parsons (1962 [1937]), mentre l’editore Rizzoli proponeva
lo strutturalismo epistemico francese con Storia della follia (Foucault 1963
[1961]) e L’archeologia del sapere (Foucault 1972 [1971]). E non solo questo. Lo stesso Mulino aveva pure aperto una breccia all’approccio del costrutti-
vismo sociale inserendo nella propria collana di Scienze sociali La realtà
come costruzione sociale di Peter L. Berger e Thomas Luckmann (1969 [1966])
e aveva introdotto manuali di metodologia della ricerca di autori stranieri: Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 29-40, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14333 30 Roberto Segatori Metodologia della ricerca sociale di William J. Goode
e Paul K. Hatt (1962 [1952]), di nuovo curato e tradot-
to da Luciano Cavalli e Anna Cavalli Servettaz; Meto-
dologia e ricerca sociologica di Paul F. Lazarsfeld (1967
[1948-1954]) e L’analisi empirica nelle scienze sociali di
Raymond Boudon e Paul F. Lazarsfeld (1969). Metodologia della ricerca sociale di William J. Goode
e Paul K. Hatt (1962 [1952]), di nuovo curato e tradot-
to da Luciano Cavalli e Anna Cavalli Servettaz; Meto-
dologia e ricerca sociologica di Paul F. Lazarsfeld (1967
[1948-1954]) e L’analisi empirica nelle scienze sociali di
Raymond Boudon e Paul F. Lazarsfeld (1969). Proseguendo nella descrizione del suo progetto, egli
ammetteva che la scelta degli autori da lui effettuata era
dettata dal proprio (inevitabile) punto di vista di ricer-
catore e che, semmai, gli restava il «rimpianto serio» di
«aver “chiamato” solo Mosca, e non anche Pareto, per
approfondire il punto di vista elitista» (Ivi: VIII). Questa panoramica, veloce e inevitabilmente parzia-
le, è utile a dare un’idea degli strumenti di cui un giova-
ne appassionato di sociologia poteva usufruire nell’Italia
di più di cinquant’anni fa. A ciò si aggiunga che i prin-
cipali sociologi italiani dell’epoca erano stati chiamati
dalla Scuola Editrice di Brescia ad approfondire le sotto-
aree disciplinari della materia nei due volumi di Questio-
ni di Sociologia (AA.VV. 1966). Maestro, studioso, intellettuale pubblico Maestro, studioso, intellettuale pubblico Certo, un uso esclusivo (ovvero senza un’immersione
nelle opere originali) dell’intermediazione/interpre-
tazione cavalliana di Marx, Durkheim, Weber, Par-
sons e degli altri autori rischiava e rischia di creare un
improprio senso di appagamento conoscitivo. Ma se
la mirabile lezione di Cavalli servì e serve ancora oggi
ad alimentare una curiosità esplorativa più avanzata e
profonda, caratterizzata dalla stessa acribia presente in
quelle pagine, allora è giusto riconoscere che lì c’è –
ancorché produttrice di possibili esiti ed orientamenti
differenti – l’impronta di un maestro. santi. Il principale dei quali era quello di trasmettere un
modello di serietà e rigore (e a tratti persino di sacralità)
nell’impegno di studio e di ricerca richiesto all’universi-
tà. Tornerò più avanti sulle ricadute positive – ancorché
in parte intimidenti – di tale clima. Il secondo misunderstanding riguardava e riguar-
da l’apparente somiglianza ma la sostanziale differen-
za tra il pensiero di Luciano Cavalli e quello di Franco
Crespi, mio maestro perugino. Entrambi possono esse-
re considerati espressione – anche se tale collocazione
è accolta da tutti e due (e per ragioni differenti) come
riduttiva – del cosiddetto “individualismo metodolo-
gico”. Ovvero, nella scelta della prospettiva sociologica
da utilizzare, tra l’individualismo e l’olismo (sempli-
ficando molto, per la prima è l’essere umano – il sog-
getto – con le sue relazioni a costruire la società; per
la seconda, è la società con le sue strutture a costrui-
re l’essere umano) sia Cavalli sia Crespi sono schiera-
ti, pur con vari accorgimenti, nella prima prospettiva. In più – e di conseguenza – entrambi trovano in Max
Weber il proprio punto di riferimento tra i classici,
condividendone l’approccio multifattoriale nell’analisi
delle dinamiche sociali e la distinzione tra relazione ai
valori e giudizi di valore. i 2. Lo studioso (incontrato da vicino). Nel 1984 la
Facoltà di Scienze Politiche “Cesare Alfieri” attiva il pri-
mo Dottorato di ricerca italiano in sociologia politica,
dopo una progettazione iniziata nel 1981 insieme a quel-
la del nascente Centro di sociologia politica. A dirigere il
Dottorato e il Centro fino al 1996 è Luciano Cavalli, che
si avvale della collaborazione dei docenti fiorentini (che
in parte lo avevano seguito da Genova) Gianfranco Bet-
tin (futura guida dei due organismi), Paolo Giovannini,
Giorgio Marsiglia e Paolo Turi. Nella costituzione inter-
universitaria del dottorato e del centro fiorentini viene
invitata in partnership anche l’Università di Perugia. Maestro, studioso, intellettuale pubblico Così i “perugini” Franco Crespi, ordinario di sociologia
generale, e il sottoscritto, all’epoca associato di socio-
logia politica, entrano a fare parte dei rispettivi collegi
docenti. Ma le somiglianze tra Cavalli e Crespi finiscono qui. Infatti, per Crespi, Weber è soprattutto il teorico che
nel primo paragrafo di Economia e società sostiene che
«la sociologia […] deve designare una scienza la qua-
le si propone di intendere in virtù di un procedimento
interpretativo l’agire sociale [dotato di senso]» (Weber
1961 [1922]: 4). E su quell’agire sociale (con un “senso” al
fondo irriducibile ad ogni “significato”, contrariamento
all’uso che ne fanno Pareto e Parsons) Crespi costruisce
la sua teoria sociologica generale, appoggiandosi all’er-
meneutica e pervenendo alla fenomenologia (Segatori
Cristofori Santambrogio 2004: 9-43). L’ingresso nell’inner circle accademico di Lucia-
no Cavalli si rivela per me pieno di sorprese. Intanto a
colpirmi c’è il “clima di lavoro” di Via Laura 48 (sede
originaria dei sociologi fiorentini di Scienze Politiche,
prima del trasferimento in altre due zone). Cavalli appa-
riva manifestamente come il dominus della maggior par-
te delle attività didattiche e scientifiche. Al suo studio
si accedeva tramite una porta blindata, retaggio fisico
e simbolico della resistenza di un professore autorevole
alla contestazione malmostosa della coda del Sessantot-
to studentesco che aveva attaccato lui e l’amico preside
Giovanni Sartori. Rimaneva però l’impressione che quel-
la porta blindata ancora in uso marcasse un senso di
severo distacco tra il titolare della stanza e i suoi inter-
locutori. Le riunioni collettive avvenivano nel rispetto di
una duplice consegna: massima deferenza verso il mae-
stro, analogo formalismo nei rapporti tra i docenti (in
cui ovviamente faceva aggio l’inquadramento universita-
rio). Per me, che venivo da un ambiente di lavoro demo-
cratico e del tutto friendly, (nell’Istituto di Studi Socia-
li di Perugia ci si dava del tu dal professore ordinario
all’assegnista), quella frequentazione costituì una secon-
da socializzazione accademica, che, accanto a intuibili
motivi di perplessità, presentava anche aspetti interes- Luciano Cavalli si ispira invece alle pagine di Max
Weber che trattano soprattutto della sociologia politi-
ca (e in particolare del potere) e degli approfondimenti
storici destinati a fornire gli spunti per la costruzione
dei modelli idealtipici, specie di quelli riferiti all’ambito
politico-istituzionale. Non sapevo – l’avrei scoperto qualche tempo dopo – che
tali scelte erano collegate alla permanenza di Luciano
Cavalli alla Columbia University tra il 1952 e il 1956 e
alle conoscenze dirette ivi maturate. Per fortuna (almeno per me) nel 1970 esce per i tipi
del Mulino, Il mutamento sociale. Sette ricerche sulla
civiltà occidentale di Luciano Cavalli (Cavalli 1970). Per
un “apprendista stregone” qual ero (ma immagino per
tanti altri studiosi di generazioni diverse), qual volu-
me presentava una sua peculiarità e un grande merito. La peculiarità era relativa al progetto scientifico-cultu-
rale che l’autore dichiarava di perseguire. Cavalli – al
momento della pubblicazione quarantaseienne – si pro-
poneva (parole sue) di Perché allora trovai di pregevole fattura e di gran-
dissima utilità quel volume di più di seicento pagine
del 1970? Perché, come scrisse Luciano Gallino, in una
recensione su Quaderni di Sociologia, «il libro si racco-
manda soprattutto come una avvincente proposta di leg-
gere in modo drammaticamente concreto alcune delle
maggiori opere del pensiero sociologico dell’ultimo seco-
lo» (Gallino 1970: 455). Far parlare gli autori prescelti [Marx ed Engels, Durkheim,
Mosca, Weber, Lynd, Parsons, C. Wright Mills] su quello
che molti chiamano, e io con loro, il mondo occidentale:
che cosa lo definisca, quale posto abbia tra gli altri «mon-
di», quali siano le sue interne contraddizioni e i suoi con-
flitti […] E da questo discorso sull’Occidente cerco di rica-
vare soprattutto idee e indicazioni per l’Italia, che è in real-
tà al centro del mio interesse. (Ivi: XVII) In altre parole, Il mutamento sociale è un testo in
cui uno studioso infaticabile si trasforma in maestro
paziente per presentare ad allievi giovani e meno gio-
vani la tradizione teorica ed empirica della sociolo-
gia. E lo fa mettendosi nella condizione di riassumere
in maniera analitica e tendenzialmente completa ogni
opera, passaggio per passaggio, degli autori proposti. 31 Maestro, studioso, intellettuale pubblico È legittimo il sospetto che Cavalli abbia voluto invi-
tare Crespi (che riporta la sociologia nell’Università di
Perugia nel 1967) memore del fatto che Roberto Michels
– prima ancora di insegnare Scienza Politica all’Istitu-
to “Cesare Alfieri” di Firenze – aveva occupato fin dal
1928 la cattedra di Economia Generale e Corporativa e
insegnato Storia delle Dottrine Economiche alla Regia
Facoltà di Scienze Politiche dell’Università di Perugia. Ma, ahimè per Cavalli, la sociologia praticata da Franco
Crespi era ed è cosa ben diversa dall’elitismo ben moti- 32 Roberto Segatori vato (anche se alla fine colluso col fascismo) di Roberto
Michels. lutivo delle ambiguità insite nei concetti weberiani sopra
richiamati e fornisce la sua risposta a due questioni: a)
sul fatto che Weber abbia ispirato o meno l’avvento di
Hitler; b) sulla compatibilità tra carisma e democrazia. Per tornare all’impegno di studioso di Luciano
Cavalli, negli ultimi vent’anni del Novecento egli si dedi-
ca a un esercizio di approfondimento ispirato da uno
scrupolo tipico dello scienziato sociale. Per restare alle
categorie metodologiche di Weber, Cavalli è consapevole
che le questioni tematiche che lo interessano nascono da
un’evidente “relazione ai valori”. Mi riferisco al fatto che,
da sempre, la sua attenzione è catturata da una serie di
ipostatizzazioni storico-politiche. Esse sono, in succes-
sione consequenziale: a) la centralità del conflitto all’in-
terno di ogni paese e fra nazioni; b) l’importanza per la
risoluzione dei conflitti del ruolo dello Sato (nazionale);
c) la necessità che a guidare gli Stati, stanti i punti a e
b, siano “grandi individualità”. È evidente che, in origi-
ne, si tratti di assiomi (personali) prescientifici che egli
razionalizzerà più avanti in nome del “realismo politi-
co” (Cavalli 2003). Proseguendo su questa strada, Caval-
li trova la chiave di volta per sostenere il punto c della
sequenza nell’opera di Max Weber, il quale si sofferma
a lungo sui concetti (supportati da evidenze storiche) di
carisma, leader carismatico, Führer- demokratie, demo-
crazia plebiscitaria. Poiché però, a sua volta, lo stesso
Weber è fatto oggetto di letture non univoche dei suoi
scritti al riguardo, per i quali ebbe anzi a subire critiche
postume, il Nostro assume il rilevante impegno di stu-
diare scientificamente e chiarire sino in fondo il pensiero
politico weberiano, anche al fine (per nulla secondario)
di supportare il proprio. f Egli dichiara esplicitamente il suo intento fin
dall’Introduzione al Capo carismatico. Maestro, studioso, intellettuale pubblico Scrive infatti: In realtà, il vasto dibattito sul carisma si è sviluppato in
grande parte sulla base delle più note definizioni e formu-
lazioni teoriche di Economia e società […] senza sbrogliare,
ricostruire e completare (con gli elementi offerti dagli scrit-
ti politici e dalle grandi ricerche) la riflessione di Weber, in
tutta la sua complessità e profondità. Ne sono derivate, a
volte, interpretazioni superficiali e anche erronee, e conse-
guenti prese di posizione, magari vivacemente polemiche,
che avevano in realtà poco a che fare con il vero Weber. […] Questo saggio risponde al proposito di cominciare a
fare ciò che da tempo si doveva fare (Cavalli 1981: 9). Ma che cosa si doveva (si deve) dunque fare? La
risposta di Cavalli si concretizza in una doppia opera-
zione: storicizzare la riflessione e le categorie weberiane
e far emergere in modo completo e coerente l’articolata
tesi del maestro di Erfurt (che, non a caso, coincide con
la propria). Storicizzare Weber dovrebbe peraltro risulta-
re un compito ovvio, considerato che egli nasce nel 1864
e muore nel 1920, mentre il nazismo si afferma nel 1933. È piuttosto la situazione della Germania immediatamen-
te dopo la Prima guerra mondiale a convincere Weber
– rileva Cavalli – ad affiancare alla proposta di demo-
cratizzazione e parlamentarizzazione, che egli condivide,
l’indicazione della necessità di una Führer-demokratie,
in considerazione del fatto che la führerlose Demokratie
(una democrazia senza capo) fosse inadatta a risolvere i
problemi fondamentali della nazione. Cavalli decide così di affrontare in quegli anni un
impegno che – specie per me ancora in fase di affina-
mento scientifico – comportava due lezioni. La prima
consisteva nell’opportunità di vedere da vicino come un
importante sociologo italiano si misurasse nel passag-
gio dalla “relazione ai [suoi] valori” allo studio oggetti-
vo degli stessi valori (in questo caso, i concetti/eviden-
ze storiche weberiani). La seconda riguardava l’essere
messo a parte, in condizioni privilegiate, dei risultati di
merito di quello stesso studio. Da qui parte il lavoro di scavo di Cavalli sui con-
cetti weberiani di “razionalizzazione”, da cui l’intorpi-
dimento delle masse, e di “carisma”, associato al leader
politico che ha la missione di risvegliare e far cresce-
re le stesse masse. È nota la posizione ambivalente di
Weber circa la razionalizzazione. Maestro, studioso, intellettuale pubblico Maestro, studioso, intellettuale pubblico avviene qui per il Cavalli che interpreta Weber un rove-
sciamento della prospettiva: sarebbero i partiti a lascia-
re i cittadini nella condizione di sudditi, e non i leader
carismatici e la formula della democrazia plebiscitaria
che svolgerebbero invece una funzione liberatoria. Il carisma è chiamato in causa, a sua volta, come
la qualità che dovrebbe avere un leader (ovvero, nell’e-
timo inglese, una guida) per condurre il suo popolo a
(ri)trovare quei fini. Cavalli dedica tantissime pagi-
ne al tema del carisma, aprendone sistematicamente la
trattazione nell’opera del 1981 e restituendola in forma
cesellata nel libro stringato del 1995. L’inizio del suo
approfondimento non può che partire dalla definizione
classica che di carisma dà Weber in Economia e società
(1961 [1922]) e che egli ripropone in apertura del testo
del 1995: «Per carisma si deve intendere una qualità
ritenuta straordinaria […] di una personalità, per cui
questa viene creduta [esser dotata] di forze e proprietà
soprannaturali o sovrumane, o almeno specificamen-
te eccezionali, non accessibili agli altri, oppure come
inviata da Dio o rivestita di potere esemplare; e, quindi,
ottiene “riconoscimento” come capo [leader]» (Caval-
li 1995: 5). Successivamente, Cavalli esplora analitica-
mente – sempre sulle orme di Weber – il significato del
concetto in ambito religioso, in cui si afferma inizial-
mente, per pervenire infine all’ambito politico, che è
quello che propriamente gli interessa: «una delle molte
incarnazioni del capo carismatico nella storia è il lea-
der politico» (Cavalli 1981: 185).f g
In Governo del leader e regime dei partiti (Caval-
li 1992), Cavalli si impegna a mostrare la validità della
sua tesi alla luce dei casi del Regno Unito, della Francia
e degli Usa, dopo aver integrato la lezione di Weber con
i contributi di Ostrogorski e (nuovamente) di Michels,
nonché aver fornito ulteriori elementi a chiarimento del
concetto di democrazia plebiscitaria. Nella stessa opera
egli sottolinea che i fattori che favoriscono la democrazia
con leader siano indubbiamente da collegare al sistema
istituzionale (presidenzialismo o semi-presidenzialismo)
e al sistema tecnologico-comunicativo dei media. Pur-
tuttavia si riconferma in lui l’idea che il fattore decisivo
resti, comunque, la situazione di crisi di un paese. La sua
critica alla cosiddetta democrazia acefala controllata dai
partiti si conclude infine con queste parole: Il principio monocratico consente di sbaragliare le oligar-
chie, oppressivo residuo del passato. Maestro, studioso, intellettuale pubblico Consente al «popolo
sovrano» di scegliere chi governerà ai vari livelli del potere,
con un’elezione preparata e semplificata nei termini neces-
sari per renderla genuinamente democratica: scelta, ten-
denzialmente, tra due candidati selezionati da un accurato
scrutinio per ciascuna carica monocratica. Consente d’al-
tronde quella governabilità a tutto tondo di cui la demo-
crazia acefala, costruita sull’opposto principio, si è dimo-
strata totalmente incapace (Ivi: 293). Viaggiando tra le puntualizzazioni weberiane diffu-
se, oltre che in Economia e società (1961 [1922]) (un libro
pubblicato postumo e sistemato dai suoi curatori), in
La politica come professione (Weber 1997 [1919]) e negli
scritti politici, Cavalli integra le doti del capo carismati-
co con quelle della vocazione, passione e missione. A questo punto, egli (sempre con Weber) è pronto a
rispondere a due interrogativi: perché una democrazia
con leader (che resta sempre una democrazia) è meglio
di una democrazia acefala, e perché i dittatori – pur
essendo dotati di carisma (vedi in primis Stalin e Hitler)
– vanno tenuti ben distinti (anzi, vanno senz’altro con-
dannati) rispetto ai leader carismatici democratici. È difficile non leggere nelle frasi appena riportate
un’eco della critica alla partitocrazia ripresa nel secondo
dopoguerra da Giuseppe Maranini, preside della Facol-
tà di Scienze Politiche “Cesare Alfieri” dal 1949 al 1968
(giusto prima delle presidenze di Giovanni Sartori e di
Luciano Cavalli), e in un percorso iniziato come quel-
lo dello stesso Cavalli a Genova e conclusosi a Firen-
ze, dopo la docenza nel 1928 all’Università di Perugia. Ancora più indicativo è il fatto che Maranini dia al suo
discorso all’inaugurazione dell’anno accademico 1949-
1950 dell’Ateneo fiorentino il titolo Governo parlamenta-
re e democrazia. Il problema è – scrive Cavalli – che il “democrati-
cismo” e il “regime dei partiti”, ovvero l’opposto della
democrazia con leader anche da lui auspicata, manten-
gono in vita e non risolvono quel burocraticismo (che
colpisce in primo luogo gli stessi partiti, come documen-
tato da Roberto Michels) che è il prodotto perverso del
processo di razionalizzazione. Maestro, studioso, intellettuale pubblico Da un lato essa, in
quanto prodotta dall’Illuminismo e tradottasi nella
prevalenza dell’uso della ragione nei processi e nell’or-
ganizzazione della vita sociale, determina (in positivo)
il passaggio della società occidentale alla modernità;
dall’altro, per sua natura, essa finisce (in negativo) col
generare una crescente burocratizzazione, fino a costi-
tuire per le persone una “gabbia d’acciaio”. A risentirne
di più è in particolare la massa, che, appiattita e alie-
nata nelle routine burocratiche (che regolano il come
delle relazioni e delle cose, ma non indicano il perché),
finisce col perdere di vista le vere necessità e, soprattut-
to, i fini più elevati dello Stato. L’attività cavalliana del periodo si traduce in par-
ticolare in libri (in assoluto, tra i suoi più importanti),
articoli e working papers (questi ultimi a circolazione
ristretta), convegni e seminari condotti nell’ambito della
nascente Sezione di Sociologia politica dell’Associazione
italiana di sociologia, di cui da subito Cavalli è uno dei
numi tutelari. Nelle pubblicazioni alle quali qui si allu-
de (oltre agli articoli, i volumi: Cavalli 1981, 1982, 1987,
1992, 1995), Cavalli puntualizza con acribia il suo meto-
do di lavoro, penetra in profondità nel pensiero di Max
Weber attraverso un’indagine sia sulle sue opere mag-
giori sia su quelle minori, punta a un chiarimento riso- 33 Maestro, studioso, intellettuale pubblico Inoltre, Pellicani deve l’ordinariato universitario
«all’accanimento con cui si batte a suo favore un mem-
bro della commissione giudicatrice» (Millefiorini 2020:
304), ovvero lo stesso Cavalli. Curiosamente, però, è
proprio nel riferimento a Weber che si registra la diva-
ricazione tra i due studiosi. Muovendo da un interesse
diverso (Cavalli privilegia di Weber i temi politici, come
lo Stato e la leadership, mentre Pellicani lo studio sull’o-
rigine del capitalismo), essi pervengono a conclusioni
nell’immediato diverse. Nella sua grande ricerca su La
genesi del capitalismo e le origini della modernità (Pel-
licani 2013) – pur concordando con Weber sul fatto che
“il modo di produzione asiatico” abbia costretto le pos-
sibilità di sviluppo in una “gabbia d’acciaio” – sostiene
la tesi che il capitalismo non nasca dall’etica protestan-
te (come per lo stesso Weber) o dall’etica ebraica (come
per Sombart), ma dall’iniziativa imprenditoriale dei ceti
borghesi, specie laddove il potere politico si trovò ad
essere limitato e il potere autocefalo passò in mano alle
città. «Non furono – per lui – i nascenti Stati naziona-
li i soggetti promotori del capitalismo in Europa occi-
dentale». Da qui, il suo convincimento secondo cui «la
limitazione del potere politico sia una condizione essen-
ziale affinché il mercato e la proprietà privata acquistino
rilevanza e autonomia» (Ivi: 308). Insomma, una visione
ben diversa da quella di Cavalli, per cui lo Stato nazio-
nale e i grandi leader politici sono gli autori, i registri e
gli attori della storia. Andando avanti, Pellicani è inve-
ce sempre più orientato verso un socialismo liberale che
assicuri “una società aperta”. Semmai l’aspetto finalisti-
co su cui entrambi tornano a manifestare intenti comu-
ni può essere intravisto nell’idea che la democrazia di
ogni sistema politico sia tale soprattutto se va a rinfor-
zare l’autonomia e la libertà dei cittadini senza trasfor-
marli in sudditi. Ho accennato al fatto che Cavalli non si sia mai
stancato di proporre e riproporre queste sue argomen-
tazioni con tutti i mezzi e in tutte le occasioni, specie
accademiche. Basti pensare agli undici Working Paper a
sua firma (Porta 2005) del Centro di Sociologia Politica
dell’Università di Firenze e del Centro Interuniversita-
rio di Sociologia Politica delle Università di Firenze e di
Perugia, in cui le parole chiave “Carisma”, “Leadership”
e “Plebisciraty Democracy” ricorrono continuamente. Maestro, studioso, intellettuale pubblico Ammiccando allo stesso
Michels (ma senza ovviamente condividerne la conclu-
sione filo-mussoliniana), egli sottolinea come l’inter-
mediazione dei partiti privi le masse della possibilità di
usufruire della potente e illuminante guida del capo, e
che la cosiddetta “democrazia plebiscitaria” vada corret-
tamente intesa come un regime in cui il leader educa e fa
crescere ogni persona (alienata nella massa) a ritrovare
la sua statura di cittadino libero e autonomo. Insomma, Cavalli ribadisce tali convincimenti in tutte le ester-
nazioni del periodo qui considerato. Delle sue sollecita-
zioni sono destinatari, in senso “grosso”, l’opinione pub-
blica generale e, in chiave scientifica, la comunità nazio-
nale dei sociologi politici, a partire in prima battuta da
quelli come me coinvolti nel circolo fiorentino. i
Resta da chiarire, a questo punto, come Cavalli, sem-
pre utilizzando Weber, riesca a motivare la differenza tra
la funzione positiva dei leader carismatici democratici 34 Roberto Segatori Radio Radicale provvede a mandarla in onda, caricando
la registrazione in rete1. e quella negativa dei dittatori e dei tiranni ugualmente
carismatici. La risposta, dettagliatamente illustrata nei
volumi del 1982 e del 1987 di cui sopra, contempla due
ragioni. La prima è che, mentre per i primi il potere è
inteso come un “servizio” a vantaggio dei cittadini (da
condurre alla riscoperta dell’autonomia) e dello Stato,
che deve ritrovare la propria missione nel mondo, per i
secondi il potere è inteso come “dominio” finalizzato a
perseguire, «con una connotazione plebea ed avventuri-
stica», i propri interessi personali o di gruppo, che spesso
confliggono (fino a portare ad esiti tragici) con la rinasci-
ta della nazione. «L’altro punto [la seconda ragione], non
propriamente indipendente, è dato dall’incapacità di que-
gli uomini [i dittatori] di penetrare le grandi tradizioni
dei loro popoli e dell’Occidente, e farsene portatori». Tra-
dizioni per le quali «i diritti autentici dell’uomo [la liber-
tà e la democrazia su tutti] fanno parte del patrimonio
acquisito della civiltà europea» (Cavalli 1981: 279). Il richiamo a queste occasioni di dibattito pubblico
suggerisce l’utilità di una breve digressione sul rappor-
to tra Luciano Cavalli e Luciano Pellicani, due sociologi
della politica italiani di riconosciuto prestigio interna-
zionale. Essi hanno più di un punto di contatto: con-
dividono un orientamento politico socialista; criticano
senza indulgenza le “due chiese” (la cattolica e la comu-
nista). 1 Radio radicale, Presentazione del libro Leadership e Democrazia, 12
maggio 1988: https://www.radioradicale.it/scheda/27115/presentazione-
del-libro-leadership-e-democrazia?i=2683798 Maestro, studioso, intellettuale pubblico Ancora più intensi e partecipati sono i convegni
organizzati dai due Centri fiorentini e dalla Sezione di
Sociologia politica dell’AIS che lo vedono come protago-
nista. Accenno qui a tre appuntamenti ai quali ho avu-
to l’opportunità di partecipare con mie relazioni e che si
sono tradotti in altrettante pubblicazioni. Tra il 5 e il 7
dicembre 1986, si tiene a San Miniato un convegno sul
tema Leadership in democrazia, decisamente stimolante
anche per il tipo di confronto attivato. Una quarantina
di studiosi si ritrovano in una grande sala, seduti intor-
no a un tavolo disposto a ferro di cavallo, per discutere
con la metodologia tipica dei meeting scientifici: diciotto
relatori a illustrare i loro paper e gli altri a fungere da
discussant. Cavalli introduce i lavori con una prevedibi-
le relazione su Potere oligarchico e potere personale nella
democrazia moderna, a cui seguono, articolate in quat-
tro sezioni, tutte le altre: da quella successiva di Lucia-
no Pellicani sino a quella finale di Gianfranco Bettin. L’impatto del convegno (che, per chi scrive e per i rela-
tori più giovani, è una vera palestra formativa) è accre-
sciuto dalla pubblicazione degli Atti (AA.VV. 1987) che
sono destinati ad altre forme di diffusione. Il 12 maggio
1988 la rivista MondOperaio organizza la presentazione
di Leadership e democrazia con l’intervento del direttore
Pellicani, nonché di quelli di Cavalli, Marletti e Bettin. Un altro convegno in cui Cavalli gioca un fonda-
mentale ruolo di stimolatore ha luogo a Perugia l’8 e il
9 marzo 1991, organizzato dallo scrivente e svoltosi con
le modalità di cui al precedente incontro di San Minia-
to, con il titolo Istituzioni e potere politico locale. Gli 35 Maestro, studioso, intellettuale pubblico esecutivi di Ciampi e di Dini, si sia passati dal gover-
no di partito al governo dei tecnici, per giungere infine
a una repubblica dal semipresidenzialismo alternante. Forse, però, la replica indiretta meno allineata alla tesi
cavalliana la fornisce un altro politologo, laureatosi (iro-
nia della sorte) al “Cesare Alfieri” di Firenze e afferma-
tosi a Torino con importanti studi sul “professionismo
politico”. Mi riferisco ad Alfio Mastropaolo, che nell’oc-
casione sviluppa un ampio discorso dal titolo Elogio del
partito politico. Maestro, studioso, intellettuale pubblico Maggiore soddisfazione a Cavalli viene
senz’altro dai numerosi mass-mediologi presenti: dal-
lo stesso Marletti, a Giampietro Mazzoleni a Rolando
Marini (uno dei primi dottori di ricerca usciti dalla sede
fiorentina e oggi Pro-Rettore all’Università per Stranieri
di Perugia). Atti del convegno (Segatori 1992) mostrano il desiderio
di Cavalli di sottoporre a verifica sul piano della politi-
ca locale la sua tesi sui limiti della “partitocrazia” e sulla
necessità di leader forti. Egli, infatti, introduce i lavori
chiedendo in primo luogo a Bettin e poi agli altri rela-
tori se il paradigma da lui auspicato, teso a privilegiare
«cariche monocratiche con elezione popolare diretta» e
in cui gli attori principali «sono gli individui (ossia, gli
uomini politici)», sia ancora soccombente, come teme, al
paradigma «caratterizzato a tutti i livelli dalla gestione
collegiale del potere» e in cui «gli attori principali sono i
partiti come gruppi autocefali chiusi» (Ivi: 23). Le risposte degli intervenuti confermano solo in
parte la fondatezza della sua avversione per il ruolo dei
partiti a livello locale. Tra coloro che si misurano con le
sue domande, Bettin ed io riportiamo dati di ricerca in
cui nelle elezioni municipali i singoli e i partiti pesano
per motivi diversi alla stessa maniera; Fiorenzo Girotti
dimostra che nei piccoli comuni gli uomini contato più
delle sigle; Paolo Segatti parla addirittura di “solitudi-
ne” dell’amministratore comunale. A compensare l’esito
non univoco di tali indagini sociologiche sulla politica
in periferia, Cavalli trova un positivo riscontro ai suoi
auspici giusto due anni dopo, quando il parlamento ita-
liano il 25 marzo 1993 approva la Legge n. 81 che intro-
duce l’elezione diretta dei sindaci e dei presidenti di Pro-
vincia. Le nuove forme della comunicazione politica stan-
no alimentando una delle condizioni fondamentali a cui
Cavalli rimette la possibilità dell’avvento di leader auto-
revoli anche in Italia. 3. L’intellettuale pubblico (tra impegno e disincan-
to). Luciano Cavalli non ha mai nascosto – nella sua
“relazione ai valori” – che al cuore dei suoi interessi ci
fosse la situazione italiana, con i suoi problemi ricor-
renti e la possibilità di uscirne. Così, accanto alla sua
sistematica applicazione da studioso, torna ad interve-
nire nel dibattito pubblico nazionale dagli anni Ottanta
del ‘900 in poi. L’aveva già fatto, nella lontana stagione
genovese, su posizioni contigue a quelle del socialista
Lelio Basso2. Aveva poi interrotto quell’esperienza per
dedicarsi agli amati studi sociologici. 2 Su questo periodo e per tutta la sua biografia fino al 2005 rinvio alle
puntualissime pagine di Paolo Turi in Bettin Lattes e Turi (2008). Maestro, studioso, intellettuale pubblico Atti e comportamenti di Bettino Craxi fanno pensare che
egli avesse anche intuito che nella nuova epoca e in corri-
spondenza con una qualche forma di presidenzialismo si
sarebbero formate vaste e complesse aree politico-culturali,
e in particolare un’area di sinistra innervata forse dai parti-
ti tradizionali ma senza egemonia di alcuno di essi, proprio
perché il suo fulcro era istituzionalmente fissato in un lea-
der. Ovviamente Bettino Craxi riteneva di poter essere lui
il primo leader di questa nuova realtà politico-culturale, e
quindi del Paese. Se le cose avessero seguito allora il corso
«naturale», ciò sarebbe verosimilmente accaduto. Ma l’im-
previsto si realizzò. Forse il leader socialista era un uomo
troppo abituato a rischiare, proprio perché il suo partito era
così piccola cosa e la posta in gioco così grande. Si espose. Commise errori. Sorpassò i limiti della legge (Ivi: 32). Alludendo al fatto che Craxi era probabilmen-
te caduto “anche” per la trappola ordita ai suoi danni
dagli avversari storici, Cavalli termina l’analisi con que-
ste parole malinconiche: «due le conseguenze principali
di quella drammatica conclusione. La prima: debolezza
strategica della Sinistra dopo Mani Pulite. La seconda,
e più grave: è stato bloccato il moto verso la repubblica
unicipite» (Ibidem). Bruciato Craxi, la speranza cavalliana che tale
“moto” potesse ripartire trova una seconda occasione con
l’avvento di Silvio Berlusconi, che non a caso di Bettino
era stato amico (pur in ragione dei personali interessi). In
riferimento alla nuova stagione, giornalisticamente ribat-
tezzata “Seconda Repubblica”, Cavalli scrive in un artico-
lo che sembra essere arrivato il tempo in cui sia «possi-
bile la formazione di correnti di opinione e, perfino, una
larga mobilitazione, in occasione di gravi crisi di carat-
tere collettivo, intorno a un progetto e a una leadership
personale o di pochi» (Cavalli 2008). Il catalizzatore della
mobilitazione ha chiaramente un nome: «l’ascesa di Ber-
lusconi al potere nel 1994 seguì quel paradigma politico
[la democrazia plebiscitaria] e n’ebbe l’impronta il suo
governo, con l’implicazione di un profondo cambiamento
del rapporto tra il leader e il suo popolo, e più in generale
tra politica e popolo» (Ibidem). E il senso del mutamen-
to non si ferma qui: basta a suo avviso che si «esamini
il più compiuto percorso “plebiscitario” di Berlusconi, che
la partita del potere l’ha giocata ben cinque volte in base
a quel paradigma, con progressivi perfezionamenti» (Ibi-
dem). Maestro, studioso, intellettuale pubblico di un leader: nelle campagne elettorali, come «personi-
ficazione del centrodestra» è «fonte di rassicurazione e
speranza» (Ibidem). È un «capo “prammatico”, ma con
un tocco di magia […] Un leader che, inoltre, sa come
usare i media: messaggi semplici, rassicuranti, sostenuti
da una suggestività personale efficace sui cittadini cultu-
ralmente partecipi del “prammatismo dinamico” cui egli
si attiene, e il sistematico ottimismo» (Ibidem). Nel volume Il primato della politica nell’Italia del
secolo XXI, Cavalli (2001) dedica un intero paragrafo
alla “Grande Riforma” che avrebbe dovuto essere appro-
vata negli anni Ottanta e al suo protagonista politico. In
particolare, egli scrive che Poi, però, anche Berlusconi fallisce nel compito
di realizzare la “Grande Riforma”, mentre la sua pre-
sa sull’elettorato (il suo carisma, che tale è almeno per i
suoi seguaci) progressivamente si appanna. Nello stesso
articolo Cavalli accenna ai due motivi che causano tale
esito critico (magari ignorandone altri, come quelli della
bassa stima goduta dal Cavaliere presso i partner euro-
pei e del trattamento conseguente riservato all’Italia
a causa sua). Il primo è relativo al fatto che Berlusconi
non si eleva mai dal suo “prammatismo” al livello di un
“Progetto-Paese”, che è uno degli atout decisivi per il
riconoscimento del vero leader politico nel pensiero del
Cavalli weberiano. La colpa di ciò è da attribuire alla
limitata disponibilità di intellettuali in grado di aiutare
il capo a predisporre quel progetto (limitata disponibilità
che dipende a sua volta dell’indebolimento dell’univer-
sità italiana) e dalla presunzione dello stesso Berlusco-
ni a fare da sé, in quanto attento soprattutto ai propri
interessi privati. A confermare l’ultima costatazione c’è
da registrare che nel tempo abbandonano Forza Italia
valenti studiosi come Giuliano Urbani e Piero Melogra-
ni. Il secondo motivo consiste nella storica mancanza di
unità di intenti delle forze parlamentari di maggioranza
e di minoranza. Un’impresa di ampio respiro istituzio-
nale, come quella mirante all’introduzione del presi-
denzialismo, presuppone, dichiara ancora Cavalli, che
«leader di governo e leader di opposizione riconoscano
la gravità della crisi, e quindi l’obbligo d’un impegno
comune, nella diversità dei ruoli, per batterla» (Cavalli
2001: 198-200). Ma che ciò in Italia non sia possibile lo
dimostra l’esito negativo dei lavori della Commissione
Bilaterale di cui sono protagonisti Massimo D’Alema e
Silvio Berlusconi, con quest’ultimo autore di numerose
giravolte (Ibidem). Maestro, studioso, intellettuale pubblico Infine, forte delle
conoscenze e delle convinzioni maturate su alcuni temi
di fondo (importanza del ruolo dello Stato e della fun-
zione dei leader, necessità del superamento del regime
partitico parlamentare tramite il presidenzialismo), tor-
na ad affacciarsi sullo spazio pubblico nazionale. La sua
attiva presenza è testimoniata da libri, numerosi articoli
su giornali e riviste, specie sull’Avanti e su MondOperaio
in continuità con le sue simpatie di vecchia data laiche e
socialiste, interviste. La Sezione di Sociologia politica dell’AIS tiene poi
a Torino, grazie all’impegno organizzativo di Carlo
Marletti, un terzo importante convegno nei giorni 8-10
maggio 1996. A quella data, molte cose sono successe
nel paese, e il titolo dell’incontro, come quello del volu-
me degli Atti (Marletti 1999), recita Politica e società in
Italia. Le questioni affrontate sono numerose, ma ruo-
tano tutte intorno al tema della transizione dello scena-
rio istituzionale, provocato dalla nuova legge elettorale e
dal passaggio dal governo Berlusconi 1 al governo Dini,
fino alle elezioni del 21 aprile dello stesso anno. Non a
caso, l’apertura dei lavori è affidata allo stesso Marletti
che si sofferma su Le interpretazioni della crisi italiana e
i confini mobili della politica (Ibidem). A Luciano Caval-
li tocca poi il compito di svolgere la relazione immedia-
tamente successiva di fronte a un numero molto eleva-
to (una quarantina) di partecipanti, ognuno dei quali
invitato ad illustrare i propri paper. Cavalli propone di
nuovo il tema della Leadership «personale» e «diffusa». Ma la situazione italiana, ancora decisamente calda, vie-
ne descritta sì in corso di trasformazione verso la per-
sonalizzazione della politica ma pure in maniera assai
problematica. Così, se Ilvo Diamanti illustra la nuo-
va mappa elettorale, accennando a molte Italie e molti
Nord, il politologo Sergio Fabbrini si chiede se, con gli In parallelo al suo auspicio di una riforma istituzio-
nale italiana sul modello francese, Cavalli si interessa,
con una curiosità che non esclude più di un’aspettati-
va, alle possibilità che Bettino Craxi, Silvio Berlusconi e
quindi Matteo Renzi impersonino la figura da lui tanto
celebrata del capo autorevole (per costoro, egli si limita a
usare l’aggettivo “carismatico” solo per alcuni momenti
e in riferimento al seguito – peraltro altalenante – degli
elettori fedeli). È invece perplesso circa l’attribuzione di
questo profilo a Romano Prodi e a Walter Veltroni, a 36 Roberto Segatori causa del loro essere espressione di partiti e raggruppa-
menti “policefali”. tute ironiche sul nascente astro fiorentino. Ecco un flori-
legio dei primi: In occasione di un
convegno su La sociologia di Luciano Cavalli, tenutosi a
Firenze nel marzo del 2005 – tradottosi poi con lo stes-
so titolo in un secondo volume a lui dedicato. Il primo
era uscito, a cura di Gianfranco Bettin (1997) –, mi capita
di intervenire mettendo il dito nella piaga con un po’ di
impudenza. «Come mai nei paesi democratici, accanto a
casi di leadership personale univocamente ritenuti ono-
revoli (Roosevelt, De Gaulle), si siano verificati casi di
leadership molto più controversi e a rischio di populismo
(per stare all’Italia repubblicana, Craxi e Berlusconi)?»
(Segatori 2008: 212). In qualche modo mi chiedo se siano
sufficienti a spiegare le parabole di costoro le principa-
li variabili indicate da Cavalli (situazioni di crisi, qualità
personali e sistema istituzionale) e se non sia il caso di
ricorrere ad altri fattori esplicativi (tipo di stratificazione
sociale, cultura civica, presenza di corpi intermedi: asso-
ciazioni, prima ancora che partiti). E concludo: Ma ormai messo sul chi vive dalle delusioni prece-
denti, sempre nella stessa intervista Cavalli non manca di
sottolineare quelle che, a suo avviso, sono le magagne per-
sonali, nonché i limiti di sistema relativi al personaggio. Renzi, di pelle mi è sempre stato abbastanza antipatico. Come modo di fare, di parlare, di porsi. Poi […] ho senti-
to come si è comportato come Presidente della Provincia,
in modo molto democristiano, clientelare. […] Continua ad
avere dei tratti che d’istinto trovo fastidiosi, un po’ trop-
po da fiorentino, ci gioca molto sul fiorentinismo e questo
può piacere a tanti, ma a me francamente piace poco. For-
se Renzi esagera un po’ con gli atteggiamenti e le battute
su misura per i fiorentini. […] Devo ripetere la mia riser-
va. Renzi non ha ancora esposto un sistema di idee che ti
permetta di dire: “Ecco un uomo che rappresenta in modo
maturo la novità e la contemporaneità in politica: sulla
misura della crisi” (Ibidem). Se si vuole allora che il ritorno della leadership in epoca
di dominio televisivo sia compatibile con la democrazia,
occorre moltiplicare, più che diminuire, le regole d’accesso ai
media (perché l’accesso sia pluralistico e avvenga in condi-
zioni di parità), come pure le regole per la selezione dei rap-
presentanti del popolo (magari su base maggioritaria, ma
con possibilità di scelta effettiva tra più opzioni) e i pesi e i
contrappesi intorno al rafforzato ruolo degli esecutivi. tute ironiche sul nascente astro fiorentino. Ecco un flori-
legio dei primi: dizionalmente schierato per il sistema “democraticistico
parlamentare” gli avrebbe permesso di perseguire il suo
disegno del “sindaco d’Italia”. E inoltre, come il Nostro
riafferma in chiusura dell’intervista citata, «probabilmen-
te [Renzi] non ha una buona macchina intorno» (Ibidem). Del Pd solo Renzi sa parlare a tutti, o quasi. Con garbo, in
genere. […] Sembra avere idee moderne. Spunti di moder-
nità, almeno… Per esempio, gli capita di dire che il partito
non può e non deve controllare ogni istituzione, ogni ruo-
lo istituzionale. Se uno viene eletto sindaco, deve appunto
fare il sindaco, e non stare sempre lì a sentire quello che
dice il partito. […] Indubbiamente è uno che ha delle carat-
teristiche e delle capacità che non sono presenti in altri
leader del partito democratico, e questo gli va riconosciu-
to. È brillante, intelligente e spesso anche innovativo nel
pensiero. Per esempio, sono rimasto favorevolmente colpito
dall’ultimo discorso fatto prima delle votazioni primarie,
ma anche da quello fatto subito dopo averle perse: ha rin-
graziato e, in un lungo discorso di mezz’ora, ha indubbia-
mente mostrato un profilo intellettuale di un certo rilievo. […] Tutto sommato Renzi sembra più avanti dei vecchi lea-
der… un’altra era geologica. È favorevole all’elezione diret-
ta di chi governerà (il sindaco d’Italia), e più in generale
alla leadership individuale; favorevole al partito leggero, e
al bipolarismo; ed esibisce sempre atteggiamenti di dialo-
go civile, Renzi. Crede che per risanare l’Italia bisognerà
contare sul volontariato alla grande, il che riconduce a quel
concetto di “mobilitazione nazionale per la crisi” a mio
giudizio al centro di ogni strategia vincente (Ivi: 217-218). [
]
(
)
Nell’atteggiamento di Cavalli verso i tre leader consi-
derati, sembra riproporsi ogni volta un’apertura di credi-
to poi seguita (a breve o a medio termine) dal malinconi-
co ritiro dello stesso credito. Questa altalenante vicenda
delle istituzioni italiane, alle prese con crisi socio-econo-
miche ricorrenti e con la limitata efficacia degli strumen-
ti risolutivi, costituisce il cruccio che accompagna la sta-
gione finale di Luciano Cavalli. Gli è di parziale conforto
la gratitudine e il riconoscimento del suo lavoro da parte
di allievi e colleghi, non privo dalla voglia di continua-
re con lui un dialogo mai interrotto. Maestro, studioso, intellettuale pubblico A lui vanno inoltre riconosciute alcune doti tipiche Deluso da Berlusconi, Cavalli guarda con attenzione
al leader allora emergente del centro-sinistra. In un’In-
tervista a Luciano Cavalli a cura di Paolo Giovannini
(2016), egli si sofferma su Matteo Renzi, all’epoca sinda-
co di Firenze, già presidente della Provincia di Firenze
(2004-2009) e appena uscito sconfitto dalle primarie del
Partito Democratico del 2012 in cui prevale Pier Lui-
gi Bersani (Renzi le vincerà nel 2013 e nel 2017). Con la
schiettezza consueta, non scevra da una buona dose di
disincanto, Cavalli alterna espliciti apprezzamenti e bat- 37 Maestro, studioso, intellettuale pubblico tute ironiche sul nascente astro fiorentino. Ecco un flori-
legio dei primi: tute ironiche sul nascente astro fiorentino. Ecco un flori-
legio dei primi: RIFERIMENTI BIBLIOGRAFICI Horkheimer M. (1967 [1947]), Eclissi della ragione, Ein-
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dell’illuminismo, Einaudi, Milano. AA.VV. (1987), Leadership e democrazia, Cedam, Padova. Inkeles A. (1967 [1964]), Introduzione alla sociologia, Il
Mulino, Bologna. Adorno T. W. (1970 [1966]), Dialettica negativa, Einaudi,
Milano. Jonas F. (1970 [1968]), Storia della sociologia, Laterza,
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Marcuse H. (1964 [1955]), Eros e civiltà, Einaudi, Milano. Bettin Lattes G. (a cura di) (1997), Politica e società. Scrit-
ti in onore di Luciano Cavalli, Cedam, Padova. – (1967 [1964]), L’uomo a una dimensione, Einaudi, Milano. Marletti C. (a cura di) (1999), Politica e società in Italia, 2
Voll., FrancoAngeli, Milano. Bettin Lattes G., Turi P. (a cura di) (2008), La sociologia
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– (2008), «La politica degli italiani», in Italianieuropei, 5,
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capitalismo: l’enigma svelato», in SocietàMutamen-
toPolitica, 11 (21): 303-312. – (1981), Il capo carismatico. Per una sociologia weberiana
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– (1982), Carisma e tirannide nel secolo XX. Il caso Hitler,
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babilmente non servono né leader deboli con partiti forti, né
leader forti con cittadini deboli. Meglio sarebbe per tutti se
ci fossero insieme leader e cittadini autorevoli (Ivi: 219). Sulle riserve complessive e finale di Cavalli giocano
probabilmente un motivo esplicitato solo parzialmen-
te e, accanto ai richiami al “carattere” del personaggio,
due ragioni generali più volte ribadite. Il motivo impli-
cito sta verosimilmente nel fatto che Cavalli, che insiste
a professarsi socialista liberale, non riesca a digerire fino
in fondo l’affermazione di un cattolico come Renzi che si
accorda con gli eredi del Pci. Le altre ragioni sono quelle
di sempre. Difficilmente il partito, ideologicamente e tra- Qualche giorno dopo Cavalli mi contatta per ringrazi-
armi dell’intervento, ma anche per ricordarmi che lui si è
sempre battuto per la crescita culturale dei cittadini e per
la formazione delle élite, insistendo con particolare enfasi
sulla funzione dell’università, da rilanciare con maggior
rigore. Gli sono grato per questa risposta e, più in generale,
per tutto quello che ha insegnato a me e a tanti colleghi di 38 Roberto Segatori Foucault M. ([1961] 1963), Storia della follia, Rizzoli,
Milano. Foucault M. ([1961] 1963), Storia della follia, Rizzoli,
Milano. sociologia politica. E mi piace pensare che a lui – democra-
tico convinto alle prese con Craxi, Berlusconi e Renzi – sia
inappropriato attribuire la massima: solo che si illude fini-
sce col restare deluso. Il suo realismo politico, a volte fin
troppo radicale ma sempre preveggente, gli ha fatto abban-
donare presto ogni facile illusione. E chissà con quale cau-
stica attenzione avrebbe seguito l’odierno dibattito italiano
sulle riforme istituzionali. – (1972 [1971]), L’archeologia del sapere, Rizzoli, Milano. Gallino L. (1970), «Recensione a Luciano Cavalli Il Muta-
mento sociale», in Quaderni di Sociologia, 4: 454-455. Giovannini P. (2016), «La crisi italiana. Intervista a
Luciano Cavalli», in Cambio, Rivista Sulle Tras-
formazioni Sociali, Anno III, 5: 217-218. Goode W. J., Hatt P. K. (1962 [1952]), Metodologia della
ricerca sociale, Il Mulino, Bologna. Citation: Lorenzo Viviani (2023) Carisma
e leadership nella sociologia della
democrazia. SocietàMutamentoPo-
litica 14(27): 41-60. doi: 10.36253/smp-
14334 Citation: Lorenzo Viviani (2023) Carisma
e leadership nella sociologia della
democrazia. SocietàMutamentoPo-
litica 14(27): 41-60. doi: 10.36253/smp-
14334 Citation: Lorenzo Viviani (2023) Carisma
e leadership nella sociologia della
democrazia. SocietàMutamentoPo-
litica 14(27): 41-60. doi: 10.36253/smp-
14334 Lorenzo Viviani Copyright: © 2023 Lorenzo Viviani. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. «[…] il mare s’è aperto, una nube vi ha scorto il
cammino, la pietra ha versato l’acqua; qui è piovu-
to la manna, ogni cosa è concorsa nella vostra gran-
dezza; il rimanente dovete far voi»
[Machiavelli, Il Principe, Cap. XXVI] 1. Democrazia e sociologia: il contributo di Luciano Cavalli. L’itinerario
intellettuale di Luciano Cavalli mette in luce il profilo di un sociologo criti-
co che in tutto l’arco della sua ricerca teorica ed empirica richiama il senso
più proprio della immaginazione sociologica di Mills, ossia quella «capacità
di riflettere su sé stessi liberi dalle abitudini familiari della vita quotidiana,
al fine di guardare la realtà con occhi diversi», leggendo ciò che avviene nella
società e nella politica al fine di «trasformare la pubblica indifferenza in inte-
resse per i problemi pubblici» (Mills 1973 [1959]: 15). Già a partire dalle sue
prime opere si trova una tensione intellettuale capace di coniugare il rigore
scientifico del sociologo e la funzione pubblica di svelamento delle manipola-
zioni di cui la democrazia è costantemente oggetto. Una postura sociologica
che porta Cavalli a prendere le distanze dalle indagini frammentarie di poco
rilievo, dalle teorie meramente speculative così staccate dalla terra da essere
paragonate “alla volante isola di Laputa”, e non di meno dalla sottomissione
ai temi di ricerca graditi al potere, specie all’industria, come tali forieri di
un acritico conformismo (Cavalli 1964). Un approccio particolarmente evi-
dente in La democrazia manipolata (1965), nella cui Introduzione si esplici-
ta la manipolazione come «la socializzazione e il controllo sociale, in quanto
strumentalizzazioni di una minoranza organizzata che detiene l’autorità in
una società-stato del nostro tempo, e, secondariamente in una istituzione o
in una sub-unità amministrativa, per fini di dominazione» (Cavalli 1965: 9). RIFERIMENTI BIBLIOGRAFICI 211-219. – (2001), Il primato della politica nell’Italia del secolo XXI,
Cedam, Padova. – (2003), Il leader e il dittatore. Uomini e istituzioni di
governo nel “realismo radicale”, Ideazione, Roma. – (a cura di) (1992), Istituzioni e potere politico locale,
FrancoAngeli, Milano. Durkheim E. (1962 [1893]), La divisione del lavoro
sociale, Edizioni di Comunità, Milano. Segatori R., Cristofori C., Santambrogio A. (a cura di)
(2004), «Sociologia ed esperienza di vita. Scritti in
onore di Franco Crespi», il Mulino, Bologna. – (1963 [1895]), Le regole del metodo sociologico, Edizioni
di Comunità, Milano. – (1969 [1897]), Il suicidio. L’educazione morale, UTET,
Torino. Turi P. (2008), Sociologia e politica nell’itinerario intel-
lettuale di Luciano Cavalli, in Bettin Lattes G., Id. (a cura di), La sociologia di Luciano Cavalli, Firenze
University Press, Firenze, pp. 23-189. – (1963) [1912], Le forme elementari della vita religiosa,
Edizioni di Comunità, Milano. 39 39 Maestro, studioso, intellettuale pubblico Weber M. (1948 [1919]), Il lavoro intellettuale come pro-
fessione, Einaudi, Milano. Weber M. (1948 [1919]), Il lavoro intellettuale come pro-
fessione, Einaudi, Milano. – (1997 [1919]), La politica come professione, Armando
Editore, Roma. – (1997 [1919]), La politica come professione, Armando
Editore, Roma. – (1958 [1922]), Il metodo delle scienze storico-sociali,
Einaudi, Milano. – (1958 [1922]), Il metodo delle scienze storico-sociali,
Einaudi, Milano. – (1961 [1922]), Economia e società, 2 voll., Comunità,
Milano. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. Non si legga in queste opere, comparandole con lo sviluppo più maturo della
sua ricerca, una forma di cesura fra una prima fase e la stagione della matu-
rità della sua ricerca sociologica. La sociologia di Luciano Cavalli non cede
mai alla tentazione di entrare a far parte della schiera dei chierici della scien-
za intesa in funzione ancillare rispetto al potere politico, e lo stesso magi-
stero educativo mantiene intatto lo spirito emancipativo della sociologia, mai
abbandonando il monito weberiano per cui «il profeta e il demagogo non si Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 41-60, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14334 42 Lorenzo Viviani addicono alla cattedra universitaria» (Weber 2004 [1919]:
30). Non è quindi un caso che i due bersagli che ricor-
rono nella sua ampia riflessione sul mutamento sociale e
sul mutamento politico siano il sociologismo e il demo-
craticismo, ossia il ricorso a interpretazioni ideologica-
mente orientate degli eventi storici fondante unicamen-
te sulle condizioni strutturali dei gruppi sociali, senza
tener conto del ruolo degli individui (Cavalli 1981a: 10),
e l’uso di principi democratici svuotati di senso, stru-
mentali alla giustificazione del potere, surrogati di “reli-
gioni laiche di redenzione” ormai anch’esse secolariz-
zate (il “democraticismo umanitario”), o manipolazioni
del concetto stesso di sovranità popolare (Cavalli 1992;
2001). ra politica, e che al contempo indaga la diminuzione del
peso del gruppo, specialmente dei partiti, nei processi di
identificazione, socializzazione e costruzione di schemi
cognitivi di interpretazione dei fenomeni politici (Manin
1997; McAllister 2007; Poguntke, Webb 2005; Karvonen
2010; Garzia 2014; Musella 2018). È in questa prospettiva che l’immaginazione socio-
logica di Cavalli si afferma in un itinerario di ricerca che
lo porta ad affrontare temi opposti al conformismo e al
superamento dei tabù di cui le scienze sociali e politi-
che sono state vittime nel secondo Dopoguerra. Retag-
gi particolarmente presenti in quei paesi e nei contesti
culturali in cui ancora aleggiavano il «complesso del
tiranno» e lo «spettro del nazismo», responsabili dell’o-
blio della leadership nei paesi continentali europei e del
costante tentativo di imbrigliarne le prerogative, qua-
si a stabilire una equivalenza tra leadership e dittatori
(Blondel 1987). Questo è lo specifico contesto cultura-
le in cui Cavalli si confronta costantemente con i clas-
sici della sociologia e contribuisce con autorevolezza a
superare le secche scientifiche di una ricezione parziale
della teoria del carisma di Max Weber, contrastando la
banalizzazione delle sue implicazioni politiche e ricon-
ducendola agli sviluppi della razionalità in Occidente. A testimonianza dell’originalità della riflessione socio-
logica di Cavalli non va sottaciuta non solo la consta-
tazione che il carisma, la leadership e il plebiscitarismo
hanno costituito una sorta di tabù lessicale per ampia
parte del secondo Dopoguerra (Marletti 2008: 194-195),
ma anche che tali termini sono stati interpretati da una
parte della letteratura come direttamente riconducibili a
sviluppi dittatoriali. Così in alcuni casi è stato accomu-
nato il pensiero di Schmitt allo sviluppo della prospet-
tiva di Weber (Habermas 1971; Lukács 1980), in altri
sono stati evidenziati i rischi di involuzione dittatoriale,
pur riconoscendo a Weber l’attenzione al mantenimento
delle garanzie costituzionali come vincolo al potere del
leader (Mommsen 1993). Al contrario, Cavalli recupera
una visione della leadership che non assume il conno-
tato moralistico di una leadership buona e una cattiva
secondo schemi ideologici, ma basa la sua ricerca teori-
ca ed empirica sullo sviluppo di tipologie, e sotto-tipo-
logie, contribuendo, da una parte, a salvaguardare il
“vero” Weber da ricezioni parziali o incomplete (Cavalli
1981a: 9), dall’altra a attualizzare il senso proprio della
leadership, e in particolare della leadership carismatica,
in quella proposta di democrazia con un leader del tut-
to distante dalle dittature e dai totalitarismi carismatici. Un Weber che per Cavalli non è esente da alcuni rilie-
vi critici che cercheremo di mettere a fuoco nel corso
del saggio, riconducibili a parti inespresse del pensiero
weberiano a tracce lasciate troppo repentinamente cade- Cavalli si pone in modo originale nell’ambito della
prospettiva del realismo politico “radicale”, recuperan-
do la lezione che va da Tucidide a Machiavelli, fino agli
elitisti classici, a Weber e oltre, sviluppando una sociolo-
gia della democrazia libera da determinismi sociologici,
da filosofie della storia e da escatologie di qualsiasi tipo,
in una chiave di costante svelamento anti-ideologico. La politica per Cavalli rientra costantemente nell’ambi-
to della weberiana lotta per la vita, per la sopravvivenza
e per il potere, fra individui e fra Stati, in cui l’agire si
compone di forza così come di astuzia, di volpi e leoni,
secondo la lezione data dal Segretario fiorentino ne Il
Principe. Un realismo che è al tempo stesso la chiave per
affidare il rapporto tra politica e masse all’azione strate-
gica della leadership e il quadro teorico per dare solidità
scientifica al cambiamento di paradigma dello studio dei
fenomeni sociali e politici improntato alla prospettiva
della «politics as leadership», la politica come espressio-
ne stessa della relazione sociale di potere di cui si com-
pone la leadership (Tucker 1981). Si tratta, in altri termini, di assumere come variabile
centrale nel processo politico la rivendicazione avanzata
direttamente dal rappresentante, il leader (e gli apparati
a sostegno della sua azione), che attraverso la propria per-
formance diviene l’attore protagonista e non un semplice
agente della rappresentanza, per di più con un riconosci-
mento non vincolato a un gruppo sociale precostituito, e
con una capacità simbolica che deriva dalle qualità perso-
nali e non dall’appartenenza a un qualche immaginario
ideologico (Pitkin 1967; Saward 2010). Cavalli si inserisce,
anzi, per molti aspetti anticipa, il dibattito sul passag-
gio dalla democrazia dei partiti alla audience democracy
attraverso lo studio del fenomeno della personalizzazio-
ne della politica e della leadership di vertice, recuperan-
do e calando nel contesto delle democrazie contempora-
nee la lezione weberiana sulla democrazia plebiscitaria. Una riflessione sociologica che torna a porre al centro il
potere e le funzioni dell’individuo in relazione alla sfe- 43 Carisma e leadership nella sociologia della democrazia ne i campi di ricerca di Luciano Cavalli siano stati molti,
così come molti sono i percorsi di ricerca da lui indicati
alle nuove generazioni di sociologi e sociologi della poli-
tica, è indubbio che la teoria del carisma e della leader-
ship segnano uno dei contributi centrali del suo magiste-
ro sociologico, a partire dalle prime riflessioni contenute
in Max Weber; religione e società (1968) e – in partico-
lare – dalla pubblicazione de Il capo carismatico (1981),
il «punto di partenza» su cui si dipana la trama della
sociologia della leadership e della democrazia di Lucia-
no Cavalli (Bettin Lattes, Turi 2008: 2). Si tratta non a caso non solo di una distinzione che
segna l’autonomia della sociologia politica dalla scien-
za politica, ma anche di una presa di distanza da quella
sociologia “della” politica che confina lo studio dei feno-
meni politici a una mera e ulteriore applicazione della
sociologia, disconoscendo la complessità della relazione
fra mutamento sociale e politico. 2. La ricezione di Max Weber: la teoria del carisma
alla prova della società contemporanea. Dopo essere sta-
to oggetto di damnatio memoriae, il carisma è tornato
a essere impiegato nel dibattito politico in una versione
che ne dilata talmente il significato da equiparare il con-
cetto a una forma di charm, a un magnetismo dell’im-
magine, a una mera abilità oratoria, quando non a una
mera «illusione (superstiziosa) di massa» (Cavalli 1981b:
170). Un tale stiramento concettuale, con il conseguen-
te venir meno della sua capacità euristica, perde di vista
la centralità sociologica del carisma e del suo processo,
vanificandosi in volta in volta in etichette labili conces-
se alla celebrità politica di turno, o tutt’al più forzando
operazionalizzazioni su meri aspetti della retorica del
leader sul proscenio mediatico. Per comprendere socio-
logicamente il carisma occorre confrontarsi con la teo-
ria che emerge dalle opere di Max Weber, ed è in questa
specifica direzione che Luciano Cavalli si colloca nell’al-
veo di chi assume la necessità di “leggere Weber” nella
sua interezza, riannodando la trama che lega la socio-
logia della religione, il processo di razionalizzazione, la
sociologia del potere, la democrazia plebiscitaria, fino
alle pagine dei suoi interventi più direttamente politici
(Mommsen 1993; Treiber 1993; Schluchter 1987; Caval-
li 1981a; Whimster, Lasch 1987; Schroeder 1998; 2020). Una tale prospettiva assume il carisma come il punto
di congiunzione della sociologia della religione con la
sociologia del potere (Treiber 2005: 362), come tale iden-
tificandolo come lo «strumento fondamentale di analisi
sociologica» all’interno della prospettiva storico-evoluti-
va del processo di razionalizzazione occidentale (Cavalli
1981b: 181). Se infatti, come
ricorda Turi (2008: 103), nella ricerca di Luciano Cavalli
il tema di fondo del progetto è quindi “carisma e demo-
crazia”, cioè, “leadership e democrazia”, il discorso sul
carisma affrontato in chiave weberiana diventa la ricerca
di come questo possa conciliarsi con le istituzioni stesse
della liberal-democrazia, trasformando la potenza rivo-
luzionaria del carisma puro in una potenza riformatrice
della politica democratica (Cavalli 1981a: 190). re, o infine alla divergenza nella valutazione di singo-
li aspetti o attori alla luce degli sviluppi della storia (ad
esempio il ruolo dei partiti). Ancora una volta non si
tratta di discostarsi da Weber, ma di portarne le intui-
zioni a una più esplicita maturazione nel contesto sociale
e politico attuale. p
L’essere pienamente inserito nell’ambito della lezio-
ne weberiana non appare solo dai temi di ricerca, ma dal
ricorso costante al metodo della sociologia comprenden-
te e al recupero del multi-fattorialismo come interpre-
tazione dei fenomeni sociali e politici. Una lezione che
rimane un magistero di stringente attualità, in partico-
lare per la sociologia politica di oggi, a partire da un’ine-
ludibile premessa per cui nell’interconnessione costante
fra società e politica non si può assegnare una completa
autonomia alla politica né, al tempo stesso, una priori-
tà alla società sulla politica. Se la politica non si spiega
con la sola politica, parimenti la sociologia politica non
può risolversi nella sola individuazione dei fattori socia-
li che condizionano l’ordine politico, dal momento che
le istituzioni stesse sono strutture sociali e spesso sono
proprio queste le variabili che influenzano la società
(Bendix, Lipset 1957; Coser 1967; Cavalli 1980). Si tratta,
più in generale, di affermare uno statuto epistemologico
della sociologia politica che Cavalli non confina “solo” ai
fenomeni politici, in continuità con i classici della socio-
logia con cui si relaziona costantemente nella sua ricer-
ca, non solo Weber, ma, fra gli altri, Durkheim, Pareto,
Michels, Lynd, Parsons, Manhheim, Mills. Appare allora
chiaro che «tutta la Sociologia è politica» e la sociologia
politica si contraddistingue proprio per l’interconnessio-
ne fra società e politica, «perché i fenomeni politici han-
no normalmente radice in più generali processi sociali
e la loro rilevanza è ordinariamente commisurata agli
effetti che producono sulla società» (Cavalli 1980: 80-81). Laddo-
ve infatti si assume la razionalizzazione come strumento
che favorisce la libera creatività umana, al tempo stesso
è in quel processo che si annida il graduale tramonto del
carisma come forza in grado di intervenire in una socie-
tà di massa. Questo è il punto su cui Cavalli si inserisce
in un dibattito di perdurante attualità sociologica, sia
per il mutamento sociale connesso al processo di globa-
lizzazione, sia per i più recenti sviluppi degli studi sul-
la democrazia, in entrambi i casi ponendosi il tema dei
limiti della pretesa (e della promessa) illuminista di agi-
re liberamente in un mondo disincantato, senza vincoli
altri se non la capacità individuale dell’uso della ragione. Come non riconoscere in tale cautela una base per l’a-
nalisi critica finanche de mitizzante di quelle tendenze sociologiche fiorenti per lo più in concomitanza con la
fine del Novecento che hanno assunto l’individuo rifles-
sivo come interprete illuminato di una società libera dal
“giogo” e dalla “colonizzazione” di forme istituzionaliz-
zate di potere attribuito alle élite. La possibilità di un
approccio alla modernità come affidamento univoco al
“progresso” scientifico e tecnologico in grado di emanci-
pare le masse si scontra non con la riproposizione di un
elitismo classico per cui la massa, indistinta e amorfa, è
incapace di procedere sul terreno della “individuazione”
(la graduale costruzione di una propria biografia per-
sonalizzata), quanto con i limiti che tale emancipazio-
ne continua ad avere nei condizionamenti della cultura,
dell’economia e delle condizioni di manipolazione messe
in atto dai processi di razionalizzazione stessa (Caval-
li 1982a). Si tratta anche in questo caso non di negare il
principio della libertà individuale, quanto di mettere in
evidenza, in un modo che richiama apertamente la pro-
spettiva critica di Mills, il paradosso della possibilità che
siano proprio le dinamiche che guidano la modernizza-
zione a creare «nella società di oggi condizioni e forze
che possono distruggere la democrazia stessa» (Cavalli
1981a: 285). Anche in questo caso Cavalli si dimostra in
piena sintonia con la lezione weberiana nell’individuare
una stretta relazione fra dinamiche della razionalizza-
zione e l’emergere del paradosso della patogenesi della
modernità, prendendo così le distanze da una acritica
fiducia nel progresso condivisa dalla teoria della moder-
nizzazione e dal marxismo (Peukert 1993: 348). Non
solo, le possibili ambivalenze della razionalizzazione si
pongono in stretta connessione con il processo di demo-
cratizzazione e con l’ingresso delle masse nella storia. In base a questa – necessariamente breve – conte-
stualizzazione iniziale, ciò che segue non vuol avere il
carattere di un mero tributo al pensiero sociologico di
Luciano Cavalli, ma l’intento è di offrire alcuni spunti
per avviare una interlocuzione costante con un Autore
che ha affrontato da sociologo temi e problemi di ine-
ludibile attualità, quali il carisma, la personalizzazione
della politica, la leader democracy, per arrivare al più
recente irrompere del populismo nella ridefinizione della
rappresentanza politica nelle liberal-democrazie. Sebbe- 44 Lorenzo Viviani Pur con la mancanza di sviluppo organico che con-
traddistingue la teoria weberiana del carisma, il nodo
centrale è costituito dalla «conciliazione fra carisma e
razionalizzazione», portando il carisma a legarsi inscin-
dibilmente alla dimensione politica attraverso i concet-
ti di democrazia con un leader (Führer-Demokratie),
democrazia plebiscitaria (plebiszitäre Führerdemokratie),
cesarismo, così come emergono in Economia e società e
negli Scritti politici, fino alla “riscrittura laica” del leader
carismatico contenuta ne La politica come professione
(Cavalli 1981b: 181; 2010: 24; Schluchter 1987: 154). Per
arrivare alla trattazione politica del carisma nelle demo-
crazie contemporanee Cavalli affronta la lettura webe-
riana della razionalizzazione della società, e in questa
si relaziona con temi centrali, ieri come oggi, per com-
prendere le dinamiche del mutamento sociale e politico,
quali il processo di secolarizzazione, la progressiva sepa-
razione delle sfere della vita, il politeismo dei valori e il
problema dell’anomia che si annida come «patogenesi
della modernità» nei processi stessi di razionalizzazio-
ne, con la pervasività della gabbia d’acciaio, da una par-
te, e il ritorno a religioni di redenzione come risposta
al vuoto di senso, dall’altra. La razionalizzazione viene
così analizzata a partire dai processi di trasformazione
della religione, fino al progressivo affermarsi in tutti gli
ambiti dell’agire umano come «ricerca e uso dei mez-
zi più adatti a raggiungere il fine proprio delle diverse
“sfere” della vita o, se si preferisce, il successo in esse»
(Cavalli 1981b: 167). Se per Weber la razionalizzazione
assume significati diversi, come razionalità strumenta-
le nella condotta di vita, ma anche come “calcolabilità”,
ossia come impiego della scienza e della tecnologia per
raggiungere i propri obiettivi, e infine come “routiniz-
zazione” del carisma (Schroeder 2020), l’aspetto su cui
Cavalli pone maggiore attenzione è come “l’illuminazio-
ne carismatica della ragione”, la fase finale del processo
di laicizzazione del carisma, porti con sé alcuni aspetti
di (apparentemente) paradossale contraddizione. Cavalli non si limita al richiamo weberiano, ma affron-
ta sociologicamente la natura “dell’uomo senza valori”,
imprigionato nella sola dimensione dell’interesse eco-
nomico come pervasivo di un quotidiano orientamen-
to all’azione, per di più continuamente esposto a pseu-
do-valori e alla manipolazione ad opera di demagoghi. Se questo è lo scenario culturale e sociale della società
razionalizzata (e ancora più di quella iper-razionalizza-
ta attuale), in agguato è il rischio di un disgregamento
sociale il cui primo effetto è la perdita del fondamento
unitario della società e dello Stato. La ragione emancipa
progressivamente la società dal ricorso a teodicee reli-
giose che giustificavano la stratificazione dei privilegi e
le disparità fra individui, promettendo un riscatto ultra-
terreno. Tuttavia la consapevolezza sociologica di tale
processo implica anche il riconoscimento del persistere
di spinte centrifughe non risolte dalla razionalizzazione
e dalla illuminazione carismatica della ragione. Oltre il
disincanto, è l’accrescersi di fenomeni di risentimento
che contribuisce a fare della irrazionalità un prodotto
stesso della razionalizzazione, segno di quel paradosso
dei processi della modernità al cui interno continuano
a operare disponibilità a nuove forme di affidamento di
tipo fideistico-religioso (Schluchter 1987). Non può non
cogliersi in questo aspetto quanto argomentato dallo
stesso Weber nel Giudaismo antico, ossia il fatto che la
condizione di oppressi nel presente viene accompagnata
dalla attesa di un riscatto nel futuro legato all’avvento di
(T
) ne di Michels – responsabile di sviluppi naturalmente
oligarchici e anti-democratici, né accettarla in termini
vaghi come Mosca, Pareto o lo stesso Durkheim, bensì
renderla possibile (Cavalli 1973: 150). La partecipazio-
ne diventa così un valore se e quando si libera dal gio-
go della tutela operata dai partiti di massa, interpreti
di una intermediazione razionalizzata e specializza-
ta, responsabile del mantenimento dei cittadini in una
condizione di sudditi, ancorché celino il proprio vero
volto celandosi dietro le retoriche del «democraticismo
umanitario» (Cavalli 2001: 217). Anche in riferimen-
to alla democratizzazione Cavalli mantiene lo sguardo
del sociologo che si interroga sulla complessa trama dei
processi sociali e politici, e non è un caso che il discor-
so sulla partecipazione, sulla democrazia e sul carisma
abbia una affinità e continuità, e non un interesse mera-
mente filologico-esegetico, con il Weber dei limiti della
illuminazione carismatica della ragione. 1 È infatti nella parte finale di Etica protestante e lo spirito del capi-
talismo che Weber pone il problema dell’esito della razionalizzazione
sugli “gli ultimi uomini”, chiedendosi se «alla fine di questo enorme
sviluppo, vi saranno profeti interamente nuovi o una potente rinascita
di principi ideali antichi, oppure, ancora – escludendo l’una e l’altra
alternativa – una pietrificazione meccanizzata. Allora, certo, per gli
“ultimi uomini” di questo sviluppo culturale potrebbe diventare veri-
tà il principio: specialisti senza spirito, gaudenti senza cuore – questo
nulla s’immagina di essere salito a un grado mai prima raggiunto di
umanità» (1965 [1905]: 306). Un interrogativo che riemerge nella sua
“tragicità” ne La politica come professione laddove Weber lascia il
monito sulla non naturale evoluzione in termini di progresso della
condizione umana, laddove afferma che «Non abbiamo davanti a noi
la fioritura dell’estate, ma in primo luogo una notte polare di gelida
tenebra e di stenti […] Quando questa notte sarà lentamente trascor-
sa chi sarà ancora vivo di coloro la cui primavera ha ora avuto una
fioritura apparentemente così rigogliosa? E che cosa sarà allora dive-
nuto interiormente di tutti loro? Amarezza o filisteismo, una sempli-
ce e ottusa accettazione del mondo e della professione, oppure – terza
ipotesi e non la più rara – fuga mistica dal mondo per coloro i quali
ne hanno il dono oppure – spesso e peggio – per coloro che vi si dedi-
cano per seguire la moda?» (2004 [1919]: 120). Nella prospettiva del realista radicale la partecipa-
zione dei cittadini alla politica, lungi dall’essere inter-
pretabile come momento emancipativo all’insegna della
«gestione sociale del potere» o come naturale approdo
alla democrazia partecipativa, assume il preciso senso
di una mobilitazione che crea le premesse per il supera-
mento delle oligarchie dei partiti ideologici e l’affermar-
si di nuove leadership (Cavalli 1973; 1981a). La parteci-
pazione politica è un tema che segna la sociologia della
democrazia in Cavalli nello svelamento delle manipo-
lazioni o delle mis-rappresentazioni che derivano dalle
ambiguità create dalla “illuminazione carismatica del-
la ragione” applicata ai processi democratici. In questo
senso, infatti, Cavalli indica nel formalismo dell’egua-
glianza “verso il basso” il vulnus che si cela dentro le
contraddizioni che caratterizzano da sempre la demo-
crazia (Cavalli 1981a: 292-293). Riconoscere weberiana-
mente i diversi livelli di competenza e di Beruf in rela-
zione alla politica, non significa infatti per Cavalli nega-
re la democrazia, né considerarla – come nella trattazio- 45 Carisma e leadership nella sociologia della democrazia to individuo (Schroeder 1987: 217). Se la religione perde
progressivamente il carattere pervasivo di fonte sovra-
ordinata di interpretazione di senso, un tale processo
non determina la soluzione dei conflitti relativi ai fini
e alle credenze, bensì li moltiplica in relazione al poli-
teismo dei valori che guida e orienta la vita degli indi-
vidui. Il dominio consapevole del mondo pone un tema
pressante, la conciliazione fra la necessità di superare il
«rifugiarsi nel regno oltremondano di una vita mistica o
nella fratellanza delle relazioni immediate tra gli indivi-
dui» (Weber 2004 [1919]: 43) e una nuova ricerca di sen-
so. Cavalli assume la consapevolezza weberiana del fat-
to che l’uomo, libero dal carisma “puro” delle origini, è
orfano di un criterio univoco di orientamento di senso
ed è chiamato a decidere autonomamente nella propria
quotidianità. La “tragicità” di questa libertà si misura
con la domanda su «quale degli dei in lotta dobbiamo
servire», nel momento in cui la secolarizzazione pone di
fronte alla assenza di Dio e di profeti nella storia. Ben
consapevole dei più recenti processi di mutamento nelle
società contemporanee, Cavalli si sofferma sulla specifica
dimensione valoriale che si pone laddove la ragione illu-
minista sia l’unico criterio chiamato a costruire senso. La relazione fra crisi e
anomia viene riproposta da Cavalli in termini di impat-
to delle situazioni di emarginazione, insicurezza e solitu-
dine sulla disponibilità della massa a riattivare elementi
latenti di disponibilità all’affidamento al carisma, come
unica forza in grado di creare una realtà comunitaria
di protezione e di senso (Cavalli 1981a: 273). Questo è il
terreno stesso in cui si riattivano le possibilità del cari-
sma, benché in forme diverse da quello della società non
razionalizzata e non secolarizzata. Il carisma torna nella
società razionale quando situazioni economiche, sociali,
religiose, al pari dei fallimenti politici, rendono i prece-
denti strumenti non più adeguati non solo alla soluzio-
ne dei problemi, ma alla riattivazione di valori in grado
di creare processi di riconoscimento, o, per recuperare
le forme del dominio di Weber, non capaci di attivare
quella credenza nella legittimità del potere che non può
ridursi alla sola esecuzione di una obbligazione formal-
mente legittima. Cavalli coglie pienamente la centralità
di questo passaggio per la teoria del carisma, assumen-
do la centralità dell’apporto di grandi personalità nelle
fasi di “crisi totali” che coinvolgono l’individuo e l’intera
società. Non solo, Cavalli è consapevole che la possibili- non operano in senso anti-moderno rispetto al processo
di autonomizzazione dell’individuo, né si ha l’inversione
dei processi di secolarizzazione delle religioni di reden-
zione, né più in generale si assiste a un qualche collasso
della razionalizzazione. La conciliazione fra carisma e
razionalizzazione ha sede proprio come portato specifico
dell’irrompere di una crisi straordinaria in una società
moderna, con la riattivazione della «qualità straordina-
ria» di un individuo, in grado di «incorporare la speran-
za», attivando un movimento rivoluzionario non solo
come cambiamento dello stato delle cose, ma in grado
di realizzare un «homo novus» (Cavalli 1982b: 16-17). Il
ritorno del carisma non equivale al ritorno del carisma
“puro” delle origini, il carisma nella sue forme magico-
religiose, così come non prevede la negazione della divi-
sione della società in sfere autonome e razionalizzate, ma
ciò che si afferma è per Cavalli «un concetto più ristretto
di carisma», che si basa sulla «qualità ritenuta straordi-
naria», ossia una qualità che richiede un riconoscimen-
to da parte delle masse, in questo senso non confinate a
un ruolo non meramente passivo, ancorché non ricono-
sciute artefici di una volontà politica autonoma rispetto
alla relazione con il leader. 2 Esemplare della non sovrapponibilità del grande eroe con la vocazione
alla leadership è la lettura che Cavalli conduce sul Coriolano di Shake-
speare ne Giulio Cesare, Coriolano e il Teatro della Repubblica (2006), Nella lezione weberiana, l’uomo liberato dai vin-
coli di credenze metafisiche e libero di usare la ragione
come strumento di emancipazione, si trova di fronte a
due prospettive, la prospettiva del politeismo dei valori e
quella del disincanto, come emerge nell’Etica protestante
e lo spirito del capitalismo e nella lezione su La politica
come professione1, scritti in fasi diverse, ma che ribadi-
scono la centralità del filo che lega il pessimismo sugli
“ultimi uomini” e, non di meno, le possibilità del cari-
sma. È infatti il richiamo e – al tempo stesso – il supe-
ramento dell’idea nietzschiana dell’Übermensch, il Supe-
ruomo che afferma la propria superiorità senza uscire
dal confine della propria individualità, che permettono
a Weber di assumere la possibilità che attraverso la lea-
dership carismatica la politica possa recuperare il suo
primato alla luce delle qualità superiori di un determina- 46 Lorenzo Viviani La disgregazione e la perdita di senso sono temi
che Cavalli ha ben presenti in relazione a quelle situa-
zioni di crisi che costituiscono il terreno delle religio-
ni di redenzione, offrendo risposta alla «fondamentale
domanda dell’uomo sul senso del mondo e della vita, a
una profonda esigenza di ordine e giustizia» (Cavalli
1981b: 184-185), a riprova del fatto che la razionalizza-
zione non elimina la ricerca di senso con la promessa del
controllo della ragione sul mondo. Non solo, infatti, la
razionalizzazione «non scaccia la religione dalla società
degli uomini», ma sono proprio i suoi corto-circuiti, gli
eccessi, che perpetuano elementi di «antichissime strut-
ture psicologiche e culturali» (Cavalli 1985: 15-16) e che
nell’era del Razionale mantengono l’Irrazionale. Oltre
a Weber nella riflessione di Cavalli si può chiaramente
leggere il richiamo all’anomia come disgregazione del
legame sociale che Durkheim identifica con la condi-
zione di mancanza di adesione a un fondamento mora-
le, e che nella riflessione sociologica successiva si pone a
fondamento dell’insorgere di fenomeni politici radica-
li, basti pensare all’affermarsi delle leadership populiste
analizzate da Germani, ma anche dell’estremismo delle
classi medie affrontato da Lipset. È proprio nella cri-
si straordinaria che le necessità trascendono la sfera dei
bisogni economici routinari e si avvia una fase di par-
ticolare tensione, sia essa «psichica, fisica, economica,
etica, religiosa o politica», che rende possibile l’affida-
mento a “leader naturali” dotati di qualità straordinarie
(Weber 1978 [1922]: 1111-1112). Su questo
aspetto Cavalli si differenzia parzialmente da Weber, for-
mulando una critica volta al completamento della sua
analisi, e non al suo superamento, per aver trascurato la
possibilità, storicamente verificatasi dopo la morte dello
stesso Weber, che in particolari condizioni i «processi
carismatici di massa si trasferissero dalla sfera religiosa
tradizionalmente intesa alla sfera politica, in base ad ide-
ologie di massa costruite come religioni laiche di reden-
zione per un’epoca di razionalizzazione» (Cavalli 1981a:
190). Nazismo e comunismo vengono così analizzati da
Cavalli come affermazioni di sistemi di credenza che ri-
sacralizzano la società, favorendo l’emergere di tiranni
carismatici, ma anche di pseudo-capi che alimentano il
“culto della personalità” con la re-infantilizzazione delle
masse. Il terzo nucleo attiene alla articolazione del pro-
cesso carismatico, dalla crisi straordinaria fino alle for-
me della successione, sviluppando in modo originale e
autonomo la lezione weberiana, e al tempo stesso com-
pletandone lo sviluppo con l’elaborazione di un para-
digma in grado di leggere i fenomeni carismatici anche
in chiave comparata (Cavalli 1986; 1987a; 1991). Infine,
il quarto nucleo attiene alla costruzione di tipologie di
leader carismatici, con l’assunzione del carisma come
concetto chiave per confutare la supposta equivalenza
fra i «dittatori plebei», fa cui i tiranni carismatici Hitler
e Mussolini, e il «capo carismatico politico propriamente
tale», il leader di Politik als Beruf di Weber, con la sua
specificità etico-culturale calata all’interno della demo-
crazia plebiscitaria (Cavalli 1992: 9). 3. Plebiscitarismo e leadership carismatica: raziona-
lizzazione e democratizzazione. La domanda su quali sia-
no le reali possibilità del carisma nella società moderna
si lega indissolubilmente allo spazio accordato alla poli-
tica nell’ambito della autonomizzazione e della raziona-
lizzazione delle sfere di vita. È infatti nel recupero del
primato della politica che si può avere la riattivazione
del carisma come soluzione alle patologie stesse della
modernità, fra cui la forza dei valori puramente stru-
mentali della burocrazia, la presenza di «interessi setto-
riali in competizione» e l’esistenza di movimenti di mas-
sa portatori di «pressioni “irrazionali”» (Beetham 1989:
13). Questo è lo specifico ambito della riflessione webe-
riana, e della sua elaborazione da parte di Cavalli, sul-
la trasformazione in senso extra-autoritario del carisma
(Weber 1978: 266), in cui «il carisma, reso per così dire
laico, è infatti al centro della ben nota proposta della
democrazia plebiscitaria» (Cavalli 1981b: 176). in particolare dove caratteristiche come l’assolutezza, l’insocievolezza, la
dismisura, l’eccesso e più in generale l’incapacità, tipica invece del rea-
lista, di cogliere il moto storico e porsi con responsabilità e anche con
una capacità di “simulazione e dissimulazione” di fronte al contesto
sociale e politico in cui il leader è chiamato a operare. È all’interno di tale processo
che si aprono le nuove possibilità per il carisma, e non di
meno è la ricezione sociologica di questa nuova relazio-
nalità fra leader e follower che ingenera il dibattito sulle
reali possibilità del carisma nelle società e nelle demo-
crazie contemporanee. p
Questo è anche lo specifico contesto in cui Cavalli
si distingue per l’originalità con cui recupera la lezione
Weber, in particolar modo attraverso la riflessione su
alcuni nuclei teorico-argomentativi che saranno decisi-
vi nello sviluppo della sua sociologia della leadership e
della democrazia. Il primo nucleo si sostanzia nel recu-
pero del ruolo «delle grandi individualità nella storia»,
con una presa di posizione critica verso quelle prospet-
tive sociologiche che affidano solo a movimenti col-
lettivi i processi di trasformazione sociale e politica. Come osservato, per Cavalli il recupero della centralità
del valore e del ruolo di tali personalità non comporta
in alcun modo l’irrilevanza dei cittadini, dato che nel-
lo stesso processo carismatico i follower non costitu-
iscono un “aggregato anonimo”, ma partecipano alla
relazione sociale della leadership e la missione stessa
del leader è valutata dall’impatto che è in grado di pro-
durre sulle masse in termini di cambiamento di valori
(Cavalli 1981b; 1982a). Nella sociologia della leadership
di Cavalli le grandi personalità non si risolvono in un
tributo all’eroe2 e non rimandano alla “teoria del gran- Carisma e leadership nella sociologia della democrazia 47 de uomo” (Carlyle 1841), a partire dall’esplicito richiamo
al fatto che non si ha nessuna contiguità con «l’indivi-
duo monumentalizzato» o con «il sovrano della storia»
(Cavalli 1981b: 181). Il riferimento costante di Caval-
li è alle qualità etico-politiche del carisma laicizzato di
Politik als Beruf, in cui emergono le qualità specifiche
del capo politico a cui si allude nella democrazia plebi-
scitaria. Il secondo nucleo riguarda invece la maggior
consapevolezza di Cavalli rispetto a Weber sul ruolo
storico delle “religioni laiche di redenzione” e sul loro
effetto nei processi di partecipazione democratica delle
masse. Si tratta di tenere in considerazione il peso del-
le ideologie totalizzanti che riportano nel regno dell’al-
diquà una promessa escatologica di salvezza, come vere
e proprie strutture a sostegno (manipolato) della clas-
se politica all’interno delle democrazie senza un leader,
il cui fondamento è da ricercarsi nel disincanto e nella
politicizzazione del risentimento delle masse. Questo
è il passaggio chiave che consente a Cavalli di assume-
re la centralità della Führerauslese, ossia della selezio-
ne dei leader, come punto di partenza per sviluppare le
differenza weberiana fra la democrazia plebiscitaria e
la democrazia senza un leader (führerlose Demokratie),
ossia fra «una democrazia subordinata a un capo e orga-
nizzata mediante la “macchina”, oppure una democrazia
senza capi, vale a dire il potere dei “politici di professio-
ne” senza vocazione, senza le intime qualità carismatiche
che per l’appunto fanno un capo» (Weber 2004 [1919]:
92). Su tale distinzione Cavalli articola il suo discorso
sociologico sulla leadership personalizzata nell’ambito
dei due tipi di democrazia, la democrazia con un lea-
der e la democrazia acefala (Cavalli 1987b; 1992; 1993;
2001; 2003), assumendo che proprio nella celebre lezione
ai Liberi studenti di Monaco del 28 gennaio 1919 debba
essere cercata la natura del capo carismatico come lea-
der politico moderno, dal momento che in essa viene
presentato il «carisma in termini laici e moderni, attra-
verso l’analisi della personalità del capo» (Cavalli 1981a:
219). È in tale opera che insieme alla ripresa dei concetti
generali del processo di legittimazione del potere, e alla
distinzione fra le due “democrazie”, viene esplicitato il
Beruf del leader carismatico calato all’interno delle dina-
miche plebiscitarie della democrazia. La passione, come
dedizione a una causa, ossia l’intima chiamata che dà il
senso alla missione interiore del leader, la responsabili-
tà nei confronti di questa causa, e la lungimiranza come
«distanza tra le cose e gli uomini» (Weber 2004 [1919]:
101-102), queste sono le tre qualità che fanno da discri-
mine alla leadership autentica. Non solo, nella lezione
weberiana sul Beruf della politica Cavalli coglie e svi-
luppa anche altri tre nodi centrali, quali la differenza
fra lo scienziato e il politico, il riferimento al realismo
politico di chi deve raggiungere il proprio fine confron-
tandosi anche con l’ambiguità dei mezzi necessari, e infi-
ne la distinzione fra i “veri” leader e gli pseudo-leader, i
leader del mero sembiante, i leader della contraffazione
d l
i
i l
d
i
d
li
i di i
ll quello che doveva in un primo momento essere per
Weber il «tipo transitivo più rilevante di legittimazione
dell’autorità» nel passaggio alla democrazia rappresenta-
tiva, con il “fatto” dell’ingresso delle masse nella politica
attraverso il suffragio universale, diviene invece il rico-
noscimento che la democrazia di massa, e potremmo
aggiungere la rappresentanza politica in una società di
massa, è intimamente e inestricabilmente incentrata sul
“principio plebiscitario”. Ma se è vero che Weber svilup-
pa un «tipo ideale, quello di una moderna democrazia in
cui le relazioni politiche significative si sviluppano intor-
no a una magistratura monocratica e ad una personalità
dotata di carisma» (Cavalli 1993: 54), ciò che crea pro-
blemi nella ricezione del carisma come fenomeno conci-
liabile con la democrazia è proprio il mancato sviluppo
di una trattazione sistematica della democrazia plebisci-
taria moderna. Non è irrilevante precisare che scrivere sul cari-
sma nel periodo in cui Cavalli affronta tali temi voleva
anche dire confrontarsi con la persistenza di un retaggio
critico, segnato anche da un marcato orientamento ide-
ologico, sulla ricezione del plebiscitarismo carismatico
weberiano, quasi che quest’ultimo fosse un’anticipazione
e un antecedente, più o meno consapevole, di una deriva
autoritaria che avrebbe aperto la strada a sviluppi ditta-
toriali (Lukács 1980; Löwith 1987; Beetham 1989). Non
solo, il carisma è stato oggetto anche di un tentativo di
derubricarne la portata, come concetto non scindibile
dal suo ancoraggio al mondo magico-religioso e come
tale inapplicabile nella modernità politica (Loewenstein
1966: 77-79), se non in specifiche realtà soggette al pro-
cesso di decolonizzazione, in una chiave interpretativa
per cui la leadership carismatica perde di rilevanza in
contesti modernizzati in cui «la fede in un essere tra-
scendente non è sufficientemente forte o generale per
fornire una base adeguata per legittimare una qualsiasi
leadership politica» (Friedrich 1961: 23). La chiave
interpretativa su cui Cavalli si inserisce è quella del ple-
biscitarismo come fatto rilevante della politica moderna
in cui si ha il passaggio dal riconoscimento del leader
come “conseguenza” della legittimità che deriva dal cari-
sma “puro”, al riconoscimento del leader come “base”
della legittimità, un processo il cui esito è l’affermarsi
della “legittimità democratica” (Weber 1978 [1922]: 266-
268). L’incompletezza dell’opera weberiana porta Caval-
li a riprendere il filo dell’analisi dipanando una trama a
tratti complessa e frammentaria fra i concetti di demo-
crazia con un leader (Führer-Demokratie), democrazia
plebiscitaria (plebiszitäre Führerdemokratie), cesarismo,
bonapartismo, con i relativi esempi, storicamente e poli-
ticamente diversi, portati da Weber. È qui che Caval-
li dà vita ai propri contributi seminali per la sociologia
politica, con la capacità di riannodare la trattazione del
carisma e della democrazia plebiscitaria in Economia e
società e negli Scritti politici, con particolare attenzione
alla “riscrittura laica” del leader carismatico ne La politi-
ca come professione. Cavalli è ben consapevole che l’analisi weberiana
della democrazia plebiscitaria e del «capo carismatico
come leader politico nella storia occidentale» lasciano
aperto il problema del «principio cesaristico della scelta
dei capi» nei regimi democratici (Cavalli 1981b: 189), e
che la concezione di democrazia plebiscitaria necessi-
ta di un ulteriore sviluppo sociologico. La democrazia
plebiscitaria è infatti il tipo più rilevante di Führerde-
mokratie, lo specifico terreno in cui avviene il passaggio
dal riconoscimento del leader come “conseguenza” della
legittimità che deriva dal carisma “puro”, al riconosci-
mento del leader come “base” della legittimità, il cui esi-
to è quello dell’emergere della “legittimità democratica”
(Weber 1978 [1922]: 266-268). Cavalli sottolinea come 48 Lorenzo Viviani e generico» di democrazia plebiscitaria contenuto in
Economia e società debba ricongiungersi con il concet-
to, pur ancora «non chiaramente distinto e tanto meno
formalizzato», di democrazia plebiscitaria moderna o
di «democrazia con un leader con potenzialità carisma-
tico-plebiscitarie» contenuto in Parlamento e governo
e, soprattutto, ne La politica come professione (Cavalli
1993: 50-51). Inoltre, a partire dal riconoscimento della
complessità del termine carisma, la «qualità straordi-
naria» può rimandare a un elemento religioso come nel
caso di Gesù, o alternativamente richiamare una quali-
tà «semplicemente eccezionale», come tale diversa dal
carisma puro delle origini (Cavalli 1992: 36). Nello specifico della trattazione di Cavalli ciò significhe-
rebbe disconoscere il rilievo da questi accordato ai pro-
cessi di formazione e selezione dei leader che richiama-
no qualità, invero del tutto moderne, quali la necessità
di coltivare la «forza della convinzione» e la «capacità di
generare fiducia», per di più contagiosa, in virtù di tale
convinzione (Cavalli 1996). Al pari della riflessione di
Weber, sarebbe ulteriormente fuorviante pensare che il
leader carismatico su cui riflette Cavalli sia “solo” il lea-
der che fa costante riferimento all’etica dei principi, o,
alternativamente, al solo perseguimento dell’etica della
responsabilità, due elementi che si completano a vicen-
da e che soltanto insieme provano il Beruf del “vero”
leader. È altresì evidente in Cavalli che il riferimento del
leader carismatico nella democrazia non è, e non può
essere, l’eroe che si muove in virtù di una assolutezza
che non assume su di sé il “senso” e il “contesto” di una
data situazione, come appare dalle doti “impolitiche” del
Coriolano di Shakespeare, non caso «del tutto inadatto
alla leadership politica nella nuova situazione di Roma»
in virtù della sua non comprensione del moto storico,
così come del farsi portatore di caratteristiche che osta-
colano la leadership, quali l’assolutezza, l’insocievolezza,
la dismisura e l’eccesso (Cavalli 2006: 113). Ancora, Cavalli definisce il perimetro di rilevan-
za della leadership nel «teatro della politica» mettendo
in risalto la funzione simbolica del potere, sviluppando
i riferimenti weberiani e offrendo una trattazione più
approfondita della capacità e necessità da parte del lea-
der di operare attraverso la forza della convinzione e la
relativa capacità suggestiva tramite il ruolo di demago-
go, termine impiegato nella sua accezione più storica-
mente fondata e depurata dalla stessa alea di negatività o
imbarazzo come già Weber aveva precisato (Weber 1978
[1922]: 1449). La funzione simbolica affidata al leader è
in questo senso “attiva”, dato che al leader non è chiesto
soltanto di assumere i valori e la cultura di un gruppo su
di sé, quanto di creare quei valori e di esserne il porta-
tore simbolicamente riconoscibile in grado di assicurare
la sopravvivenza del gruppo su di essi fondato (Elcock
2000: 25). Attraverso le pagine di Parlamento e governo
di Weber, Cavalli analizza, argomenta e arricchisce di
significato in chiave democratica il leader plebiscitario
come confidente delle masse di cui conquista la fiducia
attraverso i mezzi della demagogia. Infine non va
dimenticato il contributo delegittimante di tale concet-
to che nel corso del tempo stata offerta dalla vaghezza
di una fascinazione per lo più ricondotta al sembiante e
alla retorica del leader, con il corollario del discredito di
una mera nozione-ombrello che da concetto sociologico
è progressivamente stato banalizzato e ridotto a espres-
sione di senso comune. Cavalli si muove esplicitamente in senso contrario
a queste ricostruzioni e riprende il percorso intellettua-
le weberiano nella sua interezza, entrando a pieno titolo
a far parte di coloro che hanno contribuito alla rilettura
complessiva dell’opera di Weber e alla sua ricezione in
termini di perdurante attualità del suo pensiero socio-
logico. Nello specifico del rapporto tra carisma e demo-
crazia, Cavalli sottolinea come il concetto «molto vago 49 Carisma e leadership nella sociologia della democrazia li 1981a: 273). La funzione della leadership qui evocata
richiama non solo la necessità del leader di definire gli
obiettivi dei diversi tipi di gruppi e aiutare i follower a
raggiungerli, ma una più pervasiva capacità di costrui-
re i gruppi stessi dotandoli di una propria identità e di
un perimetro coeso di riconoscimento, di contribuire
alla loro organizzazione e mobilitazione, di diagnosti-
care i problemi e offrire soluzioni e quadri interpretati-
vi per semplificare i processi sociali e politici, così come
di offrire riferimenti simbolici di senso in caso di crisi
e trasformazioni (Tucker 1981; Kellerman 2004). La
costruzione e la conservazione del gruppo, i processi di
identificazione collettiva, la definizione della realtà, sono
le funzioni sociali che Cavalli assume come rilevanti per
la leadership, sapendo che in alcuni casi è il leader stes-
so che assume il ruolo di «“protettore” contro pericoli e
difficoltà collettive […] con poteri di intervento contro
le leggi dell’economia o le leggi medesime dello Stato»
(Cavalli 1996). i tuali di basso livello, anch’essi spostati e sradicati». Sul
riconoscimento di tali qualità è innegabile che Weber e
Cavalli si intreccino in una comune visione di cui sareb-
be sociologicamente miope indicare, per entrambi, un
vizio di origine nel non aver tenuto conto della impos-
sibilità di laicizzare la componente magico-religiosa o la
dimensione sovrannaturale dal “carisma dei moderni”. Un aspetto di cui Cavalli è ben consa-
pevole quando leggendo il Giulio Cesare di Shakespea-
re non attribuisce alla pur suadente – e in stile “asiano”
– orazione di Antonio il riconoscimento dell’atto di lea-
dership, quanto invece la forma propria del menuer che
suggestiona, manipola e sobilla la massa con lo stile pro-
prio dell’uomo comune, la cui identità che si riflette nel
popolo mettendosi al suo pari (teatralmente e politica-
mente) testimonia la specificità di un “motivo populista”,
realtà assai diversa dalle qualità della leadership come
soluzione della crisi che apparterranno, nel frangente
storico considerato, a Ottaviano Augusto. oltre la rilevanza pubblica della sua immagine, e che
riguarda la capacità di porsi dentro i processi della sto-
ria, con la consapevolezza del sociologo realista che se la
storia è alimentata da un conflitto continuo per il pote-
re e non soggiace ad alcun fine ultimo, è pur vero che
la politica necessita di una ricerca “altra” rispetto alla
mera praticabilità dei mezzi, ossia impone un «tasso di
trascendenza etica che la politica deve avere rispetto alla
fatticità del mondo» (D’Andrea 2018: 74-75). Se queste sono le funzioni della leadership cari-
smatica, nella versione laicizzata a cui Cavalli fa riferi-
mento, ciò avviene con la consapevolezza che il “come”
e la “direzione” che possono assumere tali processi di
relazione fra leader e masse nella storia sono tutt’altro
che univoci. Si ripropone un aspetto centrale in tutta la
riflessione sociologica di Cavalli, quella manipolazio-
ne di cui si compongono sia le democrazie, sia i regi-
mi totalitari, e che nel caso di questi ultimi agisce sul-
la «fuga di massa dall’insicurezza e dalla solitudine del
mondo moderno» che in particolari contesti di crisi si
attiva presso le masse (Cavalli 1996)4. Il tema dominan-
te rimane la possibilità che una crisi straordinaria pos-
sa generare alternativamente una situazione di nuova
anomia destinata a disgregare la società e la politica, o
la comparsa di una leadership in grado di risolvere la
crisi attraverso una trasformazione del sistema. Si trat-
ta quindi di valutare non solo le condizioni di contesto
sociale, istituzionale, culturale ed economico che creano
la disposizione all’affidamento a un leader, ma anche le
qualità stesse del leader che si inserisce nei processi di
politicizzazione del vuoto di senso, attribuendo un peso
significativo ancora una volta al ruolo delle individualità
nella storia. 3 Questo aspetto della centralità e del protagonismo dell’individuo in
relazione al contesto culturale e alle masse emerge in tutta la sua tra- gicità anche nell’analisi sociologica che Cavalli compie sul caso Hitler
(1982b; 1993; 2003). Cavalli ripercorre criticamente le argomentazioni
che avevano portato a negare, o a sminuire, il ruolo e le relative respon-
sabilità di Hitler come capo carismatico, affermandone il ruolo centrale
e drammatico nella storia per aver forgiato con una propria Welthan-
schauung un’attesa del popolo tedesco che, senza il ruolo del tiranno
carismatico e della «consequenziarità» rispetto alla fede nella sua fol-
le missione, non avrebbe assunto quello specifico connotato (Cavalli
1982b: 19-20). In riferimento alla centralità del leader carismatico si
deve quindi tenere primariamente in considerazione la sua capacità, nel
bene e nel male, di essere l’artefice di un «mutamento culturale in inte-
riore homine», divenendone allo stesso tempo “guida” e “modello”, anche
attraverso le forme tiranniche del “culto della personalità” (Cavalli 2003:
272-273). Questa è propria-
mente la forma che assume il carisma nella sua versione
extra-autoritaria e laicizzata propria delle democrazie,
visibile in modo esemplare nelle leadership di Churchill,
Roosevelt, De Gaulle, in cui la centralità della fiducia, in
primo luogo del leader in sé stesso e nella sua guida, e
la capacità di trasformarla in fiducia collettiva, costitu-
iscono i due pilastri del processo generativo della lea-
dership (Cavalli 2003: 31). Si deve qui una precisazione
su un aspetto che si porrà sempre più come un terreno
insidioso per cogliere la natura della leadership nel-
le democrazie contemporanee. Se infatti all’interno del
processo generativo della leadership Cavalli considera la Il leader carismatico delle democrazie moderne è
colui che weberianamente vive « “per” la politica nel
senso più alto, realizzando cioè ·un “compito interiore”,
una “vocazione”» (Cavalli 1981a: 213). Il “vero” leader è
chiamato a svolgere una serie di “funzioni sociali”, e in
questo senso se con la psicologia è possibile identificare
la rilevanza della funzione “micro” della leadership, la
dimensione chiave percorsa da Cavalli si incentra sul-
la dimensione “macro”, ossia sulla rilevanza della lea-
dership in relazione ai processi di mutamento sociale e
politico. Non si tratta di assumere una visione “funzio-
nale” parsonsiana, ma al contempo neanche di vincolare
il carisma a una mera dinamica del piccolo gruppo, per-
dendosi la specificità propria di ricostruzione di senso
affidata al leader. Il leader carismatico della democrazia
contemporanea svolge una funzione prominente in quel-
le fasi di crisi che determinano disgregazione, anomia,
emarginazione, insicurezza, solitudine, e che nelle socie-
tà moderne assumono la realtà di crisi belliche, econo-
miche, processi di sradicamento il cui effetto è rendere
le masse disponibili all’affidamento al carisma (Caval- 50 Lorenzo Viviani centralità del «carisma della parola», l’arte oratoria, così
come la capacità di operare sui e coi mezzi di comunica-
zione di massa, al tempo stesso non si può sottacere che
la sua riflessione sociologica è attenta a non confinare il
carisma alla semplice abilità tecnica del “comunicatore”. Non il “fatto” della comunicazione, ma il “fatto” del-
la relazione sociale è per leadership il fondamento della
capacità di guida e di «mobilitazione morale di massa»,
e come tale ciò che si prospetta è una più profonda inte-
razione fra la disponibilità culturale, le forme specifiche
del contesto e le qualità di direzione politica del leader
personalizzato. 4 Cavalli, oltre ad assumere come rilevanti le implicazioni psicologiche
della massa che Weber deriva principalmente da Hellpach, per analiz-
zare la relazione leader-masse fa uso degli studi di psicologia di Le Bon,
della psicanalisi di Freud, e della successiva tradizione di analisi critica
sociologica e psicologica sulle masse di Fromm, Horkheimer e Ador-
no, con particolare attenzione alla sottomissione e direzione manipolata
delle masse in contesti di crisi con sviluppi totalitari. Carisma e leadership nella sociologia della democrazia Sarebbe tutta-
via incompleto e fuorviante pensare a Cavalli soltanto
come uno studioso del pensiero sociologico di Weber,
senza valutare gli sviluppi originali offerti nel sottoline-
are le parti carenti della teoria weberiana del carisma,
e senza mettere in evidenza alcuni sviluppi della sua
riflessione, specie in relazione al tema della democrazia,
allo svilupparsi di nuove élite e al tema dei partiti. Pur
riconoscendo la presenza di “spunti” fondamentali per
l’analisi sociologica, per Cavalli ciò che in Weber appare
insufficiente e incompleto è il pieno riconoscimento del
fatto che tramite un leader carismatico, ancorché nella
versione laica del termine, la massa recupera una nozio-
ne di senso in termini di ideali e valori che costituiscono
la base morale di un sistema sociale e politico comune In più parti dell’analisi di Cavalli riecheggiano le
analisi di Mannheim sulla democratizzazione fonda-
mentale e sui rischi che si generano nelle fasi di razio-
nalizzazione dove la razionalità funzionale non rispec-
chia la razionalità sostanziale, ossia la «capacità di agire
intelligentemente in una data situazione sulla base del-
la propria capacità di penetrazione nella interrelazione
degli eventi», il che, specie nel caso di crisi sociali, rende
necessaria la capacità di saper connettere la complessità
e interpretare i cambiamenti attraverso l’appello al lea-
der (Mannheim 1972: 63-64). La «democratizzazione
sostanziale» di Mannheim esprime esattamente il rischio
che si cela nei processi di industrializzazione e di urba-
nizzazione che modificano le basi sociali della democra-
zia, creando fenomeni di insicurezza nelle classi medie
e la diminuzione di solidarietà interna al proletariato. Cosa accade dunque alla democrazia quando i processi
di integrazione sociale non sono più mediati razional-
mente da élite riconosciute (legittimate), e una serie di
movimenti centrifughi irrompono nell’ambito della irra-
zionalità che orienta il conflitto per il potere? Questo per
Mannheim è il terreno in cui può dilagare «l’insicurez-
za non organizzata», ossia lo svuotamento di senso che
deriva da fasi di crisi del legame sociale, con i relativi
effetti sulla modificazione stessa del sistema dei valori
e della struttura di personalità degli individui, conte-
stualmente al crearsi del problema della inadeguatezza
delle vecchie élite e la comparsa di nuove élite. Carisma e leadership nella sociologia della democrazia così da contrastare il vuoto creatosi nella estremizzazio-
ne individuale del politeismo dei valori, in particolare
quello ridotto a calcolo individuale delle utilità in ambi-
to economico. 4. Sul ruolo della leadership e dei partiti nella leader
democracy. Quanto osservato sulla leadership carisma-
tica nella sua versione laicizzata costituisce l’asse por-
tante della sociologia della democrazia di Cavalli. Ciò
che Cavalli coglie chiaramente è un tema che ne orienta
tutta la sua ricostruzione della natura della democrazia
con un leader, ossia il fatto che la razionalizzazione della
società porta con sé un processo di ridefinizione stessa
delle basi dello Stato, in un processo in cui si dissacra-
no i «valori comuni di base» (Cavalli 2016: 128). È così
che si tesse la trama del potere con il tema della creden-
za nel fondamento di legittimità del suo esercizio, la cui
conseguenza è la convinzione che la sola dimensione
legale-razionale priva di un fondamento etico comune
non possa porsi a tutela e mantenimento di un ordi-
ne politico. Proprio quel fondamento etico rappresenta
il “fatto” del carisma nella storia, che pur nelle diverse
forme che assume rimane costantemente la pietra d’an-
golo della legittimazione politica. In tale quadro teorico
Cavalli sviluppa il tipo della «democrazia autocefala con
un leader personalizzato», come specificazione moderna
della – eccessivamente – ampia democrazia plebiscita-
ria weberiana. Una prospettiva in cui vengono accol-
ti gli assunti dello stesso Weber sul fatto che la demo-
crazia di massa è inestricabilmente espressione di una
democrazia della «leadership in azione», così come c’è
il riconoscimento che il carisma non è estraneo a ogni
forma moderna di elezione, compresa quella democrati-
ca (Weber 1978 [1922]: 1126). Pur non potendo entrare
nell’ampia e documentata analisi comparata degli svilup-
pi delle democrazie contemporanee offerta da Cavalli, è
qui rilevante osservare come nella sua riflessione socio-
logica si affermi progressivamente una propria specifica
posizione nell’ambito di un elitismo democratico incen-
trato sul principio del riconoscimento della concen-
trazione del potere nella carica monocratica di vertice,
alla base di un pluralismo del conflitto politico centrato
sulla selezione del leader personalizzato. Consapevole delle ambiguità lasciate da Weber nel-
la scarsa organicità e nel mancato sviluppo di una com-
piuta teoria del carisma in riferimento alla democrazia,
Cavalli ribadisce che l’aspetto più rilevante del leader
carismatico delle democrazie contemporanee è la sua
capacità di entrare in relazione con l’attesa culturale del-
le masse e con il problema delle cangianti rappresenta-
zioni collettive che emergono in una società pluralizzata,
dominata da «dei e demoni», in particolar modo nelle
sue fasi di crisi. Ancora una volta Cavalli non rinuncia
alla lezione realista, e diversamente da altre interpreta-
zioni del carisma, si veda ad esempio la prevalenza del
movimento carismatico sul – successivo – emergere di
un leader (Melucci 1982; Alberoni 1977; 2007), rivendi-
ca al portatore del carisma la capacità fondativa di un
nuovo sistema di valori, una moderna metánoia, resa
possibile da una condizione di lotta e da una disponi-
bilità “effervescente” delle masse. Se la crisi «de-realizza
la “realtà di massa”», per Cavalli (1982a: 613) è il leader
che forma, agendo sulla storia, quella disponibilità e
attesa che senza il suo intervento rimarrebbe incompiu-
ta, distaccandosi nettamente da una visione di leader
come mero portavoce delle masse che di fatto recepisce,
aggrega e articola un sistema di valori e di rappresenta-
zioni collettive già formato nella società, finanche propo-
nendosi o essendo percepito come la personificazione di
un’idea3. Si delinea per il leader un compito che va ben 4 Cavalli, oltre ad assumere come rilevanti le implicazioni psicologiche
della massa che Weber deriva principalmente da Hellpach, per analiz-
zare la relazione leader-masse fa uso degli studi di psicologia di Le Bon,
della psicanalisi di Freud, e della successiva tradizione di analisi critica
sociologica e psicologica sulle masse di Fromm, Horkheimer e Ador-
no, con particolare attenzione alla sottomissione e direzione manipolata
delle masse in contesti di crisi con sviluppi totalitari. 51 Carisma e leadership nella sociologia della democrazia 5 La specificità della democrazia con un leader carismatico fa riferimen-
to alla radicalizzazione del contesto elettorale strutturato come scelta fra
due personalità e due progetti di governo, il ricorso del leader al soste-
gno nel popolo durante il mandato di governo, la possibilità di essere
confermato per un secondo mandato, o sanzionato con la non rielezio-
ne, solo tramite un voto popolare (Cavalli 1987c: 51). A questi punti
si affianca inoltre la dinamica propria del processo carismatico, con la
dinamica situazione straordinaria (di crisi o di statu nascenti), leader
chiamato, leader confermato, leader come uomo di fiducia delle masse,
leader come incarnazione di idee e valori di massa, venerazione del lea-
der, stato maggiore di devoti (Cavalli 1987c; 1995). Carisma e leadership nella sociologia della democrazia Il tipo della democrazia con un lea-
der viene elaborato a partire da una serie specifica di
punti che ne costituiscono i requisiti, fra cui i principa-
li fanno riferimento alla concentrazione del potere nel-
le cariche monocratiche di vertice, incluso il patronage,
e all’elezione diretta del leader da parte del popolo sulla
base di una competizione fra personalità (Cavalli 1987c;
1992), indicando inoltre la possibilità dello svilupparsi di
un sotto-tipo di «democrazia con un leader carismatico»
con la radicalizzazione di alcuni elementi del tipo gene-
rale (Cavalli 1987c: 51; 1995: 331)5. In Governo del leader e regime dei partiti (1992) Cavalli fa esplicito riferimento
a tre “dati” necessari perché la democratizzazione fon-
damentale possa di fatto realizzare la sovranità popolare
e l’autogoverno delle masse, specificando le condizioni
generali nell’esistenza di «una costituzione favorevo-
le, un metodo elettorale conforme», e – aspetto centra-
le dell’analisi sociologico-politica di Cavalli – un “certo
tipo di partito”, quest’ultimo risultante non solo di dina-
miche istituzionali e politico-elettorali, ma della costante
interazione con il mutamento sociale. Se per Weber la democratizzazione e la democra-
zia assumono una rilevanza come “dato di fatto” del-
la società di massa, con particolari implicazioni nel
processo di Führerauslese (Scaff 1981; Eliaeson 1998),
ciò che contraddistingue Cavalli è una maggior atten-
zione rivolta allo svilupparsi contrapposto dei due tipi
di democrazia, “con” e “senza” leader, con le diverse
implicazioni che questi hanno sulla effettiva capacità di
governo, sulla selezione dei leader, sul rafforzamento del
primato della politica sulla burocratizzazione, e sul rag-
giungimento della integrazione nazionale e della politi-
ca di potenza. In linea con l’evoluzione della prospettiva
sociologica dell’elitismo democratico, è così la politica
incentrata sulla leadership che ha la capacità di «adat-
tare e imbrigliare» le forze della razionalizzazione pre-
senti nella società moderna (Pakulski, Körösényi 2012). Non va tuttavia sottaciuto che l’analisi di Cavalli ha una
maggior focalizzazione sulle forme della democrazia di
quanta effettivamente ne abbia dedicata Weber. Abbia-
mo già sottolineato quanto, a causa della sua prematura
scomparsa, il mancato completamento e la scarsa siste-
matizzazione dell’opera weberiana abbiano influenzato
lo sviluppo di una approfondita connessione fra teoria
del carisma e sviluppi di una sociologia della democra-
zia plebiscitaria moderna con specifica attenzione a una
«nuova teoria delle élite». Carisma e leadership nella sociologia della democrazia Il tipo della democrazia con un lea-
der viene elaborato a partire da una serie specifica di
punti che ne costituiscono i requisiti, fra cui i principa-
li fanno riferimento alla concentrazione del potere nel-
le cariche monocratiche di vertice, incluso il patronage,
e all’elezione diretta del leader da parte del popolo sulla
base di una competizione fra personalità (Cavalli 1987c;
1992), indicando inoltre la possibilità dello svilupparsi di
un sotto-tipo di «democrazia con un leader carismatico»
con la radicalizzazione di alcuni elementi del tipo gene-
rale (Cavalli 1987c: 51; 1995: 331)5. In Governo del leader vi organizzati (Germani 1975), così come il più generale
attivarsi di un estremismo di centro in una fase di pro-
fonda insoddisfazione fra «coloro che si sentono tagliati
fuori dalle correnti della società moderna» (Lipset 1963
[1960]: 178). Tuttavia, se questa versione della democra-
tizzazione sembra affacciarsi, e inesorabilmente cadere,
sull’orlo dell’abisso del totalitarismo (Arendt 1966 [1951];
Mannheim 1972), la prospettiva di Cavalli si caratterizza
per una possibilità diversa, ancorché osteggiata dal per-
vicace tentativo dei «gruppi autocefali di potere», le oli-
garchie dei partiti politici, di operare lo svuotamento del
principio della leadership attraverso l’uso strumentale
della legittimazione ideologica. Per Cavalli, così come lo
era stato per Weber, è l’irresistibile processo della demo-
cratizzazione, specialmente con l’ingresso delle masse
nella Storia attraverso il suffragio universale, che crea
le condizioni per l’affermarsi della democrazia plebisci-
taria contrapposta alla democrazia senza un leader, la
democrazia dei partiti. Nello sviluppo dei due paradigmi
di democrazia, Cavalli riconduce l’affermarsi del “tipo”
della democrazia con un leader, la democrazia autocefa-
la con un leader personalizzato, alle dinamiche dei pro-
cessi di modernizzazione, con particolare attenzione alla
dimensione politico-culturale, alla democratizzazione,
al diffondersi dell’istruzione e della cultura, alla secola-
rizzazione e all’accrescersi della rilevanza dei mezzi di
comunicazione. Un tale tipo di democrazia necessita al
tempo stesso di alcune pre-condizioni di natura emi-
nentemente politica che rendono possibile l’affermarsi
di una dialettica disintermediata fra leader e popolo tale
da poter essere declinata come «democrazia immediata»
(Cavalli 1992: 42). Carisma e leadership nella sociologia della democrazia Al tempo
stesso, proprio la disintegrazione della società, l’insor-
gere di una nuova società anomica (Kornhauser 1959;
Cavalli 1981a; 1981b), e più in generale la «distruzione
delle abitudini consolidate e delle aspettative costituite»,
possono attivare quei processi di «insicurezza organizza-
ta» che Mannheim riconduce alla ricerca di un «sollievo
nella creazione di mete simboliche e attività simboliche»
(Mannheim 1968: 107; 1972: 129-130). Situazioni di que-
sto tipo favoriscono l’ascesa di leader abili nel manipo-
lare le emozioni e i simboli dell’insicurezza, sfruttando
la disponibilità delle masse ad affidarsi a sistemi orga-
nizzati che guidano il riadattamento psicologico fino,
potenzialmente, all’accettazione stessa di una “macchi-
na” totalitaria. Un tale sviluppo richiama l’insieme dei fenomeni
autoritari, non solo l’emergere del fascismo, ma la stessa
interpretazione sociologica delle leadership e dei regimi
populisti analizzati da Gino Germani in contesti privi di
capacità di integrazione politica e di aggregazione e di
articolazione degli interessi operati da soggettivi colletti- 52 Lorenzo Viviani vi organizzati (Germani 1975), così come il più generale
attivarsi di un estremismo di centro in una fase di pro-
fonda insoddisfazione fra «coloro che si sentono tagliati
fuori dalle correnti della società moderna» (Lipset 1963
[1960]: 178). Tuttavia, se questa versione della democra-
tizzazione sembra affacciarsi, e inesorabilmente cadere,
sull’orlo dell’abisso del totalitarismo (Arendt 1966 [1951];
Mannheim 1972), la prospettiva di Cavalli si caratterizza
per una possibilità diversa, ancorché osteggiata dal per-
vicace tentativo dei «gruppi autocefali di potere», le oli-
garchie dei partiti politici, di operare lo svuotamento del
principio della leadership attraverso l’uso strumentale
della legittimazione ideologica. Per Cavalli, così come lo
era stato per Weber, è l’irresistibile processo della demo-
cratizzazione, specialmente con l’ingresso delle masse
nella Storia attraverso il suffragio universale, che crea
le condizioni per l’affermarsi della democrazia plebisci-
taria contrapposta alla democrazia senza un leader, la
democrazia dei partiti. Nello sviluppo dei due paradigmi
di democrazia, Cavalli riconduce l’affermarsi del “tipo”
della democrazia con un leader, la democrazia autocefa-
la con un leader personalizzato, alle dinamiche dei pro-
cessi di modernizzazione, con particolare attenzione alla
dimensione politico-culturale, alla democratizzazione,
al diffondersi dell’istruzione e della cultura, alla secola-
rizzazione e all’accrescersi della rilevanza dei mezzi di
comunicazione. Un tale tipo di democrazia necessita al
tempo stesso di alcune pre-condizioni di natura emi-
nentemente politica che rendono possibile l’affermarsi
di una dialettica disintermediata fra leader e popolo tale
da poter essere declinata come «democrazia immediata»
(Cavalli 1992: 42). Carisma e leadership nella sociologia della democrazia Partendo dalla ricostruzione delle caratteristi-
che del “dirigente politico”, Cavalli riconduce il vulnus
principale delle organizzazioni politiche al tributo di
fedeltà che il ceto politico riserva al partito e non alla
comunità nazionale, di fatto subordinando la dimen-
sione collettiva all’interesse particolare, e instaurando
un sistema oligarchico e partitocratico dominato dalla
logica del gruppo autocefalo di potere.7 In tale contesto Weber ne La politica come professione sono particolar-
mente dure nei confronti dei partiti della pseudo-demo-
crazia, i cui notabili erano ritenuti responsabili di impe-
dire una qualsiasi ascesa di persone «dotate delle qualità
del capo», è altrettanto da riconoscere come nella demo-
crazia plebiscitaria non viene negata, osteggiata o con-
dannata la presenza dei partiti politici in sé. Negli espli-
citi riferimenti alla Gran Bretagna di Gladstone, la figura
cesaristico-plebiscitaria che irrompe nella politica come
«il dittatore del campo di battaglia elettorale», e agli Sta-
ti Uniti di Jackson, che nel 1824 «spazzò via le antiche
tradizioni», superando la direzione dei partiti da parte
dei parlamentari e introducendo la macchina plebiscita-
ria “dominata da” e a “sostegno del” Presidente (Weber
2004 [1919]: 88, 90-91), Weber accorda – ex malo bonum
– la possibilità di uno sviluppo della selezione plebisci-
taria e della relativa democrazia, proprio ad opera dei
partiti. Per il fatto stesso che «tutte le lotte tra i partiti
non si svolgono soltanto per fini oggettivi, ma anche e
soprattutto per il patronato delle cariche», e che quindi
i partiti sono «cacciatori di posti», il seguito del partito e
in specie i “funzionari” e gli “imprenditori” del partito,
sono portati a riconoscere il valore e il ruolo del leader,
in modo tale che la capacità demagogica di quest’ultimo
consenta di acquisire voti e quindi potere, così da sod-
disfare le aspettative di incentivi materiali e di carriera
dell’apparato notabilare (Ivi: 82-83). Su questo Cavalli segna una distanza rispetto a
Weber, mettendo in discussione le traiettorie di sviluppo
della democrazia plebiscitaria negli Stati moderni a par-
tire dalla natura dei partiti politici. Per Cavalli le cause
che permettono la trasformazione verso la democrazia
con un leader sono da ricercare nelle forme del muta-
mento sociale e nell’assetto istituzionale che operano a
favore del principio di personalizzazione del potere (del-
la leadership) di vertice, in parallelo all’affermarsi della
rilevanza dell’individuo nella politica attraverso la per-
sonalizzazione. 7 Cavalli riprende e sviluppa il tema della partitocrazia analizzato da
Maranini in riferimento alla natura delle oligarchie, come «tiranni sen-
za volto», la cui degenerazione ha fondamento nell’assenza «di garanzie
di democrazia o anche solo di legalità interna ai partiti», e che proprio
in Italia ha trovato un terreno particolarmente fertile. Il debole sistema Carisma e leadership nella sociologia della democrazia Carisma e leadership nella sociologia della democrazia in Italia, in specie la Democrazia Cristiana e il Partito
Comunista, ben si presta per Cavalli a cogliere questa
distorsione in riferimento al mancato sviluppo di una
cultura politica nazionale in grado di ancorare la demo-
crazia a un senso condiviso. Non solo nella democrazia
dei partiti la lealtà nei confronti delle istituzioni è stata
intermediata e di secondo grado, ma l’adesione stessa
delle masse alle diverse ideologie ha impedito un pieno
riconoscimento nel carattere nazionale, contribuendo a
quella degradazione socio-antropologica in termini di
valori e di orientamento individualista che, al momen-
to della secolarizzazione di quelle ideologie, determina
per Cavalli il trionfo dell’individualismo materialista
(Cavalli 2001). Torna a riproporsi nell’analisi sociologica
quel connubio fra organizzazioni oligarchiche fine a sé
stesse, di cui già Michels e Ostrogorski avevano descritto
le degenerazioni, e le religioni laiche di redenzione come
processo di ri-sacralizzazione (e non di emancipazione)
dell’individuo nei processi di modernizzazione. I par-
titi della democrazia acefala non sono solo esterni, ma
più nello specifico «contrapposti» allo Stato, così come
di fatto costituiscono il principale ostacolo alla realiz-
zazione della sovranità popolare, favorendo anzi la sua
«espropriazione», con l’effetto di un sistema di «domina-
zione sul popolo» (Cavalli 1992: 44-45). La democrazia
dei partiti si contraddistingue non solo per la pervasi-
va centralità del partito nella direzione di governo, ma
per l’assenza della immediatezza nel rapporto tra leader
e popolo, con la conseguente impossibilità di stabili-
re quella dialettica fra fiducia e responsabilità che è alla
base della legittimazione della politica. Ciò che ne deri-
va è l’accrescersi della sfiducia verso la classe politica, il
logoramento dell’etica pubblica, e il progressivo venir
meno del «senso di un obbligo morale verso la comuni-
tà e verso l’altro, e la gratificazione nell’adempiervi» (Ivi:
49, 220). A ciò si somma il problema della ingovernabi-
lità, dal momento che si inocula nei processi decisiona-
li un potere costante di ricatto e di veto che risponde a
esigenze particolaristiche di interessi di partito (e di sub-
partito). 6 Corsivo nel testo originale. Carisma e leadership nella sociologia della democrazia Un aspetto, quest’ultimo, di
cui Cavalli dà invece merito a Michels di aver colto la
connessione con la dinamica plebiscitaria (Cavalli 1987b:
17), pur nell’ambito di uno sviluppo che fuoriuscì dal
solco weberiano e che nella successiva adesione al fasci-
smo di Mussolini mancò di portare un reale contributo
allo sviluppo “della” e “nella” democrazia. Il punto di maggior rilievo critico di Cavalli a
Weber sta nel ruolo da accordare ai partiti nell’ambito
dello sviluppo della democrazia plebiscitaria. Se infat-
ti è indubitabile che Weber assuma la democrazia senza
un leader come la degenerazione della politica e la nega-
zione della direzione politica da parte del leader dotato
di un genuino Beruf, è allo stesso tempo da sottolineare
come questo non facesse di per sé della democrazia ple-
biscitaria una democrazia “senza” i partiti. Inoltre, se è
vero, come afferma Cavalli (1981a: 204), che le parole di 53 Carisma e leadership nella sociologia della democrazia Non solo,
nel mutato contesto politico della democrazia rappre-
sentativa «i leader diventerebbero sempre più il partito;
e il partito sarebbe diventato poco più dei leader» (Katz
e Mair 2018: 61), fino ad osservarsi un orientamento cre-
scente degli elettori “contro i partiti”, dove il voto ad un
leader può assumere il carattere di un voto “contro il suo
partito” (Cavalli 1987a; Calise 2010). Cavalli non si limita alla valutazione dell’impianto
tradizionale dei partiti, aspetto che ne renderebbe alcuni
tratti di analisi vincolati storicamente a un dato perio-
do, ma per molti versi anticipa e si inserisce da socio-
logo nei processi che ne determinano l’evoluzione verso
modelli che la letteratura ha poi definito come partiti
post-integrazione di massa, dal catch all party (Kir-
chheimer 1966) fino ai partiti di cartello (Katz e Mair
1995; Katz e Mair 2018) e alle pratiche collusive fra par-
titi mainstream da cui originano le più recenti sfide dei
partiti dell’anti-establishment e populisti. Scrivendo in
una fase più avanzata del processo di modernizzazione
rispetto a Weber, Cavalli coglie come e quanto i processi
di trasformazione sociale e culturale, dalla secolarizza-
zione all’indebolirsi della struttura di classe, dai cam-
biamenti nei processi di socializzazione al mutare delle
culture politiche, dall’impatto dei media alla rilevanza
dell’individualismo e delle nuove tematiche che assumo-
no rilevanza politica oltre i valori materialisti, fino alle
più recenti dinamiche dei processi di superamento del
sistema internazionale della Guerra fredda, con l’impat-
to della globalizzazione, siano in grado di contribuire
alla crisi dei partiti tradizionali e al loro superamento in
termini di progressiva assunzione di rilevanza del ruolo
della leadership. Tali processi alimentano una trasfor-
mazione ulteriore rispetto a quanto osservato da Weber
nel passaggio da partiti di Weltanschauung a partiti di
patronage, chiamando in causa l’orizzonte del catch all
party (Cavalli 1987a: 332), la forma progressivamente
assunta dai partiti in seguito al processo di de-ideolo-
gizzazione. Partiti contraddistinti da un’organizzazione
sempre meno incentrata sulla militanza e sul radicamen-
to territoriale, e con la specifica e prioritaria preoccupa-
zione di “prendere tutti”, ossia di raggiungere il maggior
numero possibile di consensi in un elettorato social-
mente meno definito e quindi più frammentato e vola-
tile. istituzionale italiano uscito dalla Seconda guerra mondiale ha favorito,
da una parte, una disponibilità alla pervasività dei partiti, e dall’altro
ha costruito una cornice costituzionale tesa a limitare degenerazioni
del potere delle leadership, ma non dei partiti, quest’ultimi «capaci di
difendersi dalla frantumazione con un solido, esplicito, programmatico
gerarchismo, con un controllo politico-ideologico discendente dall’alto»
(Maranini 1995: 487). Carisma e leadership nella sociologia della democrazia Occorre riflettere sul fatto che Cavalli, a
differenza di Weber, scrive in un periodo storico che ha
messo alla prova la democrazia acefala e la sua debolezza
nel fondamento dello Stato basato «esclusivamente6» sul
principio di legalità «senza più contare sulla forza legit-
timante (oltre che […] psicologicamente unificante) del
carisma» (Cavalli 1981a: 206). Le democrazie multipar-
titiche con al centro il protagonismo delle organizzazio-
ni di integrazione di massa ripropongono per Cavalli il
«soffocante ordine oligarchico» espressione della buro-
crazia dei partiti, la cui natura di «gruppi autocefali di
potere» opera come fattore disgregativo di Stato e Nazio-
ne (Cavalli 1992: 14). L’esperienza dei partiti di massa 54 54 Lorenzo Viviani Lorenzo Viviani quanto della capacità di un leader di far fronte a future
possibili contingenze. le oligarchie di partito utilizzano il proprio accesso alle
istituzioni come «risorsa di influenza» nella competizio-
ne interna ed esterna, con la conseguente pratica della
degenerazione clientelare al fine di perseguire interessi
particolari connessi alla propria riproduzione. Il principio della leadership emerge quindi non in
ragione di una utilità interna alle dinamiche di potere
del partito politico, come nella trasformazione del par-
tito americano osservata da Weber, ma più propriamen-
te in ragione di variabili sociali, economiche, culturali
e politiche che portano alla progressiva affermazione,
anche nelle democrazie parlamentari, della persona-
lizzazione della politica e della personalizzazione della
leadership di vertice. Parimenti, pur in presenza di pro-
cessi che assumono caratteristiche peculiari nel contesto
europeo, tali sviluppi della trasformazione dei partiti
hanno favorito «le fortune delle tendenze carismatiche e
plebiscitarie», ben visibili in alcuni partiti e leader socia-
listi, in specie il Ps francese di Mitterand, il Psi di Cra-
xi, il Pasok di Papandreu, proprio in corrispondenza del
venir meno della componente ideologica e del rafforzarsi
del ruolo di identità e di unità attraverso l’affermarsi di
leadership personalizzate (Cavalli 1987a: 333; 1989: 14). Si tratta di assumere la centralità del ruolo della leader-
ship di vertice in connessione al processo di personaliz-
zazione che ha contribuito e contribuisce a trasformare
la rappresentanza politica nelle democrazie occidentali,
come processo che accresce la rilevanza, il potere e le
funzioni dell’individuo in relazione alla sfera politica, e
al contempo che diminuisce il peso del gruppo, special-
mente dei partiti, nei processi di identificazione, socia-
lizzazione e costruzione di schemi cognitivi di interpre-
tazione dei fenomeni politici (Viviani 2021). Carisma e leadership nella sociologia della democrazia Carisma e leadership nella sociologia della democrazia Weber, affida al leader. Per Cavalli non si tratta infatti
di ribadire una mera proceduralità democratica di tipo
pluralista, ma di riconnettere lo sviluppo della democra-
zia alla qualità del processo di selezione della leadership,
oltre che al processo di formazione della leadership stes-
sa. Così il partito che interpreta questo processo non è
più il partito delle oligarchie, ma “il partito del leader”,
con il compito non solo di scardinare le cricche soffo-
canti e senza visione del partito burocratico, ma di far
emergere un leader «nazionale, portatore creativo della
continuità storica, etico-culturale, di un popolo», e con
il preciso compito di favorirne la selezione e sostenerlo
sia nella campagna elettorale, sia nell’attività di governo
(Cavalli 1992: 45). l favorire luoghi di elaborazione politica e culturale come
think tank e fondazioni, tali da affiancare e supportare il
leader nella sua azione di governo, secondo uno sviluppo
che rimanda a dinamiche presenti nella politica ameri-
cana. La presenza o l’assenza di questo network politico
è la linea di discrimine che rende possibile l’emergere
di una struttura “materiale e immateriale” che trova nel
leader il suo punto di sintesi e di guida, pena la fragili-
tà e il – sotto-inteso – fallimento dell’intero edifico del
potere. Cavalli, diversamente da Weber, non adotta la
prospettiva dell’ex malo bonum, non accorda ai partiti
dominati dalle oligarchie la possibilità di far emergere
un leader in ragione dei loro stessi interessi. Tanto più
questo per Cavalli non si rivela possibile in democrazie
istituzionalmente ed elettoralmente ostili al riconosci-
mento della personalizzazione della leadership di verti-
ce, come nel caso dell’Italia, anche dopo la secolarizza-
zione delle religioni laiche di redenzione, le ideologie. Proprio in riferimento alla crisi del biennio 1992-1993
in Italia (Tangentopoli), Cavalli osserva come si sia defi-
nitivamente rotto «il rapporto organico nel partito fra
oligarchie di professionisti della politica e popolazione
italiana», e come il mutamento della società contempo-
ranea non lasci più spazio a «deleghe politiche a soggetti
collettivi sacralizzati» con «fedi assolute» e relativi «miti
e riti» (Cavalli 2001: 38-39). In risposta a questi proces-
si Cavalli continua ad elaborare la propria analisi socio-
logica per dimostrare come «la personalità di un leader
può diventare un riferimento forte, e forse risvegliare il
senso d’un destino comune» (Ibidem). Carisma e leadership nella sociologia della democrazia Ciò che contrad-
distingue la sociologia della democrazia di Cavalli è la
riscrittura stessa della rappresentanza politica attraverso
un ruolo riconosciuto della leadership, con nuove basi di
fiducia e con la pluralizzazione stessa della sfera pubbli-
ca, in cui oltre ai leader e ai partiti del leader, si tesse la
trama di una nuova configurazione di associazioni, lob-
bies e movimenti che concorrono alle nuove dinamiche
di conflitto politico nella democrazia con un leader. La riflessione sociologica di Cavalli appare chiara-
mente incentrata sulla democrazia e sulla leadership,
mentre non appare un interesse specifico a sviluppare
dal punto di vista organizzativo una tipologia di partito
del leader personalizzato o del leader carismatico, dato
che l’obiettivo è mettere in rilievo come il partito sia uno
strumento a servizio del leader, e in particolare del lea-
der personalizzato di governo, il leader della «Repubbli-
ca unicipite». Non di meno, comunque, da tali analisi è
possibile sviluppare nuove tracce di ricerca per osservare
come il rapporto tra personalizzazione della leadership,
personalizzazione della politica e carisma possano assu-
mere connotati diversi, non solo nominalmente, ma dal
punto di vista della sociologia stessa dei partiti. Il partito
del leader non è infatti associabile ai partiti carismatici,
in cui il culto istituzionalizzato della personalità e l’ide-
ologia sopravanzano la rilevanza dei leader individuali,
né ai partiti personali, forme dal carattere patrimoniale
e/o espressione di una celebrity politics incentrata sul-
la mediatizzazione e sulla manipolazione del “carisma
della parola”, né infine a ipotesi di democrazia “senza”
i partiti, come reiezione generalizzata di qualsiasi for-
ma di organizzazione nella società e nelle istituzioni. La
stessa coincidenza di fatto fra leader e partito affrontata
in riferimento al caso di studio della leadership di Ber-
lusconi viene valutata da Cavalli come non rispondente
all’idea di partito del leader nel senso proprio. Fra i pun-
ti di maggior divergenza figurano alcune caratteristiche
chiave che rimandano alla leadership carismatica nella
versione laica offerta dallo stesso Weber ne La politica
come professione, in particolare il non ravvisarsi – a fian-
co del leader (Berlusconi) – di una «élite di persone che
hanno studiato, che hanno pensato, che hanno sofferto,
che sentono la “causa”, la causa che lui stesso impersona»
(Cavalli cit. in Viviani 2010: 185-186). Carisma e leadership nella sociologia della democrazia Del resto Kirchheimer aveva già osservato come la
maggior complessità dei fattori che incidono sul benes-
sere personale, unito alla minor adesione a una specifica
constituency sociale, avesse portato progressivamente gli
elettori a scegliere non in ragione di interessi particolari, Progressivamente il plebiscitarismo segna il supe-
ramento degli attori collettivi come intermediari della
democrazia e opera una “riconciliazione” della persona-
lizzazione della leadership di vertice con la teoria dell’e-
litismo democratico, a partire dalla previsione di un’au-
torizzazione che deriva dagli elettori, pur senza un loro
reale coinvolgimento nei processi decisionali, ma con il
mantenimento di una diseguale distribuzione di potere
dei governanti rispetto ai governati e la garanzia di un
livello di accountability tramite le elezioni (Best e Higley
2010; Pakulski 2013; Pakulski e Körösényi 2012). Tutta-
via, pur in linea con Schumpeter nell’ancorare la sovra-
nità popolare al metodo democratico che si esprime nel-
la selezione competitiva dei leader, non si può tralascia-
re la caratura etica che Cavalli, in piena continuità con 55 Carisma e leadership nella sociologia della democrazia Requisito neces-
sario non è solo la presenza di una élite di questo tipo
in grado di esercitare essa stessa una reale funzione di
leadership, ma il contestuale sforzo da parte del leader di 5. Per una sociologia della democrazia nel XXI° seco-
lo. Così come la lettura weberiana offerta da Cavalli non
indulge a una mera ricostruzione filologica del testo, pur
cogliendone con raffinatezza e originalità la costruzio-
ne e i problemi, la scelta di questo saggio non è orien-
tata alla semplice riproposizione di aspetti del pensiero
sociologico di Luciano Cavalli. Fra i molti temi che han-
no orientato la sua attività di sociologo e di intellettuale
si è deciso di assumere come centrale il tema del rappor-
to tra carisma e democrazia, così come maturata a par-
tire dai suoi primi scritti, fino alla più recente attività di
analisi e riflessione sui cambiamenti nella società e nel-
la politica contemporanee. Questo perché la sociologia 56 Lorenzo Viviani di Luciano Cavalli offre un quadro teorico ed empirico
di estrema attualità nel leggere i processi di trasforma-
zione della politica nella specifica ottica del sociologo,
così come consente di entrare nel dibattito stesso sulla
natura e sull’identità della sociologia politica, confron-
tandosi sul significato stesso di fare sociologia, sul ruolo
del sociologo, sui temi di studio e sulla metodologia di
ricerca di una disciplina fondamentale per lo studio del-
la democrazia. va di difficoltà esterne dovute al perdurante agitarsi del
«complesso del tiranno» nella democrazia dei partiti e
delle ideologie. Nel dialogo costante con Weber, Caval-
li “rimprovera” al sociologo tedesco il fatto di non aver
assunto alcuni aspetti pur determinanti della sua analisi
sociologica proprio in riferimento ai temi del carisma e
della democrazia, a partire dalla centralità della situa-
zione straordinaria di crisi (Cavalli 1986: 74). Inoltre,
nonostante la ben nota analisi di Weber sulle masse,
Cavalli assume i limiti di un suo eccessivo affidamento
ai «valori dell’89», ossia al riconoscimento della acqui-
sizione da parte della massa del principio della emanci-
pazione razionale dai simboli e miti della religione, con
una conseguente non sufficiente attenzione posta sulle
forme di “re-incantamento” ad opera delle ideologie, le
“religioni laiche di redenzione”, e in parallelo sulla pro-
gressiva sostituzione delle vecchie élite non con il prin-
cipio della leadership democratica ma con nuove “élite
plebee”, anche di tipo dittatoriale (Cavalli 1981a: 190-
191). Carisma e leadership nella sociologia della democrazia Se Weber accordò la trasformazione verso la demo-
crazia plebiscitaria alla macchina dei partiti, Cavalli affi-
da invece proprio alla situazione di crisi la possibilità di
conciliare il carisma con i processi di razionalizzazione
della politica contemporanea. Assumendo che l’affer-
mazione per cui «gli orfani del carisma debbono deci-
dere da sé» si rivela non corrispondente alla realtà, ciò
che contraddistingue costantemente la sociologia della
democrazia di Cavalli è il problema del senso, ossia la
necessità di costruzione di processi di “noità” (Cavalli
2001) di cui il leader carismatico, nella sua versione lai-
cizzata democratica, è il principale artefice. In una fase in cui la prospettiva della “sociologia
pubblica” è tornata a porre la questione di un rapporto
tra i sociologi e i pubblici più ampi del solo perimetro
accademico, così come la fecondità della combinazio-
ne tra il lavoro scientifico e l’impegno morale (Burawoy
2021), tanto più attuale appare la riflessione di Caval-
li sulla necessità che il rapporto tra il sociologo e la
democrazia sia segnato da un rigore metodologico che
non esclude l’impegno, la partecipazione più ampia al
dibattito pubblico, alla formazione e alla educazione alla
democrazia (Cavalli 1964). Senza cadere, e anzi rifug-
gendo dalla tentazione della sociografia e dei sociologi-
smi, l’approccio di Cavalli è quello del realista politico,
o con più precisione quello del “realista politico radica-
le”, che osserva le dinamiche di lotta per il potere nella
società, “lotta per la vita”, fra individui, gruppi, Stati, in
una prospettiva in cui lo Stato è portatore degli interessi
di sopravvivenza “primordiale” e “culturale” di un popo-
lo, e in quanto tale è un valore che identifica il “bene
pubblico” (Cavalli 2003: 41-42). Proprio questa è l’ottica
che permette di leggere il carisma recuperando il ruolo
centrale degli individui nella storia, come attori inse-
riti in una relazione sociale tanto con i follower quanto
con il contesto. Un’analisi che porta Cavalli a rifiutare
determinismi legati alla riduzione dei fenomeni politici
a mera dipendente strutturale, e al sociologismo come
spiegazione degli eventi storici in termini di condizio-
ni e gruppi sociali. Carisma e leadership nella sociologia della democrazia Un orizzonte culturale proprio del-
la sociologia politica che critica una visione meramente
residuale del ruolo dell’attore individuale nel determi-
nare il proprio agire politico, ponendo la sfida di una
ricerca sociologico-politica come parte integrante di una
«sociologia sans phrase» in grado di connettere la teoria
degli interessi alla base dell’azione dell’individuo ad altre
teorie dell’azione (Bendix, Lipset 1957: 87). i
Cavalli decostruisce e svela non solo le forme e i
meccanismi del potere, ma la più ampia giustificazione
che sorregge la democrazia dei partiti, responsabile di
ostacolare le possibilità del carisma di far fronte ai pro-
cessi di crisi delle società e delle democrazie contempo-
ranee. Non sfugge, tuttavia, che il riconoscimento del
principio della leadership non implica per Cavalli una
degradazione dell’individuo a mero suddito, anzi, ne
previene la spoliazione di sovranità, e il protagonismo
della società passa per la riscoperta di un ruolo attivo di
una fitta trama di associazioni in una chiave che inevita-
bilmente richiama il discorso sociologico sulla democra-
zia presente fin da Tocqueville. Già all’inizio degli anni
Novanta Cavalli individuava la possibilità che i nuo-
vi mezzi di comunicazione che si stavano affermando
avrebbero potuto favorire nuove forme di partecipazio-
ne, anche in quella funzione di controllo che si esplica a
partire dalla emancipazione rispetto agli attori istituzio-
nali della politica. Se per Cavalli non c’è una possibilità
partecipazionista in senso di democraticismo umanita-
rio, né tanto meno una qualche indulgenza nei confron- La consapevolezza che “tutta la sociologia è poli-
tica” è il quadro in cui Cavalli ha maturato la ricerca
sulla leadership carismatica e sulla democrazia, molto
prima che tale temi tornassero al centro della letteratu-
ra sulla presidenzializzazione e sulla personalizzazione
nelle democrazie contemporanee, e nonostante che fra
la fine degli anni Settanta e l’inizio degli anni Ottanta
la sua ricezione del tema fosse pionieristica e non pri- 57 Carisma e leadership nella sociologia della democrazia ti di quella “democrazia della strada” che propugna un
acritico affidamento alle masse della direzione politica
del paese (Weber 1978 [1922]: 1460; 1994: 231), al tem-
po stesso la forma assunta dalla partecipazione attiva dei
cittadini diventa reale proprio in ragione del sottrarsi al
vincolo della intermediazione partitica, per assumere, da
una parte, la relazione diretta con la leadership, e dall’al-
tra per dar vita a movimenti e ad associazioni di libera
espressione su determinati temi. Carisma e leadership nella sociologia della democrazia Sarebbe inoltre un erro-
re, sociologico e di manipolazione ideologica del pen-
siero, attribuire a Cavalli una lettura del plebiscitarismo
come una tendenza che indistintamente equipara ogni
sviluppo personalizzato a un processo nel senso della
leader democracy8. sibile sviluppare ipotesi ulteriori di ricerca nel campo
della sociologia politica, a partire dalle dinamiche della
leadership populista, non di rado impropriamente asso-
ciate al carisma e alla stessa leader democracy (Viviani
2017; 2021). L’uso dei mass media, il ricorso ai sondaggi,
il ruolo di staff professionalizzati e spin doctor a servizio
del leader, rischiano di alterare la natura della leadership
personalizzata, subordinando la volontà del leader alla
rincorsa dell’opinione pubblica, rinunciando al persegui-
mento della funzione stessa della leadership, e favorendo
l’emergere di «leader del sembiante» al posto di «leader
come personalità» (Cavalli 1992: 94; 1987c: 103-106). Sempre in agguato è la spirale della contraffazione del
carisma, lo pseudo-carisma, che deriva dalla costruzio-
ne pianificata dell’immagine del leader, la cui notorietà
e popolarità sono veicolate da giornali, riviste, manife-
sti, radio, film, programmi televisivi e più recentemente
dalle forme ibride di comunicazione fra vecchi e nuovi
media, sfruttando in modo manipolato le stesse forme
dell’innovazione democratica (Chadwick 2013; Sorice
2016). Un corto-circuito di contraffazione del carisma in
cui il legame plebiscitario viene alimentato dalle capa-
cità oratorie così come dalla costruzione dell’aspetto
fisico del leader (Glassman 1975). Tuttavia, cos’altro è il
carisma contraffatto se non l’antitesi stessa della presen-
za di una qualità straordinaria del leader, con il conse-
guente effetto di una finzione che inficia la caratteristica
dei “veri” leader di dirigere senza mimetizzarsi dietro le
masse? La leadership in questione si caratterizza per lo
più per una regia populista da parte di imprenditori del-
lo scontento, “mobocrati” e agitatori della politica e della
società, sapienti utilizzatori degli strumenti di manipola-
zione delle masse. y
Nel corso di tutta la sua attività di ricerca emerge
chiaramente come il connotato del “vero” leader riman-
di non solo al processo di selezione plebiscitaria, ma al
requisito indispensabile di un’etica politica che ne con-
traddistingua inequivocabilmente la natura, in continu-
ità con il Beruf weberiano, le cui dinamiche nella poli-
tica richiamano il senso che emerge nel rapporto stesso
tra denaro e ascetismo intra-mondano puritano (Cavalli
1968: 454). 8 Termine che Cavalli impiega nelle sue riflessioni più recenti per indi-
care i processi di democrazia con un leader (Cavalli 2016: 133). Carisma e leadership nella sociologia della democrazia Non si tratta soltanto di analizzare socio-
logicamente la differenza fra leader e dittatori o autocra-
ti, ma proseguendo la traccia ben visibile della sociolo-
gia della leadership e della democrazia di Cavalli è pos- 58 Lorenzo Viviani Lorenzo Viviani Calise M. (2010), Il partito personale. I due corpi del lead-
er, Laterza, Roma-Bari. rivolta», con decisioni politiche improntate «all’insegna
dell’eccitazione, dell’improvvisazione e, eventualmente,
della concessione demagogica. Altro che politica fatta
con la “testa”!» (Cavalli 1985: 29) Carlyle T. (1841), On Heroes, Hero-Worship, & the Heroic
in History. Six Lectures, James Fraser, London. In conclusione, l’immaginazione sociologica, l’at-
titudine critica e il rigore scientifico dentro e fuori l’Ac-
cademia, guidano costantemente Cavalli nella lettura dei
processi sociali e politici, dagli studi empirici sui quartie-
ri operai a Genova nei primi anni Sessanta fino alle crisi
più recenti di carattere globale. Un itinerario sociologico
in cui si è snodata la lezione di un Maestro, capace di leg-
gere e dialogare con i classici del passato e di anticipare
le evoluzioni e i campi di ricerca nel presente, offrendo a
generazioni di studiosi di sociologia la possibilità di con-
tinuare a confrontarsi con la democrazia e ad assumere la
centralità dell’apporto individuale nel “teatro della poli-
tica”, rivalutandone il significato rispetto a rappresenta-
zioni distorsive e manipolatorie. Questa la lezione di un
Maestro che ha recepito pienamente la lezione weberia-
na di adempiere «alla “richiesta di ogni giorno” – come
uomini e nella nostra attività professionale», postura che
contraddistingue chi ha trovato «il demone che tiene i fili
della propria vita» (Weber 2004 [1919]: 44). Cavalli L. (1964), Il sociologo e la democrazia, Silva Edi-
tore, Milano. - (1965), La democrazia manipolata, Edizioni di Comu-
nità, Milano. - (1968), Max Weber: religione e società, il Mulino, Bolo-
gna. - (1973), Sociologie del nostro tempo, il Mulino, Bologna. - (1980), «Sociologia politica», in L. Lotti, G. Pasquino
(a cura di), Guida alla Facoltà di Scienze Politiche, il
Mulino, Bologna, pp. 80-86. - (1981a), Il capo carismatico: per una sociologia weberi-
ana della leadership, il Mulino, Bologna. - (1981b), «Il carisma come potenza rivoluzionaria», in
P. Rossi (a cura di), Max Weber e l’analisi del mondo
moderno, Einaudi, Torino, pp. 161-188. - (1982a), «Di alcuni equivoci sul carisma», in Rassegna
Italiana di Sociologia, 23(4): 610-615. - (1982b), Carisma e tirannide nel secolo XX. Il caso Hit-
ler, il Mulino, Bologna. Carisma e leadership nella sociologia della democrazia - (1985), Acosmìa: gli anni del disordine, 1977-1981,
Working Papers Ciuspo, n. 5. Carisma e leadership nella sociologia della democrazia Il “vero” leader è colui che sa anteporre «la
grandezza della patria alla salvezza dell’anima», come
nel caso dei fiorentini ribelli al Papa ripreso da Machia-
velli (Ivi: 459). Il leader in questione è «il portatore
responsabile di una causa di rilevanza etica e come edu-
catore della coscienza popolare» (Cavalli 1995: 51), si sa
districare fra etica dell’intenzione e etica della respon-
sabilità, e – in una visione realista – la stessa leadership
riconduce, in parte, alle qualità personali del leader e,
in parte, al processo di formazione, con un contributo
rilevante della scuola e dell’università, la cui centralità è
costantemente presente nella riflessione e nell’insegna-
mento di Cavalli. Se in questo contributo non è possibile addentrarsi
nel tema della disintermediazione populista, è comun-
que rilevante segnare il netto confine fra la prospettiva
della leadership carismatica, come sociologicamente svi-
luppata da Cavalli, e la leadership populista, con la sua
particolare concezione del popolo e del potere. Occorre
inoltre ricordare come anche questa non sia liquidabi-
le come questione nominalistica. Si tratta invero della
natura diversa dei fenomeni a partire dal processo stesso
di relazione fra leader e popolo che segna, da una par-
te, l’affermazione del carisma come Grande Riforma del
sistema, e dall’altra l’avvento di processi di politicizza-
zione che usano la leva plebiscitaria per affermare un’i-
dea manipolata di “potere del popolo”, quest’ultimo ipo-
statizzato in una finzione di unità opposta agli sviluppi
stessi della personalizzazione propri dell’elitismo demo-
cratico e del realismo radicale. Del resto Cavalli sapeva
bene che in ogni fase di crisi non esiste solo la possibili-
tà del carisma, ma anche le spinte «verso la strada della In altri termini, nel valutare le forme della leader-
ship nella democrazia con un leader si assume la costru-
zione ideal-tipica del leader personalizzato della demo-
crazia autocefala, che ne rende la natura distinta da svi-
luppi altri del plebiscitarismo. A questo riguardo, con il
progressivo affermarsi nelle democrazie contemporanee
dei fenomeni di disintermediazione, personalizzazione e
di rilevanza crescente del linkage plebiscitario, la lezione
sociologica di Cavalli permette di orientarsi e di porre
ordine laddove termini come carisma o plebiscitarismo
sembrano assumere un uso distorto e banalizzante di
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der e le élite. Il contributo di Luciano
Cavalli alla sociologia politica. Socie-
tàMutamentoPolitica 14(27): 61-71. doi:
10.36253/smp-14335 Citation: Carlo A. Marletti (2023) I lea-
der e le élite. Il contributo di Luciano
Cavalli alla sociologia politica. Socie-
tàMutamentoPolitica 14(27): 61-71. doi:
10.36253/smp-14335 Carlo A. Marletti Copyright: © 2023 Carlo A. Marletti. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Un ricordo personale. Ho conosciuto Luciano Cavalli nei primi anni
Sessanta del secolo scorso, non ricordo se nel ’62 o nel ‘63, quando il cosid-
detto “miracolo economico” dispiegava i suoi effetti e si respirava un’atmosfe-
ra di ottimismo. L’Italia in quegli anni era un paese che aveva voltato le spal-
le alla guerra e alla rovinosa sconfitta subìta, anche se, riflettendoci col senno
di poi, non mancavano segni in controtendenza per capire che quello in atto
non era un processo lineare di sviluppo e modernizzazione, ma piuttosto un
cambiamento frenato, in cui sotto la superficie si agitavano energie sociali
compresse alla ricerca di uno sbocco, impedito da vari fattori e in particolare
dal gap culturale e istituzionale che caratterizzava il sistema italiano. f Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. g p
Io ero ancora studente, affascinato dalla sociologia, e grazie a una borsa
del Mediocredito piemontese, dove avevo lavorato, integrata con fondi del-
la Ford Foundation, avevo iniziato a collaborare a tempo pieno alle ricerche
sociologiche promosse da Filippo Barbano presso l’Istituto di Studi Poli-
tici Gioele Solari, diretto da Norberto Bobbio. Qualche anno prima Barba-
no, insieme a Franco Ferrarotti, aveva ottenuto la prima libera docenza in
sociologia che fosse stata indetta in Italia e com’era allora usanza nel mondo
accademico, questo titolo rappresentava la chiave per l’affidamento di inse-
gnamenti universitari. Pertanto, nell’anno accademico 1957-58, grazie all’ap-
poggio di Bobbio e altri docenti del Gioele Solari, tra cui in particolare Ales-
sandro Passerin d’Entrèves, la Facoltà di Giurisprudenza dell’Università di
Torino aveva aperto un insegnamento in questa disciplina nel corso di laurea
in Scienze Politiche, che venne tenuto per incarico da Barbano. RIFERIMENTI BIBLIOGRAFICI - (1998), «From Weber’s Political Sociology to Contem-
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Max Weber, Democracy and Modernization, Palgrave
Macmillan, London, pp. 79-92.h - (2020), «Weberian Social Theory: Rationalization in
a Globalized World», in E. Hanke, L.A. Scaff and
S. Whimster (eds.), The Oxford Handbook of Max
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declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 61-71, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14335 L’assegnazione di un incarico universitario di per sé non rappresentava
nulla di definitivo, in quanto doveva essere confermata ogni anno dal Con-
siglio di Facoltà, composto dai soli (e pochi) professori ordinari. Ma nel caso
della sociologia assumeva un significato particolare. Com’è noto, in Italia
questa disciplina era stata a lungo osteggiata tra Ottocento e Novecento da
parte degli ambienti intellettuali allora dominanti che la consideravano una
“inferma scienza” che non aveva pari dignità con altre forme di sapere stori-
co e sociale (cfr. Croce 1950; Balbo et al. 1975; Ferrarotti 2020). Per cui l’at-
tivazione di un corso di sociologia da parte di una prestigiosa facoltà come
quella di giurisprudenza dell’Università di Torino costituiva un segnale di
svolta e rivalutazione della materia. SOCIETÀMUTAMENTOPOLITICA 14(27): 61-71, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14335 62 Carlo A. Marletti Per la verità gli studenti che frequentavano le lezioni
torinesi si contavano sulle dita di una mano. Ancora nel
1959, quando io iniziai a collaborare con Barbano, mi
capitava ogni tanto di sentirmi chiedere con aria scettica
da altri studenti: «ti occupi di sociologia? Ma cos’è?». Ci
voleva un certo coraggio allora a dirsi sociologo e pen-
sare di far carriera in una disciplina alla quale gli stu-
diosi interessati erano pochi. In compenso tra loro vige-
va un’aura di “comunità dei pari”, di “padri fondatori”
d’un nuovo campo di studi e ricerche. Ed è appunto in
questo spirito che Luciano Cavalli venne allora a Tori-
no per parlare con Barbano. Tra Genova e Torino i rap-
porti accademici erano del resto cordiali. Nella capitale
ligure Filippo Peschiera, studioso di diritto del lavoro
ed economia, figura di spicco del cattolicesimo demo-
cratico genovese, aveva fondato un Istituto di Scienze
Sociali, aperto al contributo di cultori di varie discipli-
ne, dall’antropologia alla psicanalisi e in particolare di
sociologia. Oltre a Barbano, qualche anno dopo, anch’io
vi tenni alcuni seminari. Il clima era molto amichevole e
l’Istituto era una fucina di giovani talenti, come Arnaldo
Bagnasco e Alessandro “Chito” Guala tra i sociologi; e
Giorgio Sola tra i politologi, per non ricordare che quel-
li con cui ho avuto rapporti più stretti. È possibile che
avessi già incontrato anche Cavalli a Genova, ma non ho
ricordi in proposito, mentre nella memoria mi è rimasta
ben viva l’impressione che mi fece in occasione di quella
sua venuta a Torino. Filippo Barbano e Luciano Cavalli
avevano un comune interesse per gli studi sociologici ma
per altri aspetti erano fra loro alquanto diversi. Mentre
Barbano nei suoi rapporti con colleghi e studenti adot-
tava uno stile di comportamento piuttosto informale e
inclusivo, all’americana, per così dire, Cavalli era inve-
ce uno studioso tipicamente all’europea, che colpiva per
l’autorevolezza e la vastità degli interessi oltre alla sua
dimestichezza con i grandi classici del pensiero politico
e sociale. di partito. Al fine di uscire dalle strette di un dibattito
congelato dall’immobilismo degli schieramenti ideologi-
ci imposto dalla Guerra Fredda, si rileggevano i classici
dimenticati del socialismo, come Proudhon, o si riflette-
va sulle vicende complesse e contraddittorie della social-
democrazia tedesca e sulla critica del socialismo fatta da
Max Weber, che era stato fra i primi a intuire il carattere
burocratico del centralismo di partito instaurato dai bol-
scevichi dopo la Rivoluzione di Ottobre. Una tendenza
che Roberto Michels, già sindacalista rivoluzionario in
Germania e poi teorico dell’elitismo minoritario in Ita-
lia, aveva generalizzato enunciando la cosiddetta «legge
ferrea dell’oligarchia» (Michels 1912). g
La memoria, che, come sappiamo, è una costruzio-
ne sociale, non mi consente a tanti anni di distanza di
ricostruire bene quello di cui parlai allora con Cavalli,
che qualche tempo dopo avrebbe dato un’interpretazione
originale del pensiero di Max Weber sul problema della
leadership. Mi ripromettevo di andare successivamente a
cercarlo ma per vari motivi la cosa non fu possibile. Le
ricerche di Barbano a cui collaboravo mi impegnavano
molto e dovevo anche superare gli esami e preparare la
tesi di laurea in storia e filosofia, che discussi nel 1966
con Barbano come relatore e Bobbio correlatore. Barba-
no mi nominò poi assistente volontario, ma i soldi della
borsa erano finiti, non c’era modo di rinnovarla e si aprì
per me un periodo in cui dovetti adattarmi a lavori pre-
cari. Malgrado questo, nel 1970 ottenni anch’io la libera
docenza in sociologia, in una sessione in cui si addot-
torarono studiosi poi noti, come Gianni Statera e Gian
Enrico Rusconi. Fu l’ultima che il Ministero avrebbe
indetto prima dell’abolizione di questo titolo, in prece-
denza così prestigioso ma che stava ormai perdendo la
propria aura a causa della trasformazione dell’università
di élite in università di massa. Si era alla fine di un ciclo. Il clima sociale e politico
italiano era molto cambiato. E da questo punto di vista lo studio delle
forme di leadership e quello delle élites dirigenti, a cui
Luciano Cavalli ha dato un forte impulso e un fonda-
mentale contributo, restano tuttora dei topics ineludibili. Fu in quel periodo che tornai a incontrare Cavalli
ed ebbi modo di stabilire un rapporto continuativo con
lui. Erano passati più o meno quindici anni dal tempo
dell’incontro a Torino che ho ricordato. L’occasione fu
data dal dibattito – spesso autoreferenziale – che accom-
pagnò la costituzione dell’AIS, l’associazione italiana
di sociologia. In quel contesto in cui si confrontavano
vecchie e nuove generazioni di studiosi, Cavalli sosten-
ne con autorevolezza e passione la necessità di dar vita
a una sezione di sociologia politica che garantisse l’au-
tonomia disciplinare di questo campo di studi, contro
le posizioni di chi intendeva abbandonarlo ai politolo-
gi. Condividevo in gran parte le posizioni sostenute da
Cavalli. Come allievo e collaboratore di Norberto Bob-
bio non potevo che essere pienamente d’accordo sul-
la centralità del problema delle élites nello studio dei
fenomeni politici. E già da qualche tempo mi ero con-
vinto che l’approccio in termini prevalentemente istitu-
zionali e formali su proporzionale e maggioritario che
predominava tra i politologi, spiegasse solo parzialmen-
te la cosiddetta “anomalia” italiana, che dal mio punto
di vista era causata soprattutto dalla modernizzazione
incompiuta e dal ritardo culturale, particolarmente evi-
denti nell’autoreferenzialità della comunicazione politi-
ca dei partiti che aggravava il distacco fra governanti e
governati. La centralità del problema della leadership
su cui metteva l’accento Cavalli veniva perciò ad incro-
ciarsi con lo studio delle interazioni fra media e politi-
ca oggetto dei miei interessi. Il fil rouge che legava fra
loro le due tematiche era la critica di quella che usiamo
impropriamente definire come Prima Repubblica – che
sarebbe bene invece definire Repubblica dei partiti – e
la crisi di una intera classe dirigente la cui irreversibilità
sarebbe emersa chiaramente con Tangentopoli. La cri-
tica delle democrazie acefale che Cavalli riprendeva da
Max Weber, aggiornandola, andava nella stessa direzio-
ne della critica alla comunicazione autoreferenziale dei
partiti e della classe dirigente su cui stavo lavorando; e il
confronto prometteva di essere stimolante. Aderii quin-
di alla sezione di sociologia politica e successivamente
Cavalli mi incoraggiò a succedergli nella carica di cui
era stato primo coordinatore. Fu un segno di stima di
cui gli sono tuttora grato. Oggi l’orizzonte politico italiano appare nuovamen-
te annuvolato da un’instabilità cronica delle maggio 2. Cavalli e il problema della leadership carismati-
ca in Max Weber. Che la sociologia sia una non-scienza
o tutt’al più una “inferma scienza” è una concezione
riduttiva di un campo di studi e ricerche che ha le pro-
prie logiche interne di validazione e ha dato non pochi
contributi alla comprensione dell’agire sociale. Oltre al
dibattito sullo storicismo avutosi in Germania e in Ita-
lia tra la fine dell’Ottocento e la prima metà del secolo
scorso (Antoni 1940; Rossi 1974), questo punto di vista
riduttivo è tornato a riemergere nell’ambito degli studi
epistemologici e di storia delle scienze dopo la pubbli-
cazione nel 1962 del libro di Thomas Kuhn, La struttura
delle rivoluzioni scientifiche. In esso l’Autore sosteneva
che le scienze sociali – con la parziale eccezione dell’e-
conomia – non sono vere scienze in quanto sono prive
di un “paradigma” ossia, semplificando, di un insieme
di categorie e concetti fondamentali accettati come veri
e dati per scontati fin quando non ci si imbatte in un’a-
nomalia che per essere spiegata costringe a cambiarli
adottando un nuovo paradigma. La migliore risposta a
questa tesi, che allora fece molto discutere, è forse quel-
la data da Jeffrey Alexander1, secondo il quale le scien-
ze sociali e la sociologia in particolare se non hanno
un paradigma hanno però autori considerati “classici”,
che ci offrono dei modelli di pensiero, stabilendo regole
valide per la conoscenza di un dato campo di fenome-
ni. Ciò, va aggiunto, in base al meccanismo della “rivi-
sitazione”. Un classico è un autore che continua a essere
richiamato e discusso anche dalle generazioni successi-
ve a quella dei suoi contemporanei, suscitando sempre
nuove interpretazioni che aprono la via a problematiche
fondamentali in precedenza non intuite. È in questo
senso che si usa la metafora del “salire sulle spalle dei
giganti” per descrivere il modo in cui avviene l’avanza-
mento delle conoscenze nelle scienze sociali. La rilet-
tura di Max Weber, compiuta da Luciano Cavalli sul
problema della leadership, rappresenta appunto un caso
esemplificativo di questo metodo. Oggi l’orizzonte politico italiano appare nuovamen-
te annuvolato da un’instabilità cronica delle maggio-
ranze di governo, per cui molti sono tentati di riportare
indietro le lancette dell’orologio, tornando alle logiche
del proporzionalismo più o meno temperato vigente pri-
ma di Tangentopoli. Ma è un’illusione. 1 Per una contestualizzazione del dibattito sull’opera di Kuhn e la
risposta di Alexander cfr. Marletti 1991. Si vedano anche le conside-
razioni già svolte in Marletti 2008, in parte riprese, sviluppandole, nel
presente saggio. All’ottimismo e alla fidu-
ciosa attesa verso il futuro con cui negli anni Sessanta si
guardava alla modernizzazione e allo sviluppo economi-
co del paese, era subentrata l’atmosfera cupa degli anni
di piombo e del terrorismo. Nell’ambiente universitario
c’era aria di restaurazione degli assetti di potere acca-
demici. All’ultima generazione dei baroni della cattedra
stava subentrando una generazione neo-baronale anche
peggiore. In particolare, la sociologia politica rischiava
di essere schiacciata tra una concezione economicistica e
una concezione istituzionalistica degli studi e delle ricer-
che, che portava a trascurare temi fondamentali come la
leadership, le élites politiche o le forme del consenso e
della manipolazione. Lo stesso fenomeno del terrorismo
che lacerava la società italiana non avrebbe potuto essere
compreso senza un’impostazione di sociologia politica, Dopo il colloquio, a cui non fui presente, Barbano
mi aveva chiesto di accompagnarlo a una bancarella di
libri gestita da un personaggio interessante e singolare,
l’anziano anarchico Margarita, dove si potevano ancora
reperire opere ormai fuori commercio e introvabili altro-
ve di capiscuola del filone libertario. Insieme a Barbano,
che era amico del proprietario, anch’io vi avevo trova-
to ogni tanto qualche prezioso volume. Cavalli allora
acquistò dei testi di Francesco Saverio Merlino, un rap-
presentante della corrente anarchica della Prima Inter-
nazionale, le cui idee si possono brevemente riassumere
col titolo dato a una raccolta postuma dei suoi scritti,
Socialismo senza Marx (Merlino 1974). In quegli anni
questo tema si affacciava spesso nel dibattito politico
e intellettuale, ad opera sia di studiosi che di esponenti 63 Il contributo di Luciano Cavalli alla sociologia politica capace di render conto del peso di determinazioni stori-
che e ideologiche causate dal cultural gap italiano. termini formali al problema. Come Condorcet ammo-
niva, non esiste un sistema perfetto di rappresentanza
politica della volontà popolare. Ed è appunto alla socio-
logia politica che bisogna guardare per dare concretezza
alle ricerche e fare qualche passo avanti nella conoscenza
dei problemi. E da questo punto di vista lo studio delle
forme di leadership e quello delle élites dirigenti, a cui
Luciano Cavalli ha dato un forte impulso e un fonda-
mentale contributo, restano tuttora dei topics ineludibili. termini formali al problema. Come Condorcet ammo-
niva, non esiste un sistema perfetto di rappresentanza
politica della volontà popolare. Ed è appunto alla socio-
logia politica che bisogna guardare per dare concretezza
alle ricerche e fare qualche passo avanti nella conoscenza
dei problemi. Un revirement di
questo genere mostra solo i limiti di ogni approccio in 64 Carlo A. Marletti Carlo A. Marletti gioni, cosa che gli è servita per sostenere che il carisma
in generale e quello politico nello specifico, ha un fon-
damento sacrale che non deve essere scordato. Il carisma
è un dono di grazia, una “chiamata” che fa nascere una
vocazione. È noto che nella lingua tedesca il termine
Beruf ha un doppio significato, quello di “professione” e
quello di “vocazione”. Ai professionisti della politica, che
considera gente meschina, dedita soltanto alla ricerca del
proprio tornaconto, Weber contrapponeva la figura del
politico per vocazione, che lotta per realizzare le proprie
idee assumendosene la responsabilità. Una nobile figura
dell’impegno, legittimata dal riconoscimento popolare di
capo e guida, in grado di condurre una nazione a supe-
rare gravi situazioni di crisi. Non è evidentemente possibile ricostruire qui, in
poche pagine, l’itinerario percorso da Cavalli e l’ampia
e complessa ricerca da lui svolta lungo un arco quasi
trentennale di studi e riflessioni a supporto della propria
concezione della leadership. Mi limiterò ad accenna-
re ad alcuni punti importanti. Noto per i suoi studi di
storia dell’economia e soprattutto per i saggi sull’etica
protestante e il capitalismo, il Weber politico in Italia è
stato recepito alquanto tardi, come se i suoi sull’argo-
mento fossero degli scritti minori. Antonio Giolitti ave-
va bensì tradotto assai per tempo i saggi raccolti ne La
politica come professione (Weber 1948), corredati da una
approfondita nota introduttiva di Delio Cantimori, ma il
dibattito su questo testo fondamentale, denso di richia-
mi etici e sociologici alla vocazione carismatica per la
politica, non si sviluppò e l’interesse per una rilettura e
una attualizzazione del Weber politico, a parte qualche
eccezione, nasce solo nel 1981 grazie a Luciano Caval-
li, con la pubblicazione del suo primo volume dedica-
to al problema del capo carismatico2. Va notato che a
quest’opera egli ha dato un sottotitolo significativo: Per
una sociologia weberiana della leadership. Perché questa
precisazione che potrebbe sembrare ridondante? Non si
trattava di un omaggio, di una citazione di scuola, come
a volte si fanno nei confronti dei classici. Quello a cui
Cavalli si accingeva va considerato un “lavoro paradig-
matico”, ossia una individuazione di problemi non ben
risolti, di vuoti teorici e anomalie che per essere spiegate
e superate richiedono un rivolgimento categoriale e l’a-
dozione di nuove prospettive di analisi. 2 Nel 1970 uscì una traduzione degli Scritti politici, con un saggio
introduttivo di Antonio Bruno, nella quale per altro non era compre-
so il saggio sulla politica come professione. L’interpretazione di Bru-
no che accentuava gli aspetti etici e valoriali del pensiero di Weber
rispetto a quella più diffusa che metteva in primo piano il suo reali-
smo politico e il “machiavellismo”, avrebbe potuto suscitare un dibat-
tito quanto meno negli ambienti accademici, che invece mancò. Pur ispirando-
si a Weber e in un certo senso reclamandone l’eredità,
Cavalli non intendeva fare un’operazione celebrativa ma
mettere allo scoperto un quandoque latitat, una zona di
incompletezza teorica da colmare. E in proposito osser-
vava che in Weber «la trattazione del carisma politico
nella storia occidentale è poco approfondita e ben lungi
dal ricoprire il campo». Il leader politico è solo «una del-
le molte incarnazioni del capo carismatico nella storia»,
rispetto alle quali va inquadrato (Cavalli 1981: 185-186). Luciano Cavalli ha assunto e fatto propria questa
concezione weberiana della politica come vocazione e
del fondamento carismatico della leadership, ponendo-
si il problema di attualizzarla nel contesto della politica
italiana e della crisi della Repubblica dei partiti di cui,
nel passaggio dagli anni Settanta al decennio successivo
del secolo scorso, cominciavano a manifestarsi segni evi-
denti di aggravamento, anticipati in particolare dal caso
Moro e dai sempre più frequenti contrasti e scontri fra
le forze politiche di maggioranza, che aumentavano l’in-
certezza e l’instabilità dell’agire di governo. 3. L’anomalia della politica italiana e il nuovo corso
del partito socialista. Quella che opinionisti e studiosi
ritenevano fosse la principale anomalia del sistema poli-
tico italiano, era il cosiddetto “bipartitismo imperfetto”,
come Giorgio Galli (1966) lo aveva definito. Mentre le
maggiori democrazie occidentali erano caratterizzate
dalla presenza di due grandi partiti, uno di orientamen-
to conservatore e l’altro progressista che si alternavano
al governo, ciò non poteva avvenire in Italia. Nel nostro
paese c’erano bensì due grandi partiti di massa ma uno
di essi era il partito comunista. Nel contesto della guer-
ra fredda e delle decisioni prese a Yalta dalle potenze
vincitrici del secondo conflitto mondiale, che avevano
stabilito una netta divisione tra due sfere d’influenza, a
est e a ovest dello scacchiere politico europeo, in Italia
un partito comunista non avrebbe mai potuto assume-
re responsabilità di governo anche se avesse ottenuto la
maggioranza nelle consultazioni elettorali. Contravve-
nire a ciò significava rischiare contraccolpi autoritari e
guerra civile, come effettivamente accadde in Grecia. Per cui molti auspicavano la trasformazione del Pci in
un partito di massa d’ispirazione democratica e sociali-
sta che, come aveva fatto la socialdemocrazia tedesca al
congresso di Bad Godesberg nel 1959, compisse una pro-
fonda revisione ideologica e abbandonasse il marxismo
come principio ispiratore della propria linea d’azione. Il contributo di Luciano Cavalli alla sociologia politica A tutto ciò si aggiunga che Max Weber, il quale
dopo la Prima guerra mondiale aveva collaborato come
esperto alla stesura della nuova costituzione tedesca, la
costituzione di Weimar, influenzandola in senso presi-
denzialistico, era stato accusato per questo di aver aperto
la strada alla tirannide hitleriana. Per cui la discussione
sul problema appariva bloccata da una pregiudiziale ide-
ologica relativa al rapporto fra democrazia e leadership
carismatica. Beati i popoli che non hanno bisogno di
eroi, ammoniva Bertold Brecht. Ma si sa come vanno le cose nei partiti. Una delle
principali abilità che favoriscono il successo nelle car-
riere politiche, tanto nei regimi democratici che in quelli
autocratici, è la capacità di un uomo politico di rendersi
identificabile a livello di massa con dei principi ideologi-
ci, non tanto perché ci creda – anzi perlopiù i professio-
nisti della politica sono dei cinici – ma perché ci credo-
no i suoi sostenitori. È una delle caratteristiche tipiche di
ogni leadership demagogica. In via di principio il richia-
mo di Cavalli al filone della rappresentanza diretta della
volontà popolare tramite il capo, al di sopra di ogni cor-
po intermedio e lobby di potere, venne bene accolta nel
Psi. Ma quello che poi contava, alla fine, era il peso delle
correnti nella gerarchia del partito e i giochi di potere
tra di loro. Vale a dire l’esatto opposto di quello che il
richiamo di Cavalli intendeva prospettare. Di fatto, le accuse mosse a Max Weber sono quello
che oggi chiameremmo una fake news. Le cause del col-
lasso della democrazia nella Germania degli anni Tren-
ta del secolo scorso e dell’avvento al potere dei nazisti
sono molte e sono state discusse da un’ampia letteratura,
ma non dovrebbero esserci dubbi che tra le principali vi
siano la crisi economica dovuta alle condizioni capestro
della miope pace imposta a Versailles dalle potenze vin-
citrici nel primo conflitto mondiale, che avevano messo
la Germania in ginocchio; e l’incapacità dei governi a
fronteggiare l’ondata di proteste e impedire la formazio-
ne di corpi paramilitari facendo cessare le violenze da
essi compiute. La democrazia muore anche e più spesso
a causa della propria debolezza che a causa di un eccesso
di leadership. Per altro anche negli ambienti di sinistra vicini al
partito comunista, il dibattito stentava ad allargarsi. Il contributo di Luciano Cavalli alla sociologia politica Il contributo di Luciano Cavalli alla sociologia politica detto Croce e diffidente verso la sociologia. Sfogliando le
pagine non solo di Rinascita ma anche de il Mondo o l’E-
spresso ci si imbatte prima o poi in qualche polemica sul-
la sociologia come scienza o pseudo scienza americana3. E un dibattito come quello lanciato da Cavalli era troppo
sociologico per venir preso in seria considerazione. Ma ciò era concretamente fattibile e quale altra conce-
zione del socialismo adottare allora, al posto del marxi-
smo? Ad attualizzare e rendere scottante politicamente
questo dibattito, rimasto in precedenza sostanzialmente
teorico, intervenne alla metà degli anni Settanta il nuo-
vo corso del Psi inaugurato dalla segreteria Craxi, che si
proponeva di spostare la collocazione del partito da una
posizione di alleanza obbligata (e subordinata) con il
Pci, dovuta ai rapporti di forza esistenti a sinistra, a una
posizione dichiaratamente di concorrenza, non priva di
tratti aggressivi. g
p
p
Il secondo motivo di spiegazione che vorrei eviden-
ziare, è che la storia del partito comunista italiano è sta-
ta anche e in gran parte storia di leader carismatici, da
Togliatti a Berlinguer, senza dimenticare Gramsci, che per
molti aspetti è una figura di politico circonfuso da un’au-
ra di straordinarietà carismatica. Ma questa storia, che
inorgoglirebbe altre forze politiche che ne sono prive, dal
Pci è stata se non negata sottaciuta. Il “culto della perso-
nalità” stalinista denunciato da Kruscev pesava sulla sto-
ria dei partiti comunisti nati dal bolscevismo, come quel-
lo italiano. In proposito va anche ricordato che nel 1956,
in seguito all’invasione sovietica dell’Ungheria, Antonio
Giolitti fu tra gli intellettuali che si dimisero dal Pci. Luciano Cavalli partecipò, da studioso, alla discus-
sione aperta da questo nuovo corso. Come ho già accen-
nato, Cavalli sin dal primo incontro avuto con lui mi era
parso interessato al tema del “socialismo senza Marx”. Negli articoli scritti per la rivista Mondo Operaio diret-
ta da Luciano Pellicani, oltre che negli articoli pubblicati
sull’Avanti, egli richiamò l’attenzione su un filone storico
importante del pensiero socialista, un filone non marxi-
sta, che aveva teorizzato il rapporto tra il capo e le masse
come forma più avanzata di rappresentanza della volon-
tà popolare e di democrazia sociale. Direttamente o di
riflesso questa tematica ampliava la rilevanza del dibatti-
to interno al Psi sul nuovo corso e sulla figura del segre-
tario, a cui venivano attribuiti tratti carismatici. 3 Sulle “americanate” della sociologia cfr. Massironi in Balbo et al. 1975:
15-63. Nella sua rivisitazione del Weber politico, perciò,
Cavalli non si è limitato a prendere in considerazione i
saggi tradotti da Antonio Giolitti, come se si trattas-
se di una materia specialistica, separabile dal resto del
suo pensiero, né si riferì in modo particolare agli scritti
su parlamento e governo; ma ha invece dedicato ampio
spazio agli studi weberiani di storia comparata delle reli- 65 Il contributo di Luciano Cavalli alla sociologia politica Ma
se nel caso del Psi si può parlare di fisiologia dei parti-
ti come organizzazioni complesse, in quello del Pci e
dei gruppi che ad esso facevano riferimento, il dibattito
fu frenato intenzionalmente da pregiudiziali di carattere
politico. Tralasciando ogni ovvia considerazione di con-
correnza fra soggetti che occupavano una porzione dello
stesso spazio politico, ne ricorderò almeno due. Il primo
consisteva nella formazione culturale degli intellettua-
li vicini al Pci, ispirata allo storicismo assoluto di Bene- 4. Le democrazie deboli, il carisma come forza rivo-
luzionaria e il “caso Hitler”. La pregiudiziale ideologica
sul rapporto tra democrazia e leadership venne usata in
modo strumentale dai partiti, bloccando il dibattito che
finì per esaurirsi. Essa, tuttavia, poneva un problema 66 Carlo A. Marletti non privo di rilevanza, teorica e politica. Come spiegare,
infatti, l’avvento nel ventesimo secolo di regimi totalitari
come il fascismo, il nazismo e lo stalinismo per i quali
il culto del capo rappresentava uno dei massimi princi-
pi ideologici? Quella che esercitavano i leader in questi
regimi era o non era una leadership carismatica? E se sì,
come distinguere il carisma totalitario da quello demo-
cratico di un capo? Anche questo era un vuoto teorico
da colmare rispetto alla concezione weberiana della lea-
dership. Il grande maestro tedesco era scomparso prima
che il totalitarismo, dall’Italia di Mussolini alla Russia
di Stalin e alle camicie brune di Hitler, stringesse nella
sua morsa le fragili democrazie rimaste in piedi trabal-
lanti o appena spuntate fra le rovine della Grande Guer-
ra. Ma l’acuta sensibilità di Weber per l’analisi sociale
non poteva non scorgerne i primi segni, sebbene ancora
contradditori. Lo si vede nell’incertezza degli scritti sul
divenire della Rivoluzione russa e sul socialismo, in cui
Weber appare diviso nello stabilire quale sia la tendenza
vincente tra la burocratizzazione “razionale” e l’irrazio-
nalismo dei movimenti carismatici allo statu nascendi. E soprattutto lo si coglie nelle pagine affascinanti e tor-
mentate dei saggi sulla politica come professione o voca-
zione, dense di empiti profetici appena trattenuti e di
consapevole impotenza. senso di dignità umana? Si continuerà per un pezzo a
discutere su «come si diventa nazisti», per citare il sot-
totitolo italiano di una tra le migliori e più intriganti
ricerche sul problema (Allen 1965). Il contributo di Luciano Cavalli alla sociologia politica La lezione che si può
trarre da Weber, ripresa da Cavalli per rispondere a que-
sto interrogativo, è che il carisma è un’energia spirituale
straordinaria, che se non trova gli opportuni canali di
sbocco può sommergere e distruggere gli ordinamenti
sociali e politici. È in altre parole una forza rivoluzio-
naria, che può essere usata nel bene come nel male, per
imporre grandi riforme superando le resistenze di corpi
intermedi e interessi costituiti, oppure per irreggimenta-
re le masse, assoggettandole a un regime dispotico come
ha fatto Hitler, che ha usato in modo diabolico il “dono”,
ossia le doti carismatiche ricevute. i Sotto il profilo etico questo punto di vista è di gran-
de rilevanza. Il “riconoscimento” della qualità straor-
dinaria di un leader – come del resto sottolinea Cavalli
– implica un giudizio morale. Di un’energia spiritua-
le collettiva terribile come questa non ci si può fidare
di per sé, senza cautele e riserve, prescindendo dall’uso
che si può farne. Ma sociologicamente la distinzione tra
usi virtuosi e usi perversi del carisma va specificata. Nel
volume dell’81 l’attenzione di Cavalli si è concentrata
soprattutto sul problema della legittimazione carismati-
ca della leadership. E anche nel volume pubblicato l’anno
successivo, il 1982, dedicato al “caso Hitler” e al rappor-
to fra carisma e tirannide, Cavalli si è preoccupato in
primo luogo di stabilire se il dittatore nazista potesse o
meno essere considerato un capo carismatico. A questa
domanda egli risponde affermativamente, mettendo in
luce la corrispondenza tra la formazione politica di Hit-
ler e le tappe attraverso cui deve passare ogni capo cari-
smatico per essere legittimato plebiscitariamente. Che fare? La domanda rimbalzava dalle sponde del
Baltico a Berlino. Con un passo di grande suggestione,
tratto dalla Bibbia, Weber ha fatto propria la risposta del-
la scolta sulle mura a chi lo interroga dal basso chiedendo
se il tempo è venuto. È ancora buio – risponde la scolta
– l’alba non è spuntata, tornate ad attendere e continuate
intanto a svolgere il vostro lavoro, quello per cui vi senti-
te chiamati (Weber 1948: 42-43). Una scelta di sobrietà,
come Weber stesso la chiama, che però nel clima incan-
descente della Germania sconfitta nella Grande Guerra e
alla ricerca di un perché, non poteva soddisfare le atte-
se di massa, tra cui quelle degli studenti che si affollava-
no alle sue conferenze per applaudirlo ma ne uscivano
poi alquanto delusi. 4 Gli effetti perversi che si producono nelle situazioni di democrazia
debole sono analizzati da Cavalli (1992; 2001) con riferimento alla crisi
di “Tangentopoli”, nella quale un evento traumatico ha fatto collassare la
Repubblica dei partiti, impropriamente definita Prima Repubblica. For-
tunatamente ciò non ha portato alle conseguenze tragiche che avrebbe
anche potuto avere, anche se gli attentati mafiosi del ’93 mostrano che la
delegittimazione dello Stato era giunta a una soglia limite. La normale
dialettica tra le forze politiche ne fu alterata per oltre un decennio e le
ripercussioni si fecero sentire a lungo, sino a oggi. Il contributo di Luciano Cavalli alla sociologia politica Il suo merito
consiste a mio avviso nell’aver per così dire capovolto il
paradigma della leadership. Ciò che dobbiamo chieder-
ci, egli suggerisce, non è tanto se in astratto la leadership
sia da ritenersi democratica o totalitaria, quanto piutto-
sto cosa succede a una democrazia quando manca una
guida ferma e autorevole e la società e lo Stato sono lace-
rati da una molteplicità di conflitti divisivi che possono
disgregarla. Da questo punto di vista, la leadership è una
precondizione della democrazia, una risorsa fondamen-
tale che se viene a mancare prima o poi la democrazia
si debilita e collassa, aprendo la strada ad avventure e
all’effetto “diabolico” inatteso dell’uso del carisma e del-
la sua presa sulle masse4. Negli scritti prebellici di Weber il presupposto
dell’uso legittimo della forza e del potere nell’esercizio
della leadership caratterizza la sua concezione della poli-
tica in senso realistico e “machiavellico”. Anche successi-
vamente, nelle conferenze da lui tenute alle associazioni
studentesche nell’ormai imminente fine del conflitto e
subito dopo la sua cessazione, il riferimento all’uso del-
la forza è ancora presente ma temperato da connotazio-
ni etiche e dal richiamo a valori. Quello di Max Weber
in questa fase è il pensiero dell’intelligent d’una grande
potenza come la Germania, sconfitta malgrado i pri-
mati culturali e scientifici da essa conseguiti e umiliata
dai vincitori. Con la teoria del fondamento sacrale della
leadership carismatica egli guarda a una rigenerazione
morale della propria patria, al recupero della posizione
che a essa spetta tra le grandi nazioni. 5. I leader e le élite: confronto tra il pensiero di Weber
e Pareto. Luciano Cavalli nei suoi lavori ha più volte
insistito che la teoria della leadership e quella delle éli-
tes nascono da comuni esigenze di metodo e di critica,
per cui dovrebbero essere unificate in un corpus unico. Tra queste due correnti di pensiero vi sono in effetti
importanti punti di vista condivisi. Ma anche differen-
ze da non sottovalutare. Entrambe, infatti, aspirano a
guardare la politica in modo realistico e non moralisti-
co. Storicamente entrambe sono nate dalla critica al par-
lamentarismo corrotto delle vecchie classi dirigenti al
potere, incapaci di integrare nello Stato le masse popo-
lari formatesi in seguito ai processi di deruralizzazione e
di industrializzazione, che avevano sconvolto i sistemi di
valore tradizionali. Il contributo di Luciano Cavalli alla sociologia politica Quello che Weber esercitava era un
ascendente intellettuale, non un carisma leaderistico. Non è certo un piccolo risultato dimostrare che un
medesimo modello esplicativo possa venire applicato a
fenomeni di diversa e anzi opposta valenza morale. E in
un caso come quello di Hitler si potrebbe pensare che i
criteri di distinzione tra capi democratici e capi totalita-
ri siano auto evidenti e possano venire dati per sconta-
ti. Ma non dobbiamo farci ingannare dal senno del poi. Non dimentichiamo che per i contemporanei i confini
tra il bene e il male nel caso del nazismo per una gran
parte dell’opinione pubblica tedesca in quegli anni tor-
mentati del primo dopoguerra, scontati non lo erano
affatto. Quella di Hitler fu – per dirla con Brecht – una
“resistibile ascesa” che poteva e doveva essere bloccata. Se non lo è stata, ciò accadde perché la forza rivoluzio-
naria del carisma e la sua presa sulle masse venne sotto-
valutata; e perché la politica postbellica tedesca era pro-
fondamente divisa tra partiti e influenze opposte, che la
rendevano debole e incapace di reagire efficacemente. La Sappiamo bene cosa successe. Invece di un grande
leader, ispirato dal sentimento di una missione salvifica,
a cui Weber alludeva e il cui avvento suscitava aspettati-
ve quasi messianiche, ascesero al potere Hitler e i nazi-
sti. Una domanda sorge spontanea: com’è stato possi-
bile che nel cuore dell’Europa, in due paesi civili come
la Germania e l’Austria, all’avanguardia nelle scienze e
nella cultura, nel giro di non molti mesi un capopopo-
lo tirannico, venuto fuori quasi dal niente, si sia impo-
sto alle folle, conquistando un consenso quasi unanime
e scatenando uno tsunami politico, una risacca immane
di risentimento, odio e spietatezza che ha travolto ogni 67 Il contributo di Luciano Cavalli alla sociologia politica nuova costituzione di Weimar avrebbe potuto consentire
un superamento della crisi, ma era ormai troppo tardi. che tenesse insieme componenti aristocratiche come gli
Junker e i vecchi gruppi di potere ai vertici della buro-
crazia amministrativa e delle gerarchie militari, con le
nuove “élites plebee” espresse dalle masse industrializ-
zate. Il cemento di questa alleanza era l’idea dello Stato
nazione tedesco inteso come valore, di cui Bismarck si
faceva garante. Mettendo l’accento sulle logiche degenerative che
hanno luogo nelle democrazie führerlose, prive di leader-
ship, Cavalli sposta i termini del problema. 5 Cfr. Marletti 1966, Segre 1985: dedicato soprattutto al confronto sul
problema della stratificazione sociale, che per altro è uno dei pochi
testi di comparazione tra il pensiero di Weber e quello dei minoritaristi
italiani. Il contributo di Luciano Cavalli alla sociologia politica Com’è noto, prima di inte-
ressarsi alla sociologia, Pareto è stato un grande econo-
mista, considerato ancora oggi uno dei maggiori classici
in questa disciplina. La sua formazione ingegneristica
giovanile lo aveva portato ad applicare le equazioni della
meccanica idraulica al comportamento umano. Ciò che
lo colpiva maggiormente era che gli uomini, anziché agi-
re in modo logico, cioè in base al calcolo dei pro e dei
contro connessi a ogni corso di azione, lo facessero mol-
to spesso illogicamente, in base a sentimenti trascinanti
e credenze senza fondamento. Con lo scorrere del tempo i valori e i miti che legit-
timano una élite perdono il loro ascendente e la capacità
di presa sulle masse. Si ha allora una fase di decadenza:
lo spirito guerriero dei leoni si trasforma in uso della
violenza e prevaricazione dei deboli, quello delle volpi
in corruzione, slealtà e intrigo. Proprio questo per Pare-
to era ciò che stava accadendo nel tempo in cui scriveva. La vecchia élite parlamentare e burocratica delle volpi
al potere era in procinto di venire scalzata da una nuo-
va élite di leoni che si stava facendo largo con l’uso della
forza sulla scena politica. Avanzando questa previsione
Pareto pensava ai socialisti, ma il corso storico si svi-
luppò diversamente e molti furono indotti a identificare
con i fascisti la nuova élite ascendente al potere6. Pare-
to intervenne nel dibattito con un certo distacco. Come
Max Weber, del resto, egli si considerava soprattutto uno
“scienziato”, quella era la sua fede. Per lui i concetti eco-
nomici e sociologici dovevano mantenere un livello di
astrazione, servivano a spiegazioni di carattere generale
e non a essere applicati a contingenze politiche. Ma nello
stesso tempo era lusingato dal riconoscimento tributato
alle capacità predittive del suo modello. Mussolini gli
offrì la nomina a senatore a vita, ma lui, che ormai vive-
va in Svizzera, dove aveva insegnato a Losanna7, non si
recò mai in Italia per accettarla. La risposta a questo problema gli fu ispirata dal-
le riflessioni sulla funzione politica dei miti di George
Sorel, il teorico del sindacalismo rivoluzionario. Negli
ambienti socialisti era allora piuttosto diffusa l’idea che
prima o poi le masse lavoratrici avrebbero posto fine
al capitalismo incrociando le braccia e rivendicando la
socializzazione e il controllo dei mezzi di produzione. Per Sorel quest’idea dello sciopero generale non aveva
alcun fondamento reale, né alcuna possibilità di attua-
zione pratica. Il contributo di Luciano Cavalli alla sociologia politica Questo sfondo comune è però stato
interpretato diversamente, secondo il contesto di nation
building in Germania e in Italia. Nulla di questo per i minoritaristi italiani, che guar-
dano alla formazione e al ricambio delle élites come a un
problema di fisiologia – o patologia – del divenire socia-
le. In particolare, per Pareto le lotte di potere, delle quali
è intessuta la realtà politica, non hanno alcuna finalità
morale né nobile scopo di alcun genere. Sono nient’altro
che l’effetto della diseguaglianza sociale, dell’eterna lotta
per eccellere di individui e gruppi sociali che porta alla
formazione delle élites, i raggruppamenti dei migliori e
dei vincenti, che unendosi fra loro dominano sulle mas-
se. Non ci sono élites buone o cattive. C’è l’élite politica
come c’è l’élite dei ladri o delle prostitute, osserva Pare-
to fra il divertito e il beffardo. E ne Le mythe vertuiste
(1911) egli irride e sferza l’ipocrisia sociale del benpen-
santismo in materia erotica e sessuale. A differenza di
Weber, che si fa portavoce intellettuale della rigenerazio-
ne morale tedesca, Pareto non manca di tratti quasi cini-
ci, quelli dell’intelligent italiano che sotto al manto delle
nobili parole vuol vedere cosa si nasconde, quali sono
i “residui”, come egli li chiama, quello cioè che rimane
dopo aver grattato via le “derivazioni” ideologiche che lo
ricoprono e vengono usate per mascherarlo. Residui che
sono poi sempre la medesima cosa: sesso, soldi, potere. Anche se variano i mezzi con cui procurarseli e i miti
con cui li si nobilita (Pareto 1916)5. Per costruire l’idealtipo del capo politico carisma-
tico Weber ha ripreso alcuni tratti della personalità di
Bismarck, il cancelliere di ferro, che aveva servito il pro-
prio paese unificandolo al di sopra dei suoi interessi per-
sonali e dei localismi culturali o religiosi. La missione da
lui compiuta aveva un duplice carattere: quello di usare
la forza, se necessario, per superare le resistenze al cam-
biamento; e quello di creare il consenso per un’alleanza 68 Carlo A. Marletti Carlo A. Marletti del potere. Richiamandosi a Machiavelli, egli distingueva
due tipi fondamentali di élites, quella dei leoni e quella
delle volpi. Nel primo prevalgono i residui e le derivazio-
ni dello spirito guerriero, dell’eroismo, dell’onore e del
coraggio. Nel secondo prevalgono il mercantilismo, il
commercio, l’aspirazione alla ricchezza e al potere acqui-
siti tramite la compravendita dei beni e delle merci. Una visione disincantata. 6 Mario Missiroli ha raccolto in Trasformazioni della democrazia
(Pareto 1921) note e riflessioni del “grande vecchio” di Céligny, a pro-
posito delle diverse interpretazioni date alla sua previsione sull’avven-
to di una nuova élite identificabile con i leoni di Machiavelli. Parti-
colarmente significative dell’atteggiamento avalutativo adottato da
Pareto le pagine aggiunte in appendice, che descrivono i torbidi avve-
nuti in conseguenza della situazione di democrazia debole creatasi nel
dopoguerra italiano, con riferimento a casi agghiaccianti di esecuzio-
ni sommarie decretati da improvvisati “tribunali del popolo” durante
l’occupazione delle fabbriche a Torino nel 1920 (cfr. p. 141).
7 Com’è noto, Pareto tenne la cattedra di economia politica all’uni-
versità di Losanna, dopo aver conosciuto durante un viaggio in treno
Léon Walras, uno degli introduttori del marginalismo e della teoria
dell’equilibrio negli studi economici, che lo aveva poi indicato come
suo successore (Cfr. Malandrino e Marchionatti 2000). Il contributo di Luciano Cavalli alla sociologia politica I capitalisti non avrebbero mai ceduto il
potere senza lotte e senza violenza. Malgrado fosse pale-
semente non vero, però, questo mito dello sciopero gene-
rale non veniva abbandonato perché era un’idea guida
che infondeva fiducia nelle masse e nel loro potenzia-
le di controllo sui processi produttivi, spingendole alla
mobilitazione e preparandole allo scontro finale. A que-
ste considerazioni di Sorel si potrebbe aggiungere che se
ciò valeva per le masse, a spiegare il favore che quest’i-
dea conservò a lungo anche presso la cerchia degli intel-
ligent e dei dirigenti del partito socialista, nonostante la
sua non verità, interveniva quello che oggi conosciamo
come il meccanismo dell’autoinganno e che si potrebbe
sintetizzare così: nessuna propaganda è tanto efficace e
persuasiva quanto quella a cui, almeno in parte, anche i
suoi promotori vogliono credere. 6. Verso nuove dimensioni del carisma? Conclusio-
ni. Tra i temi che mi è accaduto di discutere con Lucia-
no Cavalli nel corso degli incontri avuti con lui, uno tra
quelli per me più interessanti, come studioso della comu-
nicazione politica, è il cambiamento della legittimazione
carismatica in un’epoca di intensa mediatizzazione come Pareto accolse e generalizzò ulteriormente questa
concezione soreliana. Ogni nuova élite per affermarsi e
ascendere al potere, scacciando la vecchia élite che l’a-
veva preceduta, non può riuscirci solo facendo uso del-
la forza, pur necessaria, ma deve appoggiarsi a dei miti
che infondano entusiasmo e volontà di lotta nei propri
seguaci. Era un’intuizione della politica simbolica e del
ruolo della propaganda di massa, di cui le potenze bel-
ligeranti avrebbero fatto un uso massiccio nel primo
conflitto mondiale e i regimi totalitari del XX secolo ne
avrebbero sfruttata pienamente l’efficacia. fi
Per Pareto quella che lui chiamava la «circolazio-
ne delle élites», ossia il cambiamento determinato dalla
lotta fra vecchie e nuove élites, segue un ciclo nel quale
prevalgono alternativamente l’uso della forza e quello del
calcolo come mezzi per la conquista e il mantenimento 69 Il contributo di Luciano Cavalli alla sociologia politica influenza tende a venire sottratta all’ambito del giorna-
lismo corrente e a spostarsi a livello sovranazionale8. Il contributo di Luciano Cavalli alla sociologia politica La
tenuta d’immagine sui media di una personalità poli-
tica che in precedenza era il risultato di abili strategie
orchestrate da spin doctors è ora conseguita con l’ausi-
lio di un capitale reputazionale certificato da ambienti
di élite, come quelli delle banche centrali o delle grandi
agenzie internazionali di rating, scarsamente influen-
zabili da forme demagogiche di comunicazione ad essi
esterne. Un mondo felpato, di back stage, che interagisce
preferenzialmente con le macchine redazionali di testa-
te come quelle del New York Times e del Washington
Post negli Usa o del Guardian e del Financial Times nel
Regno Unito. L’operare di questi ambienti d’élite, che
definirei “comunità esperte”9, funziona come celebrity
making system che, oltre all’attestazione di fama a sin-
gole personalità che si sono distinte e rese influenti per
le cariche ricoperte in organismi sovranazionali di varia
natura, considerato complessivamente si muove nella
direzione del formarsi di una classe dirigente transconti-
nentale di nuovo tipo.i l’attuale. La definizione corrente di carisma che viene
data dalla sociologia politica rimanda alle qualità straor-
dinarie di un leader, che generano un ascendente perso-
nale sui suoi seguaci. Ma nella società presente i media
hanno ormai da più di un secolo assunto una funzione
di “fabbrica dello straordinario” e del “grandioso”. Il
mestiere dell’informazione sembra ridursi all’attribuzione
di qualità sorprendenti, fuori dalle aspettative, a eventi e
persone del tutto ordinarie, conferendo loro uno specia-
le quanto effimero appeal. Il clamore mediatico prende
ormai il posto del dono sacrale del carisma, azzerandone
l’aura? O le cose sono più complesse? In un volumetto di sintesi del suo pensiero sul
carisma – il terzo dedicato a questo soggetto dopo le
monografie del 1981 e 1982 che abbiamo già ricordato
– Cavalli, forse anche in risposta alle mie osservazioni
sul tema, ha dedicato una critica a quello che definisce
un «fenomeno inquietante», la creazione del “falso cari-
sma”, causata dalla tendenza alla personalizzazione del-
la politica (Cavalli 1995: 92-93). Personalmente ritengo
che quello cui stiamo assistendo sia un allargamento a
nuovi ambiti di legittimazione carismatica della leader-
ship, a cui corrispondono anche nuove forme di comu-
nicazione e nuovi attori che ne sono protagonisti. Max
Weber sosteneva che il carisma, in quanto dono sacrale
è privo di connotazioni economiche. Oggi questo non
può più considerarsi vero. 8 Al giornalismo corrente è riservato sempre più spesso quello che si
potrebbe definire un “effetto eco” rispetto alle grandi narrazioni media-
tiche. Esso ormai può rivestire un ruolo primario solo in materia scan-
dalistica o nel divismo spettacolare e sportivo.
9 Quello di comunità, ha sostenuto acutamente Bagnasco, è «un con-
cetto ingombrante». Impiegato inizialmente per la critica della società
industriale, non privo di connotazioni nostalgiche per la società tra-
dizionale, ha finito col tempo di perdere in gran parte il suo potere
esplicativo, anche se ne rimangono tuttora delle “tracce”, segno di
una problematicità ancora aperta (Bagnasco 1999). Qui per “comunità
esperte” intendiamo degli aggregati sociali di tipo paretiano, nel sen-
so di raggruppamento dei migliori in un dato campo d’azione, carat-
terizzati sia da competenze di alto profilo, sia da forte competitività
interna, che nel loro agire, tendenzialmente orientato alla razionalità e
al calcolo, non seguono logiche di tipo burocratico ma meritocratico. Il contributo di Luciano Cavalli alla sociologia politica La sempre maggiore integra-
zione fra politica ed economia e in particolare l’impor-
tanza assunta dalle politiche monetarie rispetto all’agire
di governo, fa nascere nuovi tipi di leadership che sareb-
be errato definire “tecnici” tout court e sembra prelude-
re invece all’emergere di un diverso modello in cui alla
crescente domanda di competenza del leader in mate-
ria economica si accompagna una richiesta di capacità
decisionali “forti” insieme a doti di negoziazione “per-
suasiva”, un mix di qualità che va ben oltre gli standard
professionali e l’expertise di un tecnico, riproponendo,
in un contesto storicamente mutato, l’alternativa fra pro-
fessione e vocazione per la politica su cui rifletteva e si
tormentava Max Weber. Un caso esemplificativo, che ci riguarda da vici-
no, della tendenza che si è descritta, è quello di Mario
Draghi. Indipendentemente da come si giudichi il suo
operato come premier del governo di unità naziona-
le nella fase finale della XVIIIa legislatura, il profilo
dell’ex governatore della Bce presenta molti tratti, anche
se non tutti, della figura idealtipica di leader carismati-
co così come è stata delineata da Luciano Cavalli nella
sua rilettura di Max Weber. Il più importante fra essi è
quello che Weber definiva Sachlichkeit, ossia la dedizio-
ne appassionata a una causa (Weber 1948: 101). Diffici-
le negare all’ex banchiere centrale di Francoforte una
continuità d’intenti nel volersi presentare come una
figura salvifica che agisce non per il proprio tornaconto
ma per superare la crisi che minaccia l’Unione Euro-
pea, proteggendo la moneta unica dagli attacchi specu-
lativi cui viene sottoposta. In questo ruolo Draghi ha
dispiegato inattese capacità retoriche. Il suo «whatever La conseguenza di maggior rilievo che questa esten-
sione del carisma politico alla sfera economica può pro-
durre è l’indebolirsi del ruolo preminente che i media
mainstream hanno finora svolto nel processo di selezio-
ne e riconoscimento della leadership. In quella che Pip-
pa Norris definisce l’età “moderna” della comunicazione
politica (Norris 1997; cfr. anche Marletti 2016), i mezzi
di massa e il broadcasting televisivo in particolare, ave-
vano un’influenza decisiva sulla comunicazione demago-
gica con cui i leader politici si affermavano sulla scena
pubblica. Oggi, almeno per quanto riguarda le posizioni
di potere più importanti in materia economica, questa 70 Carlo A. Marletti Cavalli L. (1981a), Il capo carismatico, il Mulino, Bologna. Il contributo di Luciano Cavalli alla sociologia politica it takes», dovunque ce ne sia bisogno, riferito all’acqui-
sto di obbligazioni da parte della banca centrale europea
per impedire l’aggravarsi del divario tra economie forti
ed economie deboli, è diventato rapidamente un detto
proverbiale, accrescendo la sua reputazione di fermezza e
capacità di porsi al di sopra degli scontri d’interesse tra
falchi e colombe. Una reputazione non solo di “tecnico”
ma anche di politico, che gli è servita poi nell’esplicare
il ruolo di capo del governo in una situazione complessa
come quella italiana. – (1981b), «Il carisma come potenza rivoluzionaria», in
Rossi P. (a cura di), Max Weber e l’analisi del mondo
moderno, Einaudi, Torino, pp. 161-188. – (1982a), Carisma e tirannide nel secolo xx. Il caso Hitler,
il Mulino, Bologna. – (1982b), Leadership e democrazia, introduzione di L. Cavalli, Cedam, Padova. – (1992), Governo del leader e regime dei partiti, il Muli-
no, Bologna. – (1995), Carisma. La qualità straordinaria del leader,
Laterza, Bari. È presto però per dire se il caso Draghi rappresenti
una tendenza destinata ad affermarsi e durare o se non
si tratti piuttosto di un unicum, un’eccezione non gene-
ralizzabile. Personalità di alto profilo come la sua non si
trovano ad ogni angolo di strada. – (2001), Il primato della politica nell’Italia del secolo xxi,
Cedam, Padova. Croce B. (1950), «L’utopia della forma sociale perfetta»,
in Il Mondo, 22 gennaio 1950. Vi sono perciò segni in entrambe le direzioni. A
fronte di un modello di leadership competente e respon-
sabile, sostenuto da élites esperte, come quello imperso-
nato da Draghi, negli Stat Uniti il caso Trump mostra
che il modello contrapposto della leadership demagogi-
ca sorretta da una mediatizzazione populista è tutt’altro
che scomparso e può ancora imporsi anche nelle grandi
democrazie. Ma se per affermarsi come leader in questo
senso, giocando a scatenare il risentimento di massa e
azzerando comunità esperte e intermediazioni politiche
bisogna giungere a sfiorare un colpo di Stato e inferi-
re un vulnus costituzionale profondo, ciò significa che
il prezzo per farlo sta forse diventando troppo alto per
cui ormai pochi sarebbero disposti a pagarlo. Il che non
esclude, beninteso, un colpo di coda antistorico e il suc-
cesso di The Donald alle prossime presidenziali. Ferrarotti F. (2020), La sociologia. Inferma scienza vera
scienza, Solfanelli, Chieti. Galli G. (1966), Il bipartitismo imperfetto. Comunisti e
democristiani in Italia, il Mulino, Bologna.hi Kuhn T. Il contributo di Luciano Cavalli alla sociologia politica (1962), The Structure of Scientific Revolution,
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nomia, sociologia e politica nell’opera di Vilfredo Pare-
to, Leo S. Olski editore, Firenze.l Marletti C.A. (1987), «Influenza politica, mass media e
leadership», in AA.VV., Leadership e democrazia,
Cedam, Padova, pp. 55-86. – (1991), Fra sistematica e storia. Saggio sulle idee dei
sociologi, Angeli, Milano. – (2008), «Leadership e democrazia. L’interpretazione
neo-weberiana di Luciano Cavalli», in G. Bettin e
P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze University Press, Firenze, pp. 193-209. RIFERIMENTI BIBLIOGRAFICI – (2016), «Media e comunicazione politica in Italia», in
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pp. xii-xxxviii. – (1916), Trattato di sociologia generale, Barbera, Fire-
nze. 71 71 Citation: Gianfranco Bettin Lattes (2023)
I lati oscuri della democrazia. Socie-
tàMutamentoPolitica 14(27): 73-84. doi:
10.36253/smp-14336 Gianfranco Bettin Lattes Copyright: © 2023 Gianfranco Bettin
Lattes. This is an open access, peer-
reviewed article published by Firenze
University Press (http://www.fupress. com/smp) and distributed under the
terms of the Creative Commons Attri-
bution License, which permits unre-
stricted use, distribution, and repro-
duction in any medium, provided the
original author and source are cred-
ited. 1. Introduzione. Luciano Cavalli, da autentico Maestro, ci ha lasciato un
patrimonio di conoscenza sociologica che merita di essere rivisitato attenta-
mente. In particolare, la sua sociologia della politica, costruita tramite cate-
gorie analitiche originali e paradigmi radicati nel pensiero di alcuni teori-
ci classici ed applicati al mondo politico nella sua effettività, ci propone un
insieme di strumenti euristici che, attualizzati, appaiono di sicura efficacia
per l’interpretazione della contemporaneità. Qui ci si sofferma su alcuni stru-
menti concettuali che indagano i lati oscuri della cultura democratica. Lati
oscuri che Cavalli da studioso preclaro, animato da una genuina passione
politica e da un profondo amore per il suo Paese, ha esplorato con estremo
rigore e con straordinario acume critico. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. g
Che la democrazia sia un bene sociale fondamentale, anzi il bene sociale
per eccellenza, può oggi apparire un fatto dato per scontato, con il rischio che
ci si dimentichi della sua rilevanza fondante o, forse peggio, che si scivoli nel-
la sua banalizzazione. Tutti parlano di democrazia, tutti almeno formalmente
elogiano la democrazia, la si considera un modello vincente sugli autoritari-
smi; ma solo in pochi prestano attenzione al fatto che un sistema democratico
non è un fatto naturale, che è un bene fragile, qualcosa che va attentamente
curato e difeso da degenerazioni incombenti. Da questa consapevolezza nasce
lo studio delle condizioni sociali della democrazia, delle sue trasformazioni in
riferimento al mutamento sociale. La democrazia non si può ridurre ad una
procedura in grado di realizzare accordi e compromessi tra posizioni politi-
che e sociali diverse, piuttosto va concepita come un mezzo per far emergere,
per consolidare e per rendere operanti valori e interessi comuni a tutta la col-
lettività. Il rapporto tra sociologia e democrazia acquista oggi una particolare
rilevanza per vari ed ovvi motivi. Il contributo di Luciano Cavalli alla sociologia politica Il contributo di Luciano Cavalli alla sociologia politica – (1921), Trasformazioni della democrazia, Corbaccio,
Milano. Rossi P. (a cura di) (1974), Lo storicismo contemporaneo,
Loescher, Torino. – (1981), Max Weber e l’analisi del mondo moderno, Ein-
audi, Torino. Segre S. (1985), Weber Mosca Pareto, Angeli, Milano.l Sorel G. (2000), Scritti politici. Riflessioni sulla violenza. L’illusione del progresso. La decomposizione del marx-
ismo, UTET, Torino. Weber M. (1948), Il lavoro intellettuale come professione,
Einaudi, Torino. – (1970), Scritti politici, saggio introduttivo di A. Bruno,
Giannotta, Catania. – (1982), Parlamento e governo nel nuovo ordinamento
della Germania e altri scritti politici, Einaudi, Torino. – (1998), Scritti politici, Donzelli, Roma. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 73-84, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14336 Sociologicamente interessante è la veri-
fica di come i diversi segmenti costitutivi della struttura
sociale elaborino omogenee o divergenti rappresentazioni
sociali della democrazia. Alcuni ipotesi orientative sotto
il profilo empirico: i giovani diversamente dagli anzia-
ni; i disoccupati diversamente dagli occupati; gli istru-
iti diversamente dagli ignoranti; le donne diversamente
dagli uomini; et alia. Affine a questo è il tema cruciale
delle basi sociali della democrazia. Si può formulare l’
ipotesi sociologistica – à la Marx – che la democrazia sia
radicata presso certi strati sociali. Un’ipotesi che, come è
noto, associa l’esperienza democratica all’affermazione di
una classe borghese i cui interessi sono sostenuti e pro-
tetti dalla democrazia e dalle sue istituzioni per lo meno
fino ad un certo punto, fino a quando cioè la democrazia
stessa non permette agli operai di vincere le elezioni con
la conseguente reazione della borghesia che instaura un
regime dittatoriale per proteggersi dall’avanzata proleta-
ria. Questa prospettiva è oggi ovviamente più complessa
ed articolata. Vale a dire in un’epoca di ristrutturazione
dei differenti segmenti che compongono la stratificazio-
ne sociale e che condizionano i relativi influssi sull’agi-
re politico si deve tener conto di macroprocessi sociali e
politici; come l’affermazione dello Stato e la sua obsole-
scenza apparente nella globalizzazione e/o all’opposto
del suo rafforzamento a scapito delle autonomie locali. Ma v’è di più. La crescita dell’urbanizzazione e la conse-
guente riorganizzazione della società su base urbana; la
dilatazione del processo di individualizzazione; l’indebo-
limento politico ed economico del ceto medio e del prole-
tariato; la trasformazione in una direzione multiculturale
della società sono tutte dinamiche che condizionano il
processo democratico. f La sociologia adotta una prospettiva analitica speci-
fica e polivalente sulla questione democrazia. Un esem-
pio – classico ma tutt’altro che datato – è quello dovu-
to a Roberto Michels. La sua, come è a tutti noto, è una
ricerca empirica dedicata al partito di massa. In parti-
colare, al partito socialdemocratico tedesco che ci viene
proposto come campo empirico peculiare per verificare
la sua legge sulla leadership oligarchica nelle organizza-
zioni politiche. Si tratta di un esempio di un approccio
che svela empiricamente l’assenza di democrazia effetti-
va in un ambito istituzionale fondamentale per una cul-
tura politica autenticamente democratica. La sociologia
allora si propone come scienza che studia il funziona-
mento delle istituzioni che producono democrazia. Viviamo infatti in un’epoca di mutamento
rapido ed erratico, in ampia misura dipendente dalla globalizzazione ed infit-
tito da crisi economico-sociali ripetute, nonché da una tragica pandemia e da
guerre brutali che sconvolgono l’ordine internazionale. Inoltre, assistiamo da
tempo ad un processo di transizione degli Stati che, ad esempio, nel contesto
europeo sembrano progressivamente costretti ad un trasferimento della loro
sovranità ad un ente transnazionale sovraordinato con l’effetto prevedibile di
un’evoluzione delle forme di governo. La democrazia è stata associata stori-
camente nella cultura politica occidentale ad una dimensione di mutamento
e ad una dimensione di diffusione progressiva del potere nella società civile. Di fatto si assiste ad una dinamica di verticalizzazione istituzionale che sva- Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 73-84, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14336 74 Gianfranco Bettin Lattes luta il carattere parlamentare di molti sistemi politici. Il
principio della rappresentanza politica che lega il popo-
lo ai politici di professione si riduce spesso una formula
politica à la Gaetano Mosca. Più in generale si assiste
ad una trasformazione delle forme di democrazia ed, in
generale, ad uno sfaldamento della cultura politica demo-
cratica che infragilisce la dimensione sociale del contesto
democratico nel senso che l’associazione tra democrazia
e implementazione del principio dell’uguaglianza socia-
le non regge più. La globalizzazione – nella esaspera-
zione della sua valenza economica – reclama un ritorno
alla vera democrazia affinché la politica argini gli effetti
perversi di un’economia incontrollata nel suo funziona-
mento e nella realizzazione delle sue finalità che sono,
oltre una certa soglia, a-democratiche. Il mondo dell’e-
conomia, infatti, quando prescinde dai necessari con-
trolli, è soprattutto motore di un processo bivalente di
svalutazione della politica e di espansione delle disegua-
glianze sociali che non ha precedenti nella storia degli
Stati democratici, esponendoli a processi pericolosamen-
te involutivi. La democrazia è tale spesso solo in termi-
ni formali ma nei fatti è un sistema di potere dominato
da élites politicamente poco responsabilizzate. Non si
può più trascurare la questione di un rinnovamento del-
la democrazia idoneo a far fronte sia agli effetti perversi
della globalizzazione, sia alla diffusione delle autocrazie
nel mondo non occidentale. ca fluttuante che si modifica nel tempo, a seconda delle
circostanze storico-politiche. Lo studio sociologico del-
la cultura politica caratteristica di una società-Stato si
può orientare al dato normativo, cioè alla ricostruzione
– anche tipologica – della configurazione oggettiva dei
valori che fanno democratica una società. La sociologia
si focalizza così sulla democrazia intesa come un ambito
di vissuto sociale. È il tema delle rappresentazioni sociali
della democrazia. Sociologicamente interessante è la veri-
fica di come i diversi segmenti costitutivi della struttura
sociale elaborino omogenee o divergenti rappresentazioni
sociali della democrazia. Alcuni ipotesi orientative sotto
il profilo empirico: i giovani diversamente dagli anzia-
ni; i disoccupati diversamente dagli occupati; gli istru-
iti diversamente dagli ignoranti; le donne diversamente
dagli uomini; et alia. Affine a questo è il tema cruciale
delle basi sociali della democrazia. Si può formulare l’
ipotesi sociologistica – à la Marx – che la democrazia sia
radicata presso certi strati sociali. Un’ipotesi che, come è
noto, associa l’esperienza democratica all’affermazione di
una classe borghese i cui interessi sono sostenuti e pro-
tetti dalla democrazia e dalle sue istituzioni per lo meno
fino ad un certo punto, fino a quando cioè la democrazia
stessa non permette agli operai di vincere le elezioni con
la conseguente reazione della borghesia che instaura un
regime dittatoriale per proteggersi dall’avanzata proleta-
ria. Questa prospettiva è oggi ovviamente più complessa
ed articolata. Vale a dire in un’epoca di ristrutturazione
dei differenti segmenti che compongono la stratificazio-
ne sociale e che condizionano i relativi influssi sull’agi-
re politico si deve tener conto di macroprocessi sociali e
politici; come l’affermazione dello Stato e la sua obsole-
scenza apparente nella globalizzazione e/o all’opposto
del suo rafforzamento a scapito delle autonomie locali. Ma v’è di più. La crescita dell’urbanizzazione e la conse-
guente riorganizzazione della società su base urbana; la
dilatazione del processo di individualizzazione; l’indebo-
limento politico ed economico del ceto medio e del prole-
tariato; la trasformazione in una direzione multiculturale
della società sono tutte dinamiche che condizionano il
processo democratico. Per comprendere come l’affermazione e la trasfor- ca fluttuante che si modifica nel tempo, a seconda delle
circostanze storico-politiche. Lo studio sociologico del-
la cultura politica caratteristica di una società-Stato si
può orientare al dato normativo, cioè alla ricostruzione
– anche tipologica – della configurazione oggettiva dei
valori che fanno democratica una società. La sociologia
si focalizza così sulla democrazia intesa come un ambito
di vissuto sociale. È il tema delle rappresentazioni sociali
della democrazia. L’essenza di quest’ultimo è l’intima
ammissione del diritto di ciascuno a realizzare sé stesso
in onesta competizione con gli altri, ossia dell’uguale e
divina dignità di ogni uomo, come della diversità degli
uomini» (Cavalli 1965: 13). La democrazia normativa è
un ideale che va difeso attraverso un’analisi che valuti,
caso per caso, le sue realizzazioni storicamente concre-
te, senza chiudersi nel recinto di una riflessione teorica
astratta. Il dato con cui misurarsi è che alcuni paesi che
si dichiarano democratici sono in realtà delle democra-
zie manipolate. Nella prima metà degli anni Sessanta
quando in Italia si era appena varato il primo governo di
centrosinistra, che sembrava esprimere la maturazione
di una nuova fase di democratizzazione del nostro siste-
ma politico, Cavalli rifletteva sui lati oscuri (‘nascosti’)
della democrazia ed introduceva nel dibattito sociologico
la categoria della democrazia manipolata. I sociologi non si sono dedicati in un modo partico-
larmente impegnativo, così come sarebbe naturale aspet-
tarsi, allo studio della democrazia e delle sue basi sociali
e dei suoi mutamenti per motivi che non è possibile qui
analizzare. Luciano Cavalli rappresenta un’importante
eccezione. La sua vasta bibliografia comprova come l’in-
teresse per questa tematica fondamentale lo abbia impe-
gnato fin dai primi suoi studi. Basta ricordare Il socio-
logo e la democrazia (1964) e La democrazia manipolata
(1965). L’interesse per uno studio sociologico della demo-
crazia lo accompagnerà per l’intero suo itinerario scien-
tifico fino agli anni Duemila, in particolare con i libri:
Governo del leader e regime dei partiti (1992); Il primato
della politica nell’Italia del secolo XXI ( 2001) e con Il lea-
der e il dittatore. Uomini e istituzioni di governo nel “rea-
lismo radicale” (2003). Proporre una rivisitazione di que-
sti testi significa, oltreché rendere omaggio ad un Mae-
stro della sociologia contemporanea e ad un amico, ten-
tare di gettare luce sul suo vocabolario di studioso delle
dinamiche politiche democratiche e offrire un contributo
per decodificarne alcuni aspetti latenti ma fondamentali. Cosa si intende per democrazia manipolata? Si intende che
i principi della democrazia (liberale) sono formalmente
sanciti nelle carte istituzionali e incorporati nelle istituzio-
ni politiche, ma non vi è democrazia in senso sostanziale: i
principi sono elusi e traditi […] in una democrazia mani-
polata, certi principi sono ormai sanciti, troppo forti nella
coscienza pubblica per essere apertamente violati e distrut-
ti, troppo deboli per dare luogo ad una vera democrazia. Dai
partiti, ai consigli di quartiere, al parlamento, ai movi-
menti sociali, ai sindacati. Questo approccio si pone
anche in termini di rendimento, di efficacia, di misura-
zione del buon funzionamento della democrazia. Ma è
riduttivo affrontare sociologicamente la questione demo-
crazia facendo riferimento unicamente ad una sociologia
dei partiti politici e ad una sociologia delle istituzioni e
dei movimenti che producono democrazia. La democra-
zia è anche, e forse soprattutto, un insieme di valori. Per comprendere come l’affermazione e la trasfor-
mazione della democrazia siano strettamente legate al
quadro sociale generale, nel senso che ne sono influen-
zate ed al tempo stesso lo influenzano, si deve fare rife-
rimento anche a forme storicamente distinte assunte
dall’esperienza democratica. Un esempio significativo? La democrazia si dà certe forme e modalità di funzio-
namento in una società che sia uscita recentemente da
un’esperienza totalitaria. Assume altre forme quando,
invece, è consolidata in relazione anche alle dinamiche
politiche che le sono specifiche ed alle dinamiche econo- L’ assetto valoriale democratico ha un suo nucleo
fondante, centrale, e poi una costellazione periferi- 75 I lati oscuri della democrazia miche che la orientano. Pensiamo ad un caso importante
di processo politico che ci coinvolge oggi. La formazione
dell’Unione europea come macroprocesso di portata sto-
rica che si realizza in un momento di apparente trion-
fo della democrazia dopo il 1989. Un trionfo che però
non è privo di ombre e di inquietudini. In particolare: il
sovradimensionamento del potere burocratico nell’ambi-
to di questo processo ne indebolisce la venatura demo-
cratica e crea anche serie resistenze alla sua attuazione. È ovviamente importante, poi, considerare l’impatto del-
la tecnologia nell’ambito della comunicazione politica. I
mezzi di comunicazione di massa e la rete stanno cam-
biando profondamente la qualità e l’efficacia dell’opinio-
ne pubblica, che resta una delle variabili cruciali della
dinamica democratica, anche in una società complessa. Così come si ridefiniscono la natura e l’identità della lea-
dership e della partecipazione politica1. sua piena realizzazione empirica è legata allo sviluppo
di una metodologia democratica particolare che ad una
lettura superficiale può apparire fin ambivalente. Caval-
li definisce la democrazia come l’istituzione che assi-
cura «il massimo cambiamento sociale compatibile con
la garanzia di continuità delle relazioni sociali e delle
istituzioni politiche» che l’hanno forgiata. «In effetti la
democrazia consta delle istituzioni democratiche più lo
spirito democratico. 1 Particolarmente significativo su queste tematiche è, tra gli altri, il sag-
gio di Cavalli L. (1999), Leadership “personale” e “diffusa” in Marletti
C. (a cura di), Politica e società in Italia, vol. I, Cambiamento politico e
identità sociali, Angeli, Milano, pp. 50-72. Perciò accade che il sistema di dominazione dovrà usare
piuttosto della manipolazione che della violenza. (Cavalli
1964: 21, 22) La manipolazione democratica è più facilmente
documentabile nei paesi a democrazia avanzata, perché
la libertà rende possibile la raccolta oggettiva di prove e
di materiali di testimonianza, mentre «in quelli a demo-
crazia falsa, la prudenza chiude la bocca» (ibidem). Il
rapporto tra democrazia e forme di manipolazione può
essere indagato in modo penetrante attraverso un’anali-
si del ruolo dell’élite politica nazionale. Charles Wright
Mills ci ha dimostrato che alcuni paesi che si definisco-
no campioni della democrazia, come gli Stati Uniti, pre-
sentano all’interno di un ciclo storico-politico definito
una minoranza organizzata che controlla il potere poli-
tico ed esercita un’azione di dominio sulla maggioranza
disorganizzata. Rispetto ad un regime autoritario, in cui
la concentrazione del potere in poche mani è un fenome- 2. La democrazia manipolata. Chi ha avuto il pri-
vilegio di seguire da vicino e con continuità il dipanar-
si nel tempo del lavoro scientifico di Luciano Cavalli
ha potuto facilmente constatare che Cavalli assume la
democrazia come ‘la’ dimensione costitutiva, e insie-
me problematica, della società moderna. Cavalli, come
Weber, è intimamente convinto della superiorità norma-
tiva di questo modello, ma è altrettanto convinto che la 76 Gianfranco Bettin Lattes La manipolazione è tanto più pervasiva quanta più
costrizione è presente in una società. Le istituzioni, tra-
mite un controllo che ne snatura le funzioni, diventa-
no i canali attraverso i quali la manipolazione legittima
il sistema di dominazione. Anche in una condizione di
democrazia manipolata la volontà della maggioranza
può esistere ed agire come unico, o comunque come fon-
damentale, principio di legittimazione: solo che, di fatto,
la maggioranza è etero diretta e si adatta al dominio di
minoranze che tutelano esclusivamente interessi partico-
laristici: no ampiamente palese e riconosciuto, in una democrazia
più avanzata l’azione di dominio delle minoranze orga-
nizzate procede spesso in maniera inavvertita e dissimu-
lata, avviene cioè attraverso la manipolazione. Il concet-
to di manipolazione ci riconduce all’ «esercizio segreto
o impersonale del potere; colui che è influenzato non
riceve disposizioni esplicite circa ciò che deve fare, ma
è ciò nondimeno soggetto alla volontà di chi ha potere»
(Cavalli 1965: 51). La democrazia manipolata si associa all’esperienza
profondamente antidemocratica della dominazione. Cioè
si associa ad una particolare modalità politica tramite
cui «la minoranza organizzata non dà alla maggioranza
la chance di diventare capace di autogoverno» (ibidem). In sintesi, il potere di alcune minoranze organizzate sta
al centro delle dinamiche che producono la dimensio-
ne manipolativa della democrazia secondo un proget-
to definito ed animato dall’interesse alla conservazione
della condizione sopraordinata di queste élite, non certo
illuminate e motivate solo dalla loro esigenza di domi-
nazione. Vanno allora evidenziati tre punti sui quali non
è possibile qui sviluppare una trattazione approfondita
data la prospettiva tematica prescelta: a) la trattazione
cavalliana della democrazia manipolata si inquadra in
una riflessione sui due processi sociologici fondamenta-
li della socializzazione e del controllo sociale; b) quello
che viene analizzato e ricostruito è un modello puro di
democrazia manipolata; c) la finalità della riflessione
tipologica di Cavalli sulla democrazia e sui suoi poten-
ziali degenerativi è quella di alimentare l’impegno civi-
le, difendere la libertà dei cittadini e dunque rendere più
democratica la democrazia. Una finalità che corrisponde
ad una concezione della sociologia politica radicata nel
pensiero dei classici e di cui Cavalli è stato costantemen-
te uno strenuo paladino nel dibattito sociologico di que-
sti ultimi cinquant’anni. La democrazia manipolata non insegna e non può insegna-
re ai giovani il valore della democrazia. Può offrirgli solo
un lip-service, come dicono gli anglosassoni, a questo valo-
re. Insegnarlo sul serio, significa dire ai giovani che il valo-
re più alto di tutti è in realtà quello di pensare e di decide-
re da sé e che, come corollario, tutti hanno il diritto-dovere
di partecipare al governo della repubblica. Naturalmente
anche nella democrazia manipolata la volontà della mag-
gioranza dovrebbe indicare la via, e può perfino diventare
l’unico principio di legittimazione realmente sentito da una
parte del popolo. Ma essa non si forma mai liberamente, e
quindi si riduce sempre alla volontà dei dominatori. È il
supremo inganno, come Mosca, Michels e tanti altri hanno
ormai esaurientemente dimostrato. (ivi: 33, 34) La manipolazione è funzionale alla conservazione di
un sistema di dominazione nel senso che il potere ten-
de a conservarsi nella sua sterile autoreferenzialità e per
farlo ha tutta la convenienza a non esporsi troppo, dato
che un eccesso di esposizione potrebbe in qualche modo
renderlo più vulnerabile. Per manipolazione intendo sia quella psicologica inavver-
tita da chi la riceve, come per esempio nell’insegnamento e
nella propaganda, sia quella sociale, che esercita una pres-
sione direttiva dall’esterno sulla condotta dell’interessato,
come quando si fa pesare la minaccia del licenziamento su
un dissenziente politico [...] o la manipolazione nei partiti,
per cui una oligarchia stronca ogni tentativo di organizzare
la opposizione ai suoi voleri entro il partito, sicché i citta-
dini non possono più influire sulla formazione della poli-
tica entro i partiti; non solo, ma, paradossalmente i partiti
divengono il vero luogo della diseducazione democratica in
un paese di democrazia manipolata. (Cavalli 1964: 25) I lati oscuri della democrazia Questa stessa linea analitica la si può scorgere più
recentemente in un autore influente come Ulrich Beck
(1944-2015). La caratteristica peculiare che Beck attri-
buisce alla società contemporanea è la crisi di quel-
la capacità di garantire sicurezza che era riconosciuta
un tempo alla tradizione, ai valori culturali comunitari
e alla scienza. L’emergere della società del rischio non
ha soltanto cambiato le questioni rilevanti in politica e
reso più impellente l’esigenza di partecipazione. Essa ha
anche gettato le basi per il passaggio da un modello line-
are di democrazia ad un modello riflessivo. Come bene
è sintetizzato nell’espressione «democratizzazione della
democrazia» per Beck la nuova concezione di democra-
zia prende corpo attraverso l’applicazione dei principi
della democrazia alla democrazia stessa. l A questo scopo, Cavalli, come si è accennato, utilizza
le categorie di socializzazione e controllo sociale, derivate
dalla sociologia funzionalista parsonsiana. La socializza-
zione e il controllo sociale costituiscono i due strumen-
ti privilegiati dai titolari del potere di manipolazione, di
un potere che sa e che vuole raggiungere i suoi risulta-
ti senza un uso palese della costrizione. Ovviamente ciò
non vuol dire che i due processi si riducano tout court
a manipolazione. Al contrario, si tratta di due processi-
chiave necessari allo stesso ‘buon’ funzionamento della
società. La socializzazione consiste nella trasmissione di
determinati modelli di comportamento agli individui,
mentre il controllo sociale ha a che fare con il motivare
gli individui a conformarsi ai modelli socialmente rico-
nosciuti, anche attraverso la denuncia e la stigmatizza-
zione dei comportamenti devianti. La manipolazione
può essere esercitata manovrando alcune delle istituzio-
ni che sono fondamentali per lo sviluppo dell’autonomia
individuale e per il buon funzionamento della società: la
famiglia, la scuola, la religione, la cultura, l’arte, i mezzi
di informazione. Se non efficacemente individuate e con-
trastate, le pratiche di manipolazione possono svuotare di
autentico significato democratico le istituzioni e le prati-
che della democrazia. La democrazia viene falsificata, e
si trasforma appunto in una democrazia manipolata. È
nell’individuazione di questo rischio latente, sempre in
agguato nei sistemi politici della nostra modernità, che
il discorso sociologico può e deve dare il suo contributo
allo sviluppo dello spirito democratico. 2 Questa trasformazione, secondo quanto suggeriscono, ad esempio
Ronald Inglehart, Terry Nichols Clark e Vincent Hoffmann-Martinot,
viene alimentata da processi tipicamente strutturali: come la crescita del
livello di istruzione e del livello di reddito; l ’ampliarsi del settore occu-
pazionale legato all’alta tecnologia e alle comunicazioni; il consolidarsi del benessere diffuso e l’incrementarsi delle possibilità e dell’autonomia
individuale. Ma questo tipo di analisi non ha modo di tenere conto dei
processi che, ripetutamente, mettono in profonda crisi la società, la sua
economia e la sua cultura alimentando diseguaglianze sempre più pro-
fonde e tutt’altro che manifeste.
3 Il nuovo modello della democrazia riflessiva appare profilarsi princi-
palmente attraverso il processo che Beck chiama di «subpoliticizzazione
della politica» o di «democratizzazione della struttura». Dunque la manipolazione si
associa ad una esigenza importante dell’élite dominante
quella di esercitare il suo potere di dominazione in un
modo invisibile per la collettività dei dominati. Dato che la manipolazione procede sempre per vie
segrete, il primo problema è quello di identificarla.f i
Cavalli offre in proposito delle indicazioni empiriche
molto chiare: Il tema della democrazia manipolata viene così argo-
mentato da Cavalli tramite una metodologia empirica
che privilegia gli studi del caso e li intreccia con altre
chiavi analitiche che meritano di essere evocate perché le
adotta regolarmente nei suoi scritti. Per manipolazione intendo sia quella psicologica inavver-
tita da chi la riceve, come per esempio nell’insegnamento e
nella propaganda, sia quella sociale, che esercita una pres-
sione direttiva dall’esterno sulla condotta dell’interessato,
come quando si fa pesare la minaccia del licenziamento su
un dissenziente politico [...] o la manipolazione nei partiti,
per cui una oligarchia stronca ogni tentativo di organizzare
la opposizione ai suoi voleri entro il partito, sicché i citta-
dini non possono più influire sulla formazione della poli-
tica entro i partiti; non solo, ma, paradossalmente i partiti
divengono il vero luogo della diseducazione democratica in
un paese di democrazia manipolata. (Cavalli 1964: 25) I miei casi e i miei dati sono tratti dalla storia, dalla ricer-
ca empirica sociologica e psico-sociologica, dalla cronaca e
anche dalla letteratura, in quest’ ultimo caso solo per fini
ben circoscritti e con le preoccupazioni dovute […] I richia-
mi letterari sono usati: a) per introdurre o sviluppare ipote-
si storico-sociologiche; b) come testimonianza di usi, costu-
mi atteggiamenti comuni; c) per sottolineare i caratteri
tipici di personalità o situazioni. (ivi: 13, 18) 77 I lati oscuri della democrazia 78 Gianfranco Bettin Lattes dall’esistenza di forti disparità sociali, oggi sembra insi-
nuarsi un pericolo nuovo e forse ancora più subdolo per-
ché non facilmente visibile. una classe politica illuminata e responsabile. In entrambi
gli autori, tuttavia, si riconosce quella prospettiva anali-
tica tutta sociologica di non ridurre la politica al siste-
ma politico ma di allargarne i confini costitutivi, al fine
di cogliere le sue manifestazioni fondanti, nel loro più
ampio significato e nel loro radicamento sociale che è la
vera linfa di una cultura politica democratica. Questo nuovo pericolo consiste nella mancanza di
un processo di crescita e di maturazione di una consape-
volezza personale e collettiva sul problema della demo-
crazia politica, con il risultato che se a parole i cittadini
sembrano saper definire e caratterizzare la democrazia,
non è detto però che questa competenza cognitiva si
traduca in atti e in comportamenti ad essa congruenti. Sappiamo bene che l’assenteismo elettorale, il disinteres-
se per le vicende pubbliche e per i destini collettivi sono
diffusi in larghe porzioni della società civile e segnata-
mente nelle giovani generazioni. L’attuale “successo”
della democrazia e delle sue formule politiche può allo-
ra nascondere un pericoloso vuoto di cultura politica ed
un radicamento troppo debole dello spirito civico. Resta
senza risposta la domanda se un sistema istituzionale
guardato con sufficienza e con distacco potrà reggere e
favorire quella cultura democratica che è da sempre rite-
nuta necessaria per mantenere vivo lo spazio politico in
cui si svolge il dialogo tra le differenze e in cui le singole
individualità trovano il loro legame solidaristico con gli
altri. Il problema della democrazia manipolata si profi-
la ancora e paradossalmente insieme alla consacrazione
universale della democrazia: con quali conseguenze è
forse ancora troppo presto per dire. Sulla scia di queste considerazioni si può tentare di
individuare una sottospecie della democrazia manipola-
ta che si può etichettare come democrazia stereotipata. Si tratta di fare così un passo avanti, stimolati dall’a-
nalisi di Cavalli, focalizzata sull’azione di dominazione
dell’élite manipolante, nel senso di valutare l’effetto della
manipolazione sul milieu culturale più generale. In altre
parole, la democrazia nelle società contemporanee tende
a diventare sempre più un “tipo di società” e non soltan-
to una forma di “regime politico”, quasi a testimoniare
che la democrazia nella sua accezione più strettamente
istituzionale e politica sta ormai sullo sfondo come ele-
mento, in un certo senso, naturale e dato. I lati oscuri della democrazia Rimane tut-
tavia il dubbio che questo stato di cose possa significa-
re anche una cosa ben diversa: la crescita di un sottosi-
stema politico-istituzionale sempre più impermeabile e
sempre più lontano dalla società civile. I riferimenti alla
sfera della politica e per traslato alle implementazioni
del modello democratico assumono la forma di stereoti-
pi, cioè di conoscenze con un basso grado di consape-
volezza. È possibile che questo stato di cose possa essere
letto come l’esito di un’eccessiva chiusura e specializza-
zione del sottosistema politico, sempre più distante dalla
società civile, sempre più pressato dalle esigenze funzio-
nali della società complessa permeata dalle dinamiche
di mercato ed inserita in un processo di globalizzazione
che depotenzia la dimensione politica della vita colletti-
va con le sue procedure lente, eccessivamente burocra-
tizzate. Questo fenomeno di una democrazia data per
scontata e poco praticata può anche dipendere da altre
cause. Esso può essere visto come l’esito imprevisto, ma
forse inevitabile, dell’eccessivo ed incontrastato suc-
cesso della cultura politica democratico-liberale che ha
provocato una sorta di “oggettivazione” delle procedure
democratiche. Le procedure e le istituzioni democrati-
che, infatti, si sono trasformate in un ambiente “natu-
rale”, che spesso sfugge alla consapevolezza individuale
ed allora, in quanto tale, viene assunto come stereotipo. Esiste, in parole più chiare, il rischio che quella che ad
un primo sguardo sembra essere una definitiva vittoria
della democrazia e della cultura democratica nasconda,
invece, al suo interno l’assenza di una convinta presa di
posizione sul problema della democrazia. Se in passato i
pericoli per la democrazia nascevano da ideologie anti-
democratiche, alimentate da organizzazioni totalitarie e 3. La democrazia acefala. Nella vastissima biblio-
grafia cavalliana si possono agevolmente individuare le
tappe del suo progressivo riflettere sulla democrazia in
un arco di tempo che va dal 1964 ai primi anni Due-
mila (Cavalli 2005). Ove si voglia sintetizzare la lettura
che il Cavalli maturo ha dedicato alla moderna demo-
crazia appaiono emergere due tipi ideali contrapponi-
bili: a) la democrazia acefala (o democrazia senza lea-
der); b) la democrazia con leader. Questi due tipi ideali
di democrazia vengono associati a differenti studi del
caso, rispettivamente: a) alla repubblica parlamentare
italiana, alla quarta repubblica francese ed alla führer-
lose Demokratie tedesca postbellica criticata da Weber;
b) alle repubbliche presidenziali degli Usa e della Fran-
cia e al tipo di democrazia plebiscitaria preconizzato da
Weber, ossia una democrazia con un capo effettivo. I lati oscuri della democrazia Oggi, per la prima volta, si comincia a riflettere
democraticamente sulle stesse basi della democrazia e si
arriva a metterle in discussione, una situazione del tutto
nuova dato che in genere «le basi della democrazia sono
come delle precondizioni dei processi democratici, sot-
tratte (in larga misura) al mutamento stesso. Esse ven-
gono trattate come se fossero eterne»3 (Beck 1999: 30). Secondo il sociologo tedesco, appare impossibile limitare
i diritti fondamentali dei cittadini al solo ambito politi-
co-statale. Andrebbe allora avviata una sorta di rifles-
sività in senso orizzontale della democrazia, vale a dire
un processo che sia in grado di estenderla a tutte le sfe-
re dell’agire. Un auspicio questo che appare non esente
da elementi di ingenua utopia. Comunque mi permetto
di sottolineare un significativo parallelismo tra lo sfor-
zo analitico di Cavalli e quello di Beck. Entrambi, cer-
cano l’essenza della democrazia oltre la politica stessa,
oltre il circuito partitico-parlamentare o, se si preferisce,
elettorale-rappresentativo, per auspicarla e per ritrovarla
in ambiti strettamente sociali (scienza, famiglia, lavo-
ro, etc.). Ovviamente, per Beck si tratta di far emergere
tutto un fiorire di forme nuove di partecipazione demo-
cratica, per Cavalli invece di individuare prima di tutto
i modi subdoli con cui una minoranza organizzata può
manipolare la democrazia, nell’intento perverso di eser-
citare il suo dominio in forma non palesemente costrit-
tiva ma attraverso socializzazione e controllo sociale. In
questo modo, dopo un disvelamento, si potrà passare
ad una fase costruttiva che reclama naturalmente una
sostituzione delle minoranze dominanti e l’avvento di Certamente, non è facile individuare la manipolazio-
ne, anche perché le sue forme cambiano per effetto del
mutamento sociale e degli sviluppi tecnologici. Occorre
sforzarsi di analizzare il mutamento e gli adeguamenti
che impone alla democrazia alla luce delle nuove diffi-
coltà che può produrre. Invece, forse troppo frettolo-
samente, molti sociologi sostengono che il mutamento
sociale contemporaneo, oltre ad aver ridimensionato
l’attrazione verso i regimi autoritari, ha radicalmente
rimosso ogni rischio di manipolazione e di svuotamen-
to dall’interno della democrazia. A loro parere, il muta-
mento sociale procede non soltanto nella direzione di
cambiare le forme della democrazia, ma soprattutto nella
direzione di una crescente e inarrestabile democratizza-
zione della società. I cambiamenti vengono unicamente
interpretati come trasformazioni che stanno lentamente,
ma profondamente, ridefinendo la politica contempora-
nea in una chiave pienamente democratica2. I lati oscuri della democrazia È
la Führerdemocratie che si libera dalle perniciose pasto-
ie di una democrazia burocratizzata. Va aggiunto che
Cavalli procede nell’esplorazione comparativa di questi
casi alla luce della convinzione che lo Stato nazionale è
l’istituzione principe in grado di affrontare le sfide poste
al mondo moderno. Una convinzione radicata in una
cultura politica storicamente maturata in certi segmen-
ti della società ma che non è riuscita a diffondersi ade- 79 I lati oscuri della democrazia Cavalli ha a cuore soprattutto lo studio della crisi
del sistema politico italiano, crisi che riconduce alla par-
titocrazia, o meglio a quella che lui definisce la demo-
crazia dei partiti. La democrazia dei partiti rappresenta
un sottotipo – empiricamente analizzabile – che si con-
trappone al tipo fondamentale di democrazia di matri-
ce weberiana: la democrazia con leadership. L’analisi di
Cavalli è severa ed espressa in uno stile particolarmente
incisivo che, a tratti, assume un tono quasi profetico. I
partiti sono gruppi nei quali si entra per cooptazione che
operano in modo autoreferenziale e che sono governa-
ti da oligarchie che perseguono, in sintonia con la loro
caratterizzazione, interessi strettamente particolari. guatamente nella comunità nazionale per la responsabi-
lità dei grandi partiti e delle ideologie, internazionali o
sovranazionali, che li ispiravano. Cavalli ci ricorda che κεφαλή è un termine che indi-
ca il capo, la sommità. Questo stesso termine veniva usa-
to in senso traslato per indicare il leader. Il termine va
inteso dunque non semplicemente in un senso formale
di posizione apicale ma implica una valenza attiva, di
responsabilità e di direzione. Max Weber, in Economia
e società parla di autocefalia del gruppo sociale, descri-
ve l’autocefalia degli organi di autorità cittadini e pun-
tualizza che l’autocefalia indica «che il capo e l’apparato
amministrativo sono stabiliti in conformità agli ordina-
menti propri del gruppo, non già – come nel caso dell’e-
terocefalia – da individui estranei». Dunque, l’autocefa-
lia si intreccia con l’autodeterminazione e con la libertà,
risorse imprescindibili per una democrazia. Ovviamente
l’alfa privativo in composizione con il termine, o meglio
l’aggettivo ακέφαλος, allude all’assenza del concetto
espresso dal termine positivo. La democrazia acefala,
all’interno del processo più generale della razionalizza-
zione che anima la modernità, è pseudo democrazia e
rappresenta un processo degenerativo che svuota di sen-
so la sfera politica4. società, I, «Teoria delle categorie sociologiche», Edizioni di Comunità,
Milano, pp.269 e ss.
8 Si veda l’illuminante tavola che confronta analiticamente gli elemen-
ti dei tipi della democrazia autocefala con leader con gli elementi che
caratterizzano la democrazia acefala secondo il paradigma cavalliano in
Turi P. (2008), Sociologia e politica nell’itinerario intellettuale di Luciano
Cavalli in Bettin Lattes G. e Turi P. (a cura di), La sociologia di Luciano
Cavalli, Firenze University Press, Firenze, p. 153. 4 Weber in Politik als Beruf parla di «democrazia senza capi», ovvero
« dominio dei politici di professione senza vocazione, senza le qualità
intime carismatiche che creano un capo». Si veda: Giolitti A. (a cura di)
(1980), Il lavoro intellettuale come professione, Einaudi, Torino, p.99.
5 Cfr., ad esempio, Cavalli L. (1981), Capi democratici contro democrazia
acefala, in Id. Il capo carismatico. Per una sociologia weberiana della lea-
dership, Il Mulino, Bologna, pp.185 e ss.
6 Cavalli L. (1992), Leadership in democrazia: due paradigmi, in Id.
Governo del leader e regime dei partiti, Il Mulino, Bologna, pp.43 e ss.
7 Cavalli fa qui riferimento anche alla riflessione weberiana sulla «col-
legialità della direzione suprema» in opposizione al governo monocra-
tico ed agli esempi riportati sulla collegialità dei ministeri e negli orga-
ni governativi di vertice basati su coalizioni di partito; cfr. Weber M.
([1922] 1980) La collegialità e la divisione dei poteri, in Id. Economia e 4 Weber in Politik als Beruf parla di «democrazia senza capi», ovvero
« dominio dei politici di professione senza vocazione, senza le qualità
intime carismatiche che creano un capo». Si veda: Giolitti A. (a cura di)
(1980), Il lavoro intellettuale come professione, Einaudi, Torino, p.99. 6 Cavalli L. (1992), Leadership in democrazia: due paradigmi, in Id.
Governo del leader e regime dei partiti, Il Mulino, Bologna, pp.43 e ss.l I lati oscuri della democrazia La classe politica che governa incoraggia
la perdita di sensibilità per il bene comune perché lei
stessa, paradossalmente, è interessata alla dispersione
di un patrimonio di valori indispensabili alla vita civi-
le, autenticamente democratica. La democrazia acefala9
ha una sua storia ed un suo radicamento culturale che
vanno individuati ed interpretati se, nell’ambito di una
concezione della sociologia politica intesa come forma di
conoscenza utile ‘per’ sviluppare la democrazia si vuole
dare, nonostante tutto, alla democrazia sostanziale anco-
ra qualche chance di realizzazione. q
In Governo del leader e regime dei partiti (1992) e in
Il primato della politica nell’Italia del secolo XXI (2001)
Cavalli, con riferimento principalmente al caso italiano,
riflette sulla formazione e sull’influenza di una nuova
cultura politica trans-partitica e problematica che defi-
nisce «democraticismo». Si tratta di un’ideologia debole
rispetto alle grandi ideologie del Novecento che si ori-
gina sulle macerie del loro crollo ma che, comunque, si
collega, in maniera complementare e in parte, ai valori
che le ispiravano. Il democraticismo è una forma di eti-
ca politica, o meglio un travestimento etico della politi-
ca, che orienta pubblicamente l’agire della classe politi-
ca secondo una concezione della democrazia che viene
bene definita anche nel vocabolario Treccani nei termi-
ni di una «ostentazione o falsa affermazione di principi
democratici e di attaccamento alla democrazia». La clas-
se politica particolaristica, priva di una visione e di un
progetto politico fondante ed innovativo, alimenta la cri-
si del sistema politico italiano e più in generale la man-
canza di un autentico sviluppo democratico e sociale
nel nostro Paese; e ciò accade perché non è orientata dai
valori citati e dagli altri principi costitutivi della cultura
politica democratica. Si limita a propagandarli per legit-
timare comportamenti che li svuotano di ogni possibili-
tà di inveramento storico con conseguenze imprevedibili
sotto il profilo della integrazione della comunità nazio- Il paradigma cavalliano svela l’ambiguità di un siste-
ma politico che svuota di ogni potere decisionale effet-
tivo il meccanismo istituzionale deputato dalla Costitu-
zione, dato che le decisioni vengono prese dai partiti e
in particolare dalle oligarchie che li governano. I segre-
tari dei partiti con la loro posizione apicale sono «i veri
potenti» con «tratti di potere monocratico più impor-
tante che nella figura del Presidente del Consiglio». I lati oscuri della democrazia La Costituzione legittima una concentrazione dei poteri 80 Gianfranco Bettin Lattes nel Parlamento a scapito dell’esecutivo e a beneficio dei
partiti che di fatto governano. Il capo dell’esecutivo è
solo un primus inter pares, scelto tramite una contratta-
zione fra i partiti. Analogamente per i ministri che sono
il frutto di una lottizzazione fra i partiti di maggioranza
in proporzione alla loro forza elettorale. Il programma di
governo è definito dalla maggioranza. I partiti controlla-
no i rispettivi gruppi parlamentari e li condizionano nelle
loro funzioni. Il principio consociativo che ispira il siste-
ma politico ingloba in una logica di compromesso anche
i partiti all’opposizione. La durata dei governi quasi mai
coincide con la legislatura perché viene stabilita dai par-
titi in funzione dei loro rapporti reciproci nonché in rela-
zione a situazioni contingenti. La collegialità delle scelte
di governo impedisce ai cittadini elettori di attribuire
delle responsabilità ai governanti e ai loro partiti. In bre-
ve, dal multipartitismo e dalla molteplicità degli interessi
e dei punti di vista che animano un panorama partito-
cratico differenziato deriva uno stato di perniciosa ingo-
vernabilità. «Quindi le decisioni sono lente, spesso intem-
pestive, scarsamente appropriate; e, comunque, la loro
ricomposizione in un programma di governo ben di rado
può rappresentare un definito coerente progetto – tanto-
meno, essere ispirato a lungimiranza» (ivi: 50). La com-
petizione politica, data la moltitudine degli attori impli-
cati nella gestione del potere, si ispira «alla ricerca dell’af-
fermazione personale egocentrica» che promuove leader
fasulli. L’ingovernabilità si collega anche «all’assenza di
singoli uffici genuinamente monocratici, che consentano
una decisione personale, in piena responsabilità». partiti, e al loro sfruttamento – che, in definitiva, colpisce
ogni cittadino, anche chi, nel ruolo di cliente, per qualche
aspetto se ne avvantaggi. La registrazione di questa realtà,
associandosi al trionfante politeismo etico e all’individua-
lismo materialistico ed edonistico proprio dell’epoca, d’al-
tronde accelera nelle coscienze la progressiva evanescenza
di ogni identificazione con lo Stato e con la nazione (di
ormai fittizia esistenza) e quindi del fondamento mora-
le della vita associata: l’ingovernabilità crescente, rivela,
ormai, anche un processo disgregativo in atto. (ivi: 52) 4. Il democraticismo. La democrazia acefala orga-
nizza artatamente «un sistema della irresponsabilità
organizzata» che impoverisce ogni fede nelle istituzio-
ni pubbliche. I lati oscuri della democrazia f I partiti così intesi si contrappongono dunque allo Stato e,
d’altra parte, non determinano l’inveramento della sovra-
nità popolare, ma la sua espropriazione, e si collocano
addirittura nella dimensione della dominazione sul popolo
[…] Di fronte all’unitaria realtà della democrazia autocefa-
la con leadership personalizzata sta dunque l’unità fittizia
e tendenzialmente disgregata della repubblica parlamenta-
re partitocratica, che, non avendo in sé né la sua misura di
valore né l’autogoverno della dialettica leader-popolo, pos-
siamo ben chiamare “acefala”. La sua drastica e drammati-
ca incompiutezza – questo è il punto centrale – è la contro-
partita ineluttabile del dominio dei partiti come “gruppi di
potere autocefali” (autocefali peraltro in senso distorto, per-
ché privi essi stessi di una interna dialettica leader- base). (Cavalli 1992: 44,45) Se ci si sofferma sul tipo della democrazia acefala,
indagato da Cavalli in molti suoi studi5, il riferimento
al libro Governo del leader e regime dei partiti apparso
nel 1992, in piena crisi della Prima Repubblica, nel mez-
zo del dibattito per la riforma della Costituzione e nel-
la transizione verso un nuovo ordinamento istituziona-
le, appare imprescindibile6. Il contesto istituzionale per
eccellenza nel quale si sviluppa la democrazia acefala è
quello delle democrazie parlamentari pure. Nell’ambito
dei casi storici prescelti Cavalli non vede semplicemen-
te «il prevalere del legislativo sull’esecutivo, o addirittura
una caratterizzazione in termini di governo assemble-
are» ma sulla scia di Weber e della critica alla partito-
crazia di cui Giuseppe Maranini è stato strenuo alfiere,
definisce la democrazia acefala come una democrazia
priva di autentica leadership7. L’inclinazione autoreferenziale dei partiti viene raf-
forzata da costituzioni e da leggi elettorali che legitti-
mano con forza un multipartitismo dispersivo che fa del
partito una realtà sociologica forte a scapito della incisi-
vità di un’istituzione fondamentale come lo Stato, che
invece viene invaso e depredato dai partiti tramite lo
sviluppo irrefrenabile di clientele. Cavalli disegna allora
“un abbozzo di paradigma” articolato in sei elementi al
cui fondamento troviamo l’insussistenza della relazio-
ne leader-corpo elettorale. La pochezza della leadership
provoca la distanza-distacco tra classe politica e cittadi-
nanza cui corrisponde un uso deformato delle istituzioni
di governo8. Come si diceva gli elementi che spiegano il
paradigma dell’impoverimento della democrazia sono
molteplici ed intrecciati fra di loro. Eccone una sintesi. 9 Cfr. Cavalli L. (1992) La democrazia acefala d’Italia, in Id. Governo del
leader e regime dei partiti, pp.219-258. I lati oscuri della democrazia In particolare, si
sofferma su tre punti che illustrano alcuni macroprocessi
interdipendenti e significativi per il mutamento-degene-
rativo della cultura politica democratica. Il primo punto
riguarda la crisi del «valore tipicamente paterno dell’au-
torità che aveva ispirato storicamente l’articolazione
gerarchica delle istituzioni, cominciando dalla famiglia
per giungere allo Stato. L’eclissi di questo valore ha quin-
di operato in modo disgregativo su ogni istituzione, come
è apparso per la prima volta evidente nella grande con-
testazione giovanile, e non solo giovanile, degli anni Ses-
santa e Settanta»10 (ibidem). Il secondo punto richiama le
conseguenze inintenzionali derivate dall’ideologia libe-
ral-democratica, paladina di valori chiave quali l’ugua-
glianza e il pluralismo, entrambi centrali per la moder-
nità secolarizzante. Le fedi religiose tradizionali ed anche
le religioni secolari, come il marxismo, si sono indebolite
in una forma forse irreversibile. La mentalità delle molti-
tudini ha abbracciato il pluralismo laico in sintonia con
l’ inclinazione al relativismo dei valori svincolata sempre
più da un’etica comune ed orientata prevalentemente da
interessi personali ispirati da «egoismo materialista ed
edonista» (ivi: 46, 47). Si spiega così il terzo punto. Nel-
la tradizione valoriale liberal-democratica occupava un
posto centrale l’individualismo. La secolarizzazione ha
reinterpretato il messaggio centrato sulla realizzazione
individuale depurandolo, e così immiserendolo, di ogni
tensione morale. L’altruismo propagandato dal democra-
ticismo, fa sì che «uguaglianza e solidarismo, in partico-
lare, ricevono da molti un omaggio di superficie, mero
lip-service, per non contraddire la cultura egemone e i
suoi guardiani» (ivi: 46, 47) che sono motivati unicamen-
te da un freddo calcolo costi/benefici in vista di un arric-
chimento materialista di natura volgarmente consumista. Viene dunque denunciata una modalità di pensa-
re e di agire politicamente deteriore, priva di coscienza
storica, appiattita sul presente e sul perseguimento di
interessi particolaristici: questo dato negativo è l’effet-
to primario dell’assenza di lungimiranza dei politici di
professione e della casta partitocratica. I corti orizzonti
della classe politica si riflettono sulla mentalità colletti-
va allontanandola da un senso del bene pubblico, aspet-
to essenziale per nutrire una democrazia sostanziale. La
debolezza di coscienza civica trova probabilmente le sue
ulteriori determinanti sia nella scuola, sia nei mezzi di
comunicazione di massa, sia nella sfera economica che
propongono obiettivi in grado di travolgere la morale ed
il senso profondo dell’agire politico democratico. 10 Nel 1968 si manifesta un processo di crisi valoriale sicuramente fon-
damentale che però andrebbe interpretato con il dato politico, lato sensu
inteso, in virtù del quale le nuove generazioni contestavano un tipo di
autorità che era autoritaria e non autorevole. Questo tipo di autorità si
nascondeva dietro valori che non praticava nella loro effettività e gestiva
i propri ruoli spesso esercitandoli con abusi inaccettabili. I lati oscuri della democrazia Ma
anche loro risentono del condizionamento da parte del
partito nel senso che i vari capi corrente pretendono la
loro parte di potere e dunque non di rado limitano il
potere del loro segretario e la sua autonomia di azione. Ma v’è di più nel senso che bisogna anche tenere conto
dell’impatto di orientamenti valoriali di fondo, stratifi-
catisi storicamente nella coscienza collettiva, ma indebo-
liti e fin stravolti sia dalla cultura partitocratica e dalla
classe politica che si sovrappone ormai con i leader di
partito, sia dalla propensione al politeismo e all’estremo
relativismo dei valori che definisce la nostra modernità. I comportamenti della classe politica provano irrefutabil-
mente la capitale diminuzione dello Stato nazionale: corpo
passivamente abbandonato alla direzione eteronoma dei 81 I lati oscuri della democrazia nale. Un esito certo è quello della diseducazione civica
e di un progressivo distacco tra i partiti e la società dei
cittadini. Leggiamo Cavalli: talmente diffusi nella società che declinano solo sotto
la pressione di processi di cambiamento per i quali non
è improprio parlare di rivoluzioni. Le determinanti dei
processi di mutamento radicale nella più parte dei casi
sono plurali nel senso che derivano da ambiti differenti. Ad esempio, dal sistema economico-produttivo e/o dal
sistema politico nonché dalla crisi di istituzioni che sono
deputate alla produzione ed alla conservazione dei valori
come famiglia, scuola, Stato, Chiesa. Nella cultura dei partiti e, in interazione, in larga par-
te della cultura popolare, elementi affini o complementari
delle vecchie ideologie si sono incontrati e in qualche modo
fusi tra loro e con altri elementi preesistenti e generalmente
deteriori della memoria collettiva, come il ricordo stereo-
tipato della disfatta fra il ‘40 e il ’45. Anche per l’enorme
potenziale di controllo sociale di cui le vecchie forze poli-
tiche ancora dispongono, alcuni comandamenti della nuo-
va cultura sono professati con fede apparente da un gran
numero di italiani: tutti si dichiarano democratici, pacifi-
sti, ugualitaristi, solidaristi ad oltranza. Il concetto di tra-
gedia storica è assente, sostituito da una vaga ottocente-
sca fiducia nelle sorti pacifiche e progressive dell’umanità. (Cavalli 1992: 244) Cavalli ci avverte che i valori hanno una capacità di
presa non omogenea, vale a dire che ci sono valori più
importanti di altri che rappresentano dei veri e propri
«cardini dell’organizzazione sociale». I lati oscuri della democrazia I leader e la
classe politica che li contorna così come sono privi di stu-
dio, di morale e di realismo hanno depotenziato ed avvi-
lito «i valori dello Stato e della nazione, come comunità
di storia, cultura e destino».f Cavalli, inoltre, sottolinea tra gli effetti perversi che
si collegano alla mentalità democraticistica l’incapacità
della classe politica di affrontare la questione criminale
e una irresponsabile svalutazione dell’impatto devastan-
te delle mafie sulla integrazione dello Stato e sulla sua
immagine. Si tratta di un aspetto che è di estrema attua-
lità: non può non stupire come il problema non rientri
da tempo tra i punti che qualificano l’agenda politica di
governo e come, pure non figuri nell’ambito dei proget-
ti politici delle diverse forze politiche. Di conseguenza i
cittadini che hanno ancora a cuore la buona politica e il
bene della loro comunità assistono impotenti In queste stesse pagine Cavalli riprende la sua seve-
ra rivisitazione sul caso italiano e propone la catego-
ria specificativa del «democraticismo umanitario». Una
delle variabili determinanti di questa neo-ideologia,
che meglio sarebbe etichettare come sub-ideologia, è la
scomparsa delle ideologie che animavano le differenti
visioni politiche nella Prima Repubblica. Cavalli ci par-
la in proposito di marxismo e di cristianesimo sociale e
dei «loro incroci sperimentali». La loro influenza persiste
nonostante questi due orientamenti siano evaporati nel
loro impatto anche perché, merita di aggiungerlo, si sono
disciolti i partiti che ne erano paladini. Che cosa avviene
secondo Cavalli? Che All’intreccio di insufficienza e, anche “permeabilità” isti-
tuzionale, mentalità “democraticistica” e clientelismo arti-
colato (collettivo e personale), esercitato nelle due varianti
dell’intervento e del non intervento, che opera oggettiva-
mente a favore del complesso fenomeno mafioso e camorri-
stico, in un ambito socioculturale particolarmente segnato
dall’eclissi dei valori pubblici (e quindi di ogni etica pub-
blica), d’altronde già prima particolarmente deboli per note
ragioni storiche […] La mentalità “democraticistica” ha
quindi fortemente contribuito a rendere inefficace l’azione
contro un nemico organizzato, che conduce una vera guer-
ra contro lo Stato e per il suo proprio contrapposto domi-
nio, ispirandosi a principi che niente hanno in comune
con la nostra concezione europea di civiltà, anzi ne sono la
negazione. I lati oscuri della democrazia Cavalli puntualizza: A quest’insieme di valori, o atteggiamenti, che i propagan-
disti riassumono nel concetto di democrazia va dato un
nome più appropriato. La parola giusta ha già una tradi-
zione, dall’Alfieri a Gramsci, e il Battaglia oggi la definisce
“vacua affermazione (spesso fatta con accenti estremistici)
di principi democratici”. Questa parola è “democratici-
smo”. Nel nostro contesto, tuttavia, il significato si allarga
e, ad un tempo, si precisa. Di questo insieme di atteggia-
menti di massa, il “democraticismo” denuncia la carenza
di razionalità, studio e etica responsabile; e ne mostra il
fondamento reale di residui ideologici e di timido confor-
mismo. Nello stesso tempo il “democraticismo” costituisce
l’ultima importante “giustificazione” dei partiti, largamen-
te comune, e influenza (con effetti ovviamente negativi) le
loro politiche; e, proprio perché comune, consente nuove
possibilità di incontro e collaborazione – anche tra “ ex-
nemici”. (ivi: 244-245) Quando si parla di valori ci si riferisce ad elemen-
ti normativi profondamente radicati, di lunga durata e 82 Gianfranco Bettin Lattes sistema politico: l’instabilità dei governi, l’apatia politi-
ca con un astensionismo elettorale crescente e, per non
citare altro, i fenomeni corruttivi dilaganti nell’ambito
delle massime istituzioni pubbliche. Questo percorso
trasformativo del sistema politico reclama come pre-
condizione un processo di rieducazione politica degli
italiani che transiti dal principio ideologico al principio
di realtà. La società nella sua evoluzione complessiva
reclama, a dire di Cavalli, una razionalizzazione della
rappresentanza e della partecipazione e della sua guida
politica, aprendo a delle possibilità di un’ ulteriore razio-
nalizzazione della selezione dei leader di governo. In bre-
ve, sono circostanze – che purtroppo a distanza di un
ventennio – sembrano ancora in una fase embrionale, o
meglio di improbabile realizzazione in un sistema politi-
co che dimostra crescente propensioni implosive. A questi dis-valori si affianca la pretesa smodata di pote-
re e di prestigio, una pretesa che con la politica in sen-
so proprio nulla a che fare. Le forze politiche, o meglio,
i leader di partito non hanno saputo e non hanno voluto
impedire l’impatto disgregante dei macroprocessi involu-
tivi sopracitati sulla governabilità dello Stato. 11 Meritano un’attenta lettura le pagine di antropologia politica che
Cavalli dedica alla crisi etico-politica della nazione e alla “snazionaliz-
zazione” delle masse in Il primato della politica nell’Italia del secolo XXI,
e il ruolo svolto da quella particolare variabile che Cavalli definisce «lo
spirito della disfatta». 12 Si veda tra gli altri: Cavalli L.(a cura di) (1973), Materiali sull’Italia in
trasformazione, Il Mulino, Bologna; e dello stesso autore (1974) Socio-
logia della storia italiana,1861-1974, Il Mulino, Bologna; (1976) L’Italia
promessa. Riflessioni sulla crisi nazionale, Il Mulino, Bologna; (1985)
Acosmìa. Gli anni del disordine,1977-1981, Facoltà di Scienze Politiche
“C. Alfieri”, Firenze. I lati oscuri della democrazia Oggi, a distanza da almeno cinque lustri da
tale diagnosi, sembra opportuno domandarsi se non sia
invece un dato di realtà la capacità dei cittadini, socializ-
zati ad una cultura politica democratica – pur instabile e
deludente – di adottare i sedimenti di un insieme di ele-
menti ideologici dispersi e di assemblarli per dare con-
senso ad uno Stato in grado di inverare i valori fonda-
mentali di equità e di libertà. Lo straordinario impegno
della società civile che tramite diverse forme associative
sa farsi carico di problemi sociali importanti, ignora-
ti dalla politica, gestendoli e risolvendoli nell’interesse
generale, dimostra che la sub-ideologia “democraticisti-
ca”, viva nella società civile, include anche aspetti positi-
vi e non semplicemente parole vuote. I partiti italiani importanti, dal 1945,sono stati partiti che
avevano posto sé stessi come massimo valore. Cioè il parti-
to fine a sé stesso. Sono quindi disposti a una riforma come
quella del passaggio dalla “democrazia dei partiti” alla
“democrazia con un leader” solo quando questa riforma
sembra favorire i loro interessi di potere. Essendo il partito
il massimo valore, infatti, l’interesse pubblico viene sempre
subordinato […] Così è che i progetti di riforma istituzio-
nale ed elettorale si piegano alle priorità strategico-tattiche
dei partiti (Cavalli [Viviani] 2010: 188) I lati oscuri della democrazia (ivi: 252, 253) I sistemi ideologici delle due parti dissolvendosi hanno
lasciato liberi degli elementi comuni o affini, che, presso i
reduci di quelle esperienze storiche, si sono organizzati in
una elementare visione della vita, e relativa etica, intorno
all’idea-valore di solidarietà umana (senza confini). È quel
che usualmente si chiama catto-comunismo, per riguardo
alla sua duplice origine culturale, anche se a ben considerar-
ne le caratteristiche, sarebbe più conveniente la denomina-
zione democraticismo umanitario, perché, sempre in nome
della comune umanità, si vorrebbero introdurre democra-
zia e solidarietà, considerate inseparabili, in ogni rapporto
sociale e in ogni istituzione e fra istituzioni, ivi comprenden-
do gli Stati. E chi non condivide questa visione, e l’etica rela-
tiva, è senz’altro un reprobo. (Cavalli 2001: 39, 40). Nel 2001 ricollegandosi alle analisi di Governo del
leader e regime dei partiti, Cavalli in Il primato della
politica nell’Italia del secolo XXI mentre dichiara di voler
qui «contribuire all’individuazione di temi e problemi su
cui dovrebbe insistere l’indagine dei nuovi ricercatori»
(Cavalli 2001: VII) disegna una proposta di riforma che
superi il regime dei partiti tramite una leadership effet-
tiva motivata da un progetto-Paese e capace di attivare
una dialettica innovativa leadership/elettorato in gra-
do di ridare respiro all’autonomia dello Stato nazionale. La proposta è quella della “repubblica unicipite” frutto
di una riflessione sociologica e storica pluriennale del
Nostro. La dialettica leadership/elettorato è indispensa-
bile per un superamento dell’insofferenza verso la vec-
chia politica testimoniata da diversi aspetti critici del Il catto-comunismo, i cui spazi di influenza reale
sono stati comunque sempre contenuti, ha fatto il suo
tempo per motivi che non è il caso qui di esplorare. Cavalli ci suggerisce però una pista di indagine interes-
sante sugli effetti del crollo delle grandi ideologie su un
sistema politico frantumato ed instabile e, soprattutto,
popolato da pseudo-leader e da politici che interpreta-
no la loro ‘professione’ in modo impoverente, privo di
ogni etica volta al bene comune. Ancora più interessan- 83 I lati oscuri della democrazia te è una riflessione, bisognosa di indagine empiriche,
su come a livello del cittadino comune il crollo delle di
ideologie abbia prodotto un meccanismo di recupero
di alcuni elementi valoriali essenziali ed indispensabili
per un minimo di coscienza collettiva democratica. Un
minimo valoriale senza il quale sarebbe lo stesso sistema
politico a dissolversi. I lati oscuri della democrazia In sostanza merita una verifica se
sia vera la diagnosi cavalliana, fortemente pessimistica,
secondo cui «il “democraticismo umanitario” opera sia
per dividere ancor più gli italiani, e indebolire presso i
suoi seguaci quel poco che resta di intima appartenenza
allo Stato nazionale, sia per imporre all’Italia una rotta
politica che non ha nel bene del Paese la sua stella pola-
re» (ivi: 41). Oggi, a distanza da almeno cinque lustri da
tale diagnosi, sembra opportuno domandarsi se non sia
invece un dato di realtà la capacità dei cittadini, socializ-
zati ad una cultura politica democratica – pur instabile e
deludente – di adottare i sedimenti di un insieme di ele-
menti ideologici dispersi e di assemblarli per dare con-
senso ad uno Stato in grado di inverare i valori fonda-
mentali di equità e di libertà. Lo straordinario impegno
della società civile che tramite diverse forme associative
sa farsi carico di problemi sociali importanti, ignora-
ti dalla politica, gestendoli e risolvendoli nell’interesse
generale, dimostra che la sub-ideologia “democraticisti-
ca”, viva nella società civile, include anche aspetti positi-
vi e non semplicemente parole vuote. Questa analisi che si è fondata, come si è ricordato,
su una molteplicità di studi, focalizzati sul caso italiano e
sviluppati con la metodologia da sociologo della storia12,
viene ripresa anche nelle riflessioni più recenti di Cavalli
che meritano di essere richiamate a conclusione di questo
excursus perché bene illuminano i limiti in cui è incap-
pato il nostro sistema politico e la sua veste democratica. La mancanza di un repubblicanesimo civile come collan-
te nazionale trova la sua causa perversa nel fatto che te è una riflessione, bisognosa di indagine empiriche,
su come a livello del cittadino comune il crollo delle di
ideologie abbia prodotto un meccanismo di recupero
di alcuni elementi valoriali essenziali ed indispensabili
per un minimo di coscienza collettiva democratica. Un
minimo valoriale senza il quale sarebbe lo stesso sistema
politico a dissolversi. In sostanza merita una verifica se
sia vera la diagnosi cavalliana, fortemente pessimistica,
secondo cui «il “democraticismo umanitario” opera sia
per dividere ancor più gli italiani, e indebolire presso i
suoi seguaci quel poco che resta di intima appartenenza
allo Stato nazionale, sia per imporre all’Italia una rotta
politica che non ha nel bene del Paese la sua stella pola-
re» (ivi: 41). Ne consegue che Caval- 84 Gianfranco Bettin Lattes li sottolinea, costantemente, il collegamento tra crisi
e conflitto e l’insopprimibile presenza della lotta e del
dominio nelle relazioni sociali secondo l’insegnamen-
to realistico di Weber. Molte delle sue energie intellet-
tuali sono state dedicate allo studio dell’emergere, della
gestione e del superamento della crisi nell’ambito della
legittimità democratica. Non compete però a chi scrive
queste pagine affrontare in modo adeguatamente appro-
fondito qui tale tematica così rilevante nella sociologia
politica cavalliana. Compete, invece, concludere queste
riflessioni ricordando la straordinaria statura scientifica,
morale ed umana di Luciano Cavalli. La sua indagine
sui lati nascosti della democrazia, qui appena abbozzata,
non lascia intravvedere orizzonti luminosi a breve, ma
non deve mai lasciarci senza speranza. Sembra certo che
questa fase della democrazia così claudicante lo abbia
deluso e addolorato ma è altrettanto certo che non lo ha
mai visto rassegnato e domato nella sua passione civica. Piace allora pensarlo ancora tra i suoi libri, nel suo stu-
dio sui tetti di Firenze, da dove continua ad insegnarci
che in un mondo che impedisce, per ora, la vittoria del-
la ragione «la sola via degna dell’uomo libero è sempre
quella antica e stupenda del dissenso solitario, la via del
Waldgänger» (Cavalli 1965: 261) e a raccomandarci che
«anche queste pagine devono chiudersi con una pacata
nota di richiamo alla saggezza, che chiuda l’anello del
nostro discorso. I ribelli non devono dimenticare mai,
nemmeno nelle fasi più aspre della lotta politica, che l’u-
nica mèta umana si chiama democrazia: ossia una diffi-
cile bilancia tra innovazione e conservazione» (ivi: 263). – (1985) Acosmìa. Gli anni del disordine,1977-1981,
Facoltà di Scienze Politiche “C. Alfieri”, Firenze. i
– (1992), Il Governo del leader e regime dei partiti, Il
Mulino, Bologna. – (1992), Leadership in democrazia: due paradigmi, in
Id. Governo del leader e regime dei partiti, Il Mulino,
Bologna.f – (1999), Leadership “personale” e “diffusa” in Marletti C. (a cura di), Politica e società in Italia, vol. I, Cambia-
mento politico e identità sociali, Angeli, Milano, pp. 50-72. – (2001), Il primato della politica nell’Italia del secolo XXI,
CEDAM, Padova. – (2003), Il leader e il dittatore. Uomini e istituzioni di
governo nel “realismo radicale”, Ideazione, Roma.i – (2005), Umberta Porta (a cura di), Bibliografia 1955-
2005, Facoltà di Scienze Politiche “C. Alfieri”, Firenze. i
Giolitti A. (a cura di) (1980), Il lavoro intellettuale come
professione, Einaudi, Torino. Turi P. Ne consegue che La politica, in Italia, è sempre stata debole, nonostante i
partiti siano stati molto forti. Il primato dei partiti è stato il
contrario del primato della politica, perché i partiti, o alme-
no quelli che contavano di più, erano portatori di culture
classiste, internazionaliste e sovranazionali […] È oppor-
tuno ribadire che le personalità politiche provenienti dai
tradizionali partiti non possono verosimilmente esercitare
un’appropriata leadership nella crisi. Perché quei partiti
hanno formato e selezionato i loro quadri in base al prin-
cipio di cui si è detto: il partito è il più alto valore. (ivi: 190) 5. Per concludere in modo non definitivo. Sembra
allora verosimile che, nonostante tutto, il principio di
realtà – un principio che sta a cuore a Cavalli perché
senza questo principio l’agire politico perde la sua nobil-
tà morale e la sua efficacia – possa di fatto ancora ope-
rare e includere idee/valori che operano liberamente tra
i cittadini al di fuori dalle logiche perverse ed autorefe-
renziali che orientano il sistema politico e la classe par-
titocratica che lo occupa. L’uso manipolativo delle stesse
idee/valori da parte dei leader di partito – specialmente
preoccupati di catturare voti – e da parte di un’opinione
pubblica apatica, che per il bene pubblico non ha alcun
interesse, non impedisce che un settore importante del-
la società civile e aree istituzionali virtuose adottino un
operare civico responsabile che sembra avere come «stel-
la polare» (ivi: 101, 116), in modo più o meno consape-
vole, «il bene del Paese» (ibidem), superando così i limi-
ti politici derivati da una perniciosa “partitizzazione”
dell’idea di nazione11. Giunti a questo punto di una conclusione neces-
sariamente non definitiva, si impone un interrogativo
cruciale. La sovrapponibilità tra il modello pubblico di
governo ed il modello partitocratico è una strada irri-
mediabilmente senza uscita per il sistema politico, per
dei veri leader e per i cittadini che aspirino ad un’au-
tentica democrazia? La speranza non abbandona Caval-
li secondo cui un partito con un leader può formarsi in
situazioni di crisi e così tendere ad una trasformazione
dell’ordine sociopolitico generale nella direzione di uno
sviluppo della democrazia sulla base di «un progetto
Paese» adeguato alle sfide del nostro tempo. Il concet-
to di crisi, ora evocato, viene adottato e posto al centro
della riflessione sociologica – teorica ed empirica. Ne consegue che (2008), Sociologia e politica nell’itinerario intellet-
tuale di Luciano Cavalli in Bettin Lattes G. e Turi P. (a cura di), La sociologia di Luciano Cavalli, Firenze
University Press, Firenze. Viviani L. (a cura di) (2010), Società e politica nell’Italia
della crisi. Riflessioni di Luciano Cavalli, in «Società-
MutamentoPolitica», p.183-193. Weber M. ([1922] 1980), La collegialità e la divisione dei
poteri, in Id. Economia e società, I, «Teoria delle cat-
egorie sociologiche», Edizioni di Comunità, Milano. Citation: Paolo Giovannini (2023) La
democrazia manipolata nella società
digitale. SocietàMutamentoPolitica 14(27):
85-88. doi: 10.36253/smp-14337 Citation: Paolo Giovannini (2023) La
democrazia manipolata nella società
digitale. SocietàMutamentoPolitica 14(27):
85-88. doi: 10.36253/smp-14337 Paolo Giovannini Copyright: © 2023 Paolo Giovannini. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Nel 1965 – più di mezzo secolo fa – Luciano Cavalli pubblicava con le
Edizioni di Comunità un libro di forte respiro critico: La democrazia mani-
polata. Per me, allora suo studente, fu forse la lettura decisiva nell’orientarmi
verso il lavoro sociologico: perché vi lessi sì il rigore dello scienziato socia-
le, ma anche l’impegno sociale e politico di denunciare – da cittadino – le
carenze e i tradimenti della democrazia. L’impianto del volume è decisamente elitista, anche se non di comple-
ta ortodossia. Una classe dominante (una minoranza organizzata) esercita
un potere legittimo (costruito su un potere di fatto) sulla gran massa della
popolazione: con diverso equilibrio tra società a diverso tasso di democrazia,
ma comunque dove il rapporto rimane sempre tra élite dominante e mas-
se dominate. Un rapporto di potere che non si esercita però solo in forma
diretta e palese, ma anche indiretta e inavvertita, con l’uso dei più raffinati
strumenti della manipolazione. Questa, in sintesi, si può definire come una
tecnica, o meglio un insieme di tecniche, che tende a ottenere obbedienza e/o
a condizionare l’azione degli individui senza che di queste pratiche si avverta
chiaramente provenienza e finalità – fino al punto di ritenerle, con maggiore
o minore consapevolezza, frutto di propria autonoma determinazione1. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. Socializzazione e controllo sociale sono i due processi fondamentali che
caratterizzano l’azione manipolativa. Ad ognuno sono preposti specifici atto-
ri sociali e istituzionali (famiglia e scuola nel primo, polizia e magistratura
nel secondo), ma in realtà i due processi sono solo analiticamente distingui-
bili; in linea generalissima, possiamo dire che l’uno supporta l’altro nei suoi
parziali fallimenti. Nel libro, il riferimento fondamentale cui Cavalli applica la sua indagine
è quella particolare configurazione storica che è la società-stato. 1 In un rapporto diseguale di potere, i politologi distinguono tre diverse modalità di relazione
dominanti/dominanti: influenza, persuasione, manipolazione (Morlino 2021). RIFERIMENTI BIBLIOGRAFICI Beck U. (1999), L’epoca delle conseguenze secondarie e la
politicizzazione della modernità, in Beck U., Gid-
dens A., Lash S., Modernizzazione riflessiva: politica,
tradizione ed estetica nell’ordine sociale della moder-
nità, Asterios Editore, Trieste. Cavalli L. (1964), Il sociologo e la democrazia, Silva Edi-
tore, Milano. – (1965), La democrazia manipolata, Edizioni di comu-
nità, Milano. – (1973), Materiali sull’Italia in trasformazione, Il Mulino,
Bologna. – (1974) Sociologia della storia italiana,1861-1974, Il
Mulino, Bologna.l – (1976) L’Italia promessa. Riflessioni sulla crisi nazionale,
Il Mulino, Bologna. – (1981), Capi democratici contro democrazia acefala, in
Id. Il capo carismatico. Per una sociologia weberiana
della leadership, Il Mulino, Bologna. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a C’è, innanzi-
tutto, come dato tecnologico di base (sempre in pro-
gress comunque), la massiccia disponibilità di sensori e
di connessioni che permettano di raccogliere quantità
esponenzialmente crescenti di dati di ogni tipo e dimen-
sione, sia individuali che collettivi; c’è, secondo, una cre-
scente raffinatezza nell’uso di Cloud Computing in grado
di gestire e manipolare quell’enorme patrimonio di dati
(Big Data); terzo, c’è la possibilità/capacità di collegare
e mantenere in rete persone, cose e luoghi coinvolti nei
processi decisionali di qualunque livello; c’è, quarto, la
tracciabilità e calcolabilità delle azioni di singoli indivi-
dui come di organismi collettivi (Talia 2018): c’è, quin-
to, una interconnessione e una collaborazione tra siste-
mi tali da consentire una raffinata «personalizzazione di
massa» dell’azione manipolativa (Calise, Musella 2019);
infine, ed è la condizione di maggior rilievo, si fa cre-
scente uso di strumentazioni capaci di apprendimento
autonomo (AI - Artificial Intelligence). f Non è possibile naturalmente dar conto di tutte
quelle trasformazioni che hanno modificato così profon-
damente il contesto interno e internazionale delle socie-
tà-stato cui si riferiva idealtipicamente (ma non solo)
Luciano Cavalli. Né è possibile fare anche soltanto cen-
no ai nuovi assetti sociali, economici e culturali che oggi
disegnano un quadro assai diverso dagli anni ‘60 del
‘900. Sceglierò allora di prendere in considerazione solo
quella variabile – la tecnologia – che a mio parere risulta
oggi in più stretta relazione con il problema della mani-
polazione in società democratiche – naturalmente senza
con questo voler negare la rilevanza di altre trasforma-
zioni cui ho fatto cenno. 3. In una certa misura la manipolazione è sempre
stata uno strumento utilizzato dal potere quando altri
mezzi di comando si mostravano inefficaci o erano indi-
sponibili. Ma il passaggio da una società di pubblici a
una società di massa cambia radicalmente e direi qualita-
tivamente il processo manipolativo. C’era raffinata mani-
polazione nell’orazione funebre di Marco Antonio sulla
tomba di Cesare, certo, ma rivolta a un pubblico relativa-
mente ristretto di Romani, a portata di voce. Come c’era
manipolazione nell’uso del pettegolezzo da parte dei vec-
chi residenti del quartiere di Winston Parva nei confron-
ti dei nuovi arrivati (Elias, Scotson 2004 [1965]), così che
inconsapevolmente si sviluppasse in loro un sentimento
di inferiorità utile per stabilire e mantenere una relazione
diseguale di potere. Ma anche qui il gioco si svolgeva in
una piccola comunità di una piccola città2. Ma il discor-
so è tutt’altro che astratto. In realtà è costante l’esemplificazione con la situa-
zione italiana, specialmente dei due decenni che avevano fatto seguito alla
fine della Seconda guerra mondiale. 2. Ma non seguiremo Cavalli in questa pur interessantissima analisi. Perché, a distanza di più di cinquant’anni, l’interrogativo che si impone – di
fronte ai cambiamenti intervenuti – è se nelle società cosiddette democrati-
che siano aumentati o al contrario si siano ridotti i gradi di libertà indivi- SOCIETÀMUTAMENTOPOLITICA 14(27): 85-88, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14337 86 Paolo Giovannini duale e collettiva, e se, e in quale misura, con quali stru-
menti, con quale estensione e con quali finalità le élite
dominanti facciano ricorso a pratiche di manipolazione. La ragione principale per la quale l’analisi è limitata alle
società democratiche sta nel fatto che sono queste demo-
crazie a fare un uso più sistematico degli strumenti della
manipolazione. Mentre infatti i regimi autoritari o ditta-
toriali dispongono quando necessario di strumenti coer-
citivi in grado di ottenere obbedienza (se non consenso)
dai propri sudditi; nelle democrazie la ricerca del con-
senso è un elemento essenziale, che forze in competizio-
ne (politiche, economiche, culturali, ecc.) si contendono
con ogni mezzo consentito dalle regole democratiche. inconsapevole manipolazione. Siamo a un salto di quali-
tà. Non solo perché la strumentazione disponibile diventa
enormemente più potente e di più generale applicazione,
ma anche perché – come vedremo – finisce per toccare
quell’ambito così essenziale per il funzionamento della
democrazia che è la libertà personale. p
Richiamo rapidamente quelli che sono gli elementi
fondamentali del processo di digitalizzazione utilizza-
bili (e utilizzati) a scopo manipolatorio. 2 Per inciso, Norbert Elias avanza comunque l’idea che tale meccani-
smo (il “pettegolezzo”) possa trovare vita anche su scala più ampia, per
esempio tra Stati nazionali. 3 Per certi versi, viene alla mente la classica ricerca di Paul Lazarsfeld,
Bernard Berelson e Hazel Gaudet, The People’s Choice (1944), dove si
sosteneva che la comunicazione dei mass media, per esempio la pro-
paganda politica, non arrivava direttamente alla gente, ma passava per
così dire attraverso il filtro delle istituzioni tradizionali (famiglia, paren-
tela, reti amicali) che ne reinterpretava i messaggi (The Two-step Flow of
Communication). La democrazia manipolata nella società digitale qualcosa a che fare col governo del pubblico, dello Stato,
delle istituzioni politiche sovranazionali, certo non sia-
mo lontani dal modello di postdemocrazia ipotizzato da
Crouch (2003). Che però oggi ha bisogno di una rifor-
mulazione che guardi non ai soli aspetti economico-
finanziari. Crouch aveva in mente soprattutto l’enorme
potere sovranazionale delle multinazionali. Ma oggi il
discorso va esteso alle molte «autorità indipendenti»,
come le chiama Fruncillo (2020), che determinano con il
loro potere manipolatorio destini individuali e percorsi
sociali senza disporre di alcuna legittimazione, diretta o
indiretta (Morlino, Sorice 2021). Né andrebbero dimen-
ticate, ma lo posso soltanto accennare, le potentissime
piattaforme digitali che dominano nella commercializza-
zione on line, imponendo in ogni modo la loro presen-
za nel sistema dei social (e non solo), con metodi solo in
parte controllabili e con pesantissime intrusioni (profila-
zione, targetting idividualizzato, ecc.) nella sfera privata
dei cittadini – molti dei quali, a dire il vero, manifestano
una soddisfatta accettazione del sistema. Sul piano pub-
blico, tutto questo avviene nella totale impotenza e ina-
zione delle autorità di sorveglianza e controllo di fronte
al sovrastante potere delle sette o otto piattaforme digi-
tali che dominano il panorama mondiale4, condizionan-
do e manipolando in mille modi il partecipato consenso
di masse enormi di consumatori. complessità del sistema di comunicazione diventa sem-
pre più difficile individuare la “fonte” e valutare la “rile-
vanza” del processo manipolativo. Perché già allora si
poteva registrare la diffusione di reti sociali sempre più
ramificate, dove viaggiavano informazioni, valutazioni,
idee, fatti, ecc. che – secondo Crespi – stavano intaccan-
do robustamente il potere manipolatorio dei tradizionali
mezzi di comunicazione di massa. Questa parziale rottura di una situazione comunica-
tiva pressoché monopolistica ha indotto, un po’ affretta-
tamente, studiosi e politici a considerarla nei suoi aspetti
positivi. Politicamente, poteva aprire la strada a ipotesi
di democrazia elettronica (Budge 2008); in ogni caso si
poteva pensare che i media digitali potessero costituire
almeno parzialmente un’alternativa ai media mainstre-
am (Freschi 2019). La storia, a me pare, si è svolta diver-
samente. Presto – anzi, con straordinaria velocità – i
media digitali hanno occupato massicciamente il terri-
torio dell’informazione (e della manipolazione) dell’opi-
nione pubblica. A sua volta, l’universo dei social è sta-
to conquistato con estrema facilità, la società di massa
ha perso anche le sue ultime caratteristiche distintive,
lasciando il posto a l’«idiotismo delle arene digitali» (per
parafrasare Marx). 4 Per citare le maggiori: Apple, Microsoft, Google, Amazon, Facebook,
Lime, Alibaba. Ben diverse le
condizioni nella moderna società di massa, dove il potere
nelle sue varie articolazioni ha potuto (e dovuto) via via
utilizzare nuovi linguaggi, nuove tecniche, differenzia-
te strategie di comunicazione, fino a quella rivoluzione
digitale che oggi mette a disposizione delle élite domi-
nanti potenti strumenti di consapevole (quasi sempre) o i
In sintesi, l’era digitale offre alle élite dominanti la
straordinaria possibilità di combinare, intercambiare,
utilizzare congiuntamente enormi quantità di dati pro-
venienti da mondi sociali, economici, biologici, fisici
e altri possibili, molto al di là di qualunque precedente
disponibilità di informazioni. Il salto, ripeto, è qualitati-
vo, direi rivoluzionario. Perché mai nella storia moderna
privati e pubblici hanno avuto a portata di mano qual-
cosa di paragonabile in termini quantitativi e qualitativi
di informazioni e conoscenze potenzialmente utili per
orientare l’azione sociale. Una condizione che oggetti-
vamente sarebbe positiva, per la gamma quasi infinita
di scelte che rende possibili ad ognuno di noi. Ma forse
bisognerebbe dire “teoricamente” positiva. Perché l’era
digitale, nei fatti, ha presto generato tecnologie in gra-
do di dominare, governare, utilizzare, finalizzare questo
accumulo informativo al fine opposto, quello di indiriz-
zare e condizionare le scelte individuali e collettive senza
che chi le compie ne sia avvertito. 4. Quali scenari si aprono allora nei sistemi demo-
cratici con l’estendersi e l’articolarsi del processo di
digitalizzazione? Non è facile rispondere a questo inter-
rogativo. Come già notava Crespi non molto tempo fa
(1997, ma sembrano passati anni luce) al crescere della 87 La democrazia manipolata nella società digitale La democrazia manipolata nella società digitale L’adesione acritica alla nuova realtà, il soddisfat-
to asservimento di enorme masse di sudditi del digitale
hanno completamente mutato il modello comunicativo e
le stesse strategie della manipolazione. Siamo in presen-
za – come sostiene Origgi (2018) – di una estesa «vulne-
rabilità cognitiva» della gente, irresistibilmente affasci-
nata dalle nuove tecnologie digitali, cui si accompagna
secondo l’Autore una cieca fiducia nei mezzi e nelle rela-
zioni che con esse si stabiliscono. Un giudizio severo ma
che sicuramente coglie una parte di verità. Forse però
Origgi trascura un elemento. E cioè che i più tradiziona-
li mezzi di comunicazione di massa (stampa e televisioni
innanzitutto) non sono scomparsi dalla scena (anche se
da tempo sfiduciati), ma hanno in realtà stabilito solide
interdipendenze con il mondo dei social (Morlino, 2021). Per cui il gioco manipolativo si snoda oggi su due livelli,
quello dei mass media, ancora forte anche se indebolito,
e quello dei social, che gode invece di una fiducia presso-
ché incondizionata3. 5. Per chiudere. Forse nelle società democratiche,
dove pure è andato silenziosamente avanti un processo
di restrizioni di fatto delle libertà personali (Giovannini
2021), qualche possibilità di resistenza la si può ancora
individuare. Ma la minaccia è ormai globale, e l’assedio
si è fatto stringente in ogni parte del mondo. Le nuove
tecnologie, i social networks, l’incontrollato strapote-
re dei padroni del digitale (in gran parte privati), han-
no assunto dimensioni globali, travalicato ogni confine,
ottenuto lo stesso cieco successo e lo stesso soddisfat-
to asservimento in buona parte delle loro popolazioni. Rimane la speranza (non senza qualche fondamento
nelle condizioni sociali e culturali di questi strati delle
popolazioni) che da qualche parte (i poveri del mondo? le nuove generazioni?) si faccia forte la richiesta di un
uso diverso del potere digitale, che ampli e non riduca la
libertà di azione e di scelta della gente. Un secondo punto riguarda poi la mancanza di un
qualunque controllo democratico su chi detiene in ulti-
ma analisi le fonti del potere. Se la democrazia ha ancora Ma voglio chiudere davvero con le ultime righe del
libro di Luciano Cavalli, e con il suo drammatico appel-
lo al ribelle. La democrazia manipolata nella società digitale Scrive Cavalli: La figura del ribelle ha dato il sale alla storia occidenta-
le: il dissenter inglese del XVII secolo, il carbonaro italia- 88 Paolo Giovannini no dell’inizio del XIX secolo, il partizan, il maquisard, il
patriota del nostro secolo, sono state le varie incarnazioni
storiche di questa figura che hanno contribuito in modo
essenziale ai più alti sviluppi della nostra civiltà. Il ribel-
le è sempre ricomparso nei momenti cruciali per la nostra
libertà […] la libertà può in generale essere riscattata con
la resistenza civica giorno per giorno, che si attua nel-
la protesta sistematica contro la violazione dei patti, nel-
la testimonianza resa coraggiosamente alla verità, nella
rivendicazione testarda e puntuale del diritto, nel disve-
lamento spietato di ogni sopraffazione e di ogni minaccia
alla libertà. (Cavalli 1965: 262-263) Citation: Ambrogio Santambrogio (2023)
Carisma dei valori e carisma dei fini. SocietàMutamentoPolitica 14(27): 89-97. doi: 10.36253/smp-14338 Ambrogio Santambrogio Copyright: © 2023 Ambrogio Santam-
brogio. This is an open access, peer-
reviewed article published by Firenze
University Press (http://www.fupress. com/smp) and distributed under the
terms of the Creative Commons Attri-
bution License, which permits unre-
stricted use, distribution, and repro-
duction in any medium, provided the
original author and source are cred-
ited. Devo a Luciano Cavalli molti importanti insegnamenti, che mi ha tra-
smesso in particolare durante il mio periodo di dottorato a Firenze. Forse il
più importante può essere detto così: i concetti sociologici importanti sono
come fili da tirare. Sollecitandoli, emerge la matassa di altri concetti con cui
sono intrecciati e al cui interno, nonostante una prima apparente sensazione
di disordine, è possibile ricostruire percorsi più o meno coerenti. Tutte le sue
lezioni, che ricordo ancora bene, si ispiravano a questo principio: si partiva
da un tema definito, ma poi, in maniera del tutto naturale, emergeva la com-
plessità delle questioni cui quel tema era legato e dalla quale era impossibile
prescindere. Naturalmente, senza cadere nell’indeterminato: la complessi-
tà andava messa in luce e gestita. La matassa, con il suo groviglio, rimaneva
lì, ma si capiva che altri, e altrettanto interessanti, fili spuntavano, ognuno
meritevole di essere a sua volta sollecitato. Intanto, i suoi ragionamenti assu-
mevano la forma di un percorso da seguire, di una strada non senza alterna-
tive, ma ragionevolmente percorribile. Penso sia un insegnamento che Caval-
li, a sua volta, ha tratto dai classici, e in particolare da Weber. Ricostruire
nessi tra i fenomeni, indicando percorsi plausibili, anche se sempre falsifica-
bili, tenendo sempre in mente l’insieme cui i fenomeni appartengono: questa,
in fondo, è l’idea. Voglio provare ad esercitarmi in questa direzione, come
fosse un modo per mostrare a me stesso che quell’insegnamento non è anda-
to perso. Tiro il filo “carisma” in un percorso che, dopo aver fatto emergere
dalla problematica matassa del pensiero weberiano il concetto di “razionali-
tà”, prova a riflettere sul rapporto tra carisma e democrazia. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. 1. Il concetto di razionalità in Weber. 1 Secondo Brubaker, «il concetto di razionalità è il tema unificante della teoria sociologica di
Weber. Gli studi empirici di Weber, apparentemente eterogenei, convergono su un’unica aspira-
zione di fondo: identificare e spiegare lo sviluppo della “forma specifica e peculiare di razionalità” RIFERIMENTI BIBLIOGRAFICI Budge I. (2008), Direct Democracy, Oxford University
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r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. Un classico è un autore che permette
molte letture, così che diversi contesti storici possono riconoscersi in altret-
tanto diverse chiavi interpretative. Nessuna è quella definitivamente corret-
ta: la questione di cosa abbia davvero detto Weber – o Marx, o Durkheim,
o Habermas – mi sembra futile e fuorviante: ogni interpretazione distorce e,
soprattutto nel nostro lavoro, l’aspetto davvero interessante sta, per l’appunto,
nelle distorsioni, laddove diventino produttive. Ritengo però che una possibi-
le chiave di lettura capace di entrare nella “matassa” Weber possa partire dal
concetto di razionalità1. La sua metodologia può essere kantianamente vista SOCIETÀMUTAMENTOPOLITICA 14(27): 89-97, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14338 SOCIETÀMUTAMENTOPOLITICA 14(27): 89-97, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14338 90 Ambrogio Santambrogio Ambrogio Santambrogio come una critica della ragion sociologica; e, allo stesso
modo, nell’interpretazione weberiana, le trasformazioni
epocali interne alla cultura occidentale hanno diretta-
mente a che vedere con le diverse interpretazioni storiche
del concetto di ragione. Ritengo che Weber non abbia
una concezione sostantiva della ragione, che non ci dia
cioè né una definizione precisa di ragion pura e neppu-
re una di ragion pratica. La ragione non è una facoltà
in grado di dirci in assoluto cosa sia vero e, soprattutto,
cosa sia giusto. La sua lettura del formalismo kantiano
passa attraverso la critica che Nietzsche fa di ogni meta-
fisica, ma mantiene, al tempo stesso, l’impegno teso ad
evitare ogni forma di relativismo e di nichilismo radica-
le. Ciò che fa di Weber uno scienziato sociale e non un
filosofo sta nel fatto di collocare il concetto di ragione
dentro le azioni umane, nel tentativo di vedere quale sia
il ruolo che essa gioca – e potrebbe (o, persino, dovreb-
be) giocare – nella nostra vita sociale di tutti i giorni. La
centralità che nella sua sociologia assume l’agire sociale,
in definitiva il senso intenzionato dai soggetti, ritengo sia
una diretta conseguenza di questo tentativo. Abbiamo qui l’identificazione del diverso ruolo che
Weber dà alla ragione teorica e a quella pratica: la prima
– usata dal sociologo – si esercita su quella pratica – usa-
ta dall’uomo comune. I quattro tipi ideali sono lo stru-
mento razionale con cui la teoria indaga sul rapporto tra
autonomia e passività dentro l’agire, in pratica sulla dose
di razionalità pratica che i soggetti riescono volta a vol-
ta a mettere in campo. La razionalità appare per Weber
in prima battuta come espressione di consapevolezza e
di chiarezza. All’interno dell’agire sociale, l’agire tipi-
camente affettivo e quello tipicamente tradizionale sono
maggiormente passivi e non consapevolmente orienta-
ti; nei due tipi ideali di agire razionale, invece, il senso
appare all’attore in modo più consapevole e la sua inten-
zione gli è più chiara. Tutto ciò non significa che l’attore
sia in generale razionale: secondo Weber, la gran parte
delle nostre azioni è irrazionale, nel senso appena detto,
e solo raramente agiamo in modo consapevole e chiaro. Si tratta naturalmente di un insieme sfumato, che non è
caratterizzato da nette contrapposizioni: si possono fare
esempi di azioni il cui senso è fortemente caratterizzato
da uno dei quattro tipi ideali, ma la realtà è sempre un
miscuglio – una matassa appunto –, al cui interno però
di norma prevalgono i due tipi ideali non razionali. g
q
Come è noto, l’oggetto fondamentale della sociologia
weberiana è il senso che intenzionalmente gli attori dan-
no alle loro azioni nei contesti sociali. Su questo oggetto
si riversa il tema della razionalità: quando un attore dà
intenzionalmente un senso alle sue azioni, quanto c’è di
razionale nelle sue intenzioni? Aprire l’ombrello quando
si mette a piovere non è un’azione in senso proprio: c’è
un aspetto di reattività che soffoca la natura autonoma
e soggettiva dell’intenzionalità. Probabilmente chiun-
que, avendo un ombrello a disposizione, si comportereb-
be allo stesso modo. Non solo, ma anche in molte altre
nostre azioni, dove la nostra intenzionalità potrebbe
essere meno reattiva e manifestarsi in modo più eviden-
te, non è detto che il senso sia un nostro autentico pro-
dotto. Se faccio un regalo a Natale a mia moglie, aspetti
legati alla tradizione possono influire sulla mia scelta. Per non parlare poi delle emozioni, che muovono gran
parte delle nostre azioni. Come scrive lo stesso Weber,
«l’agire reale si svolge nella gran massa dei suoi casi in
una oscura semi-coscienza o nell’incoscienza del suo
“senso intenzionato” […] istintivamente o in conformità
all’abitudine» (Weber 1981 [1922]: 19, corsivi miei). Di
conseguenza, «l’agire effettivamente, e cioè pienamen-
te consapevole e chiaro, è in realtà sempre soltanto un
caso limite» (Ibidem, corsivi miei). che caratterizza la civiltà occidentale moderna rispetto a tutte le altre»
(Brubaker 2006 [1984]: 1, traduzione mia). 2 Per una discussione di questo punto, cfr. Santambrogio 2008. Sebbene «ciò non
toglie che la sociologia elabori i suoi concetti mediante
una classificazione del possibile senso intenzionale come
se l’agire di fatto procedesse in modo consapevolmente
orientato in base ad un senso» (Ibidem, corsivi miei). g
In estrema sintesi, l’idea di razionalità si sposa for-
temente con quella di autonomia, da cui derivano con-
sapevolezza e chiarezza: più l’intenzione è autonoma, e
quindi consapevole e chiara, più è espressione di razio-
nalità. Si tratta ora di capire in che senso i due tipi di
azione razionale siano tali. Mi ha sempre incuriosito
il modello di agire orientato al valore: in che senso è
razionale? Non può esserlo in relazione alla razionalità
del valore di riferimento: se così fosse, verrebbe meno
il pluralismo dei valori. Se uno di questi fosse raziona-
le, si collocherebbe su di un piedistallo che lo distingue-
rebbe nettamente dagli altri2. Mi sembra che riflettendo
su questo punto possa essere cercato il cuore dell’analisi
weberiana: il mondo moderno mette in luce l’inevitabi-
le irrazionalità etica del mondo. Una volta venuti meno i
dogmi della religione o della metafisica, non è più possi-
bile muovere le nostre azioni dentro un contesto di vali-
dità oggettiva. Appare l’inevitabile dimensione dell’auto-
nomia e della scelta soggettiva: ognuno di noi è tenuto a
dare un senso al mondo dal momento che non ci è più
possibile acquisire un senso oggettivo della realtà par-
tendo da una dimensione ad essa esterna, in particolare
di tipo trascendente. Così come il sociologo non ha un criterio razionale
per decidere se la sua interpretazione è vera, allo stesso
modo l’attore sociale non ha un criterio razionale per 91 Carisma dei valori e carisma dei fini per scopi voluti e considerati razionalmente, in qualità di
conseguenza» (Ivi: 21-22). Ora l’azione è razionale se il
soggetto prende in considerazione le aspettative che pro-
vengono dall’esterno come “condizioni” o come “mezzi”
utili per raggiungere uno scopo. Il rapporto con il mon-
do è ora sensibile alle conseguenze, e non è dominato
dalla pura coerenza. Una menzogna, per fare un esem-
pio, può essere ritenuta un utile mezzo per raggiungere
un fine, anche se incoerente con il valore incondizionato
della verità. Si perde in coerenza, si guadagna in adegua-
tezza; e al valore di intenzione si sostituisce il valore di
successo. sapere se la sua azione è giusta. La prima proposizione
rimanda all’idea weberiana di una metodologia intesa
come critica della ragione sociologica, su cui qui non mi
soffermo. La seconda proposizione rimanda all’idea di
una critica alla ragion pratica, che invece vorrei appro-
fondire. Diversamente da Kant, Weber non ci dà un
criterio per riconoscere quando un’azione è giusta. Egli
rifiuta l’universalismo formale di Kant. Ma allo stesso
tempo, rifiuta l’idea che la realtà sia senza senso. La que-
stione è posta in modo molto più radicale. All’uomo non
è dato sapere con certezza: se qualcuno sostenesse che il
mondo è senza senso avrebbe accesso a una conoscen-
za altrettanto falsa di chi ritiene di avere accesso a tale
senso. Il nichilista e l’uomo senza Dio commettono lo
stesso errore del metafisico e dell’uomo di fede. Questo
mi sembra il cuore tragico e stimolante della concezione
weberiana, una concezione che lo avvicina, secondo me,
all’esistenzialismo. Abbiamo così due modelli di razionalità: il primo
identificabile con il binomio “chiarezza” e “coerenza”;
il secondo con quello “chiarezza” e “adeguatezza”. Sono
due modelli estremi, che nella loro purezza si escludo-
no: faccio così perché voglio essere coerente con ciò che
credo, e poi vada come vada; oppure, faccio così perché
è il modo più efficace per raggiungere un fine che mi
sono posto. Dentro questa matassa concettuale, vorrei
sottolineare tre aspetti. 1. Il primo modello consente di
isolare ogni singola azione dal contesto in cui è inseri-
ta: ogni azione è un assoluto di cui può essere identifi-
cato il giudizio assoluto che ne è alla base. L’adeguatezza
invece, diversamente dalla coerenza, implica un’atten-
zione alla catena di scopi tra loro connessi, al contesto
in cui l’azione è inserita. 2. Di conseguenza, la coerenza
può essere testimoniata; l’efficacia misurata. Nel primo
caso, non importano le conseguenze e il giudizio razio-
nale si esercita, in modo assoluto, rispetto alla fedeltà in
un valore incondizionato. Nessuna misurazione è possi-
bile perché non c’è alcuna attenzione alle conseguenze. 3. Infine, c’è razionalità laddove si sente la necessità di
esprimere un giudizio rispetto ad alternative: così come
è possibile pensare che possano esserci diversi mezzi uti-
li per raggiungere uno scopo, allo stesso modo esistono
molti scopi compatibili con lo stesso valore. Se le azioni
fossero senza alternativa, si ricadrebbe inevitabilmente
nell’esempio dell’ombrello e della pioggia. In che direzione allora si esercitano la chiarezza e la
consapevolezza razionali? Per Weber, sempre a mio pare-
re, il giudizio di razionalità si esercita sempre su di un
rapporto: razionale è la consapevolezza che riusciamo a
inserire nel giudizio che diamo intorno ad un rapporto. Più o meno razionale rispetto allo scopo è il giudizio che
diamo a proposito del rapporto tra un mezzo e un fine;
e, ma qui aumenta il grado di interpretazione persona-
le, più o meno razionale rispetto al valore è il giudizio
che diamo nei confronti del rapporto tra uno scopo e
un valore. Abbiamo quindi tre termini distinti – mezzo,
fine e valore –, così da poter riconoscere due rapporti. Il concetto di fine fa da ponte, collegando, in maniera
molto elastica, mezzo a valore. I due diversi modelli di
razionalità si distinguono così piuttosto nettamente. La razionalità rispetto al valore è caratterizzata dal
requisito della coerenza rispetto ad un valore incon-
dizionato: «è sempre un agire secondo “imperativi” o
in conformità a “esigenze” che l’agente crede gli siano
poste» (Ivi: 22-23). L’agire è razionale per due motivi:
1. perché è chiaro ed evidente il senso intenzionato; 2. perché si ritiene che il senso sia coerente rispetto ad un
valore sentito come imperativo, cioè esterno all’attore. Quest’ultimo attiva un giudizio di coerenza sul rappor-
to esistente tra la sua azione, cioè lo scopo che intende
conseguire, e il valore di riferimento, ma non certo un
giudizio di razionalità sul valore in sé, che non è deriva-
to dalla ragione. Il giudizio di razionalità si basa su un
rapporto, è può essere collocato all’interno di un conti-
nuum, che va da un massimo ad un minimo di coerenza. Pur essendo modelli contrapposti, sono però stretta-
mente intrecciati. Ad esempio, ha senso essere del tutto
sinceri se poi scopro che non serve a nulla? E, al contra-
rio, che senso ha conseguire uno scopo per poi scoprire
che mi è del tutto indifferente? Con le parole di Weber Dal punto di vista della razionalità rispetto allo scopo, la
razionalità rispetto al valore è sempre irrazionale – e lo
è quanto più eleva a valore assoluto il valore in vista del
quale è orientato l’agire […] (la pura intenzione, la bellez-
za, il bene assoluto, l’assoluta conformità al dovere). Ma
l’assoluta razionalità rispetto allo scopo è anche soltanto
un caso limite, di carattere essenzialmente costruttivo. (Ibi-
dem, corsivi miei). Nel caso dell’agire razionale rispetto allo scopo, il
senso è determinato «da aspettative dell’atteggiamento
di oggetti del mondo esterno e di altri uomini, impie-
gando tali aspettative come “condizioni” o come “mezzi” 92 Ambrogio Santambrogio Ambrogio Santambrogio Il carisma, in quanto espressione dello straordina-
rio, e questa volta in sintonia con Durkheim, è in stret-
ta sintonia con il sacro, con quella dimensione trascen-
dente da cui deriva il senso ultimo cui si affida il senso
quotidiano. L’evoluzione del carisma in Occidente segue
una propria particolare traiettoria, che Weber ricostru-
isce. Esso subisce un progressivo processo di secolariz-
zazione e di razionalizzazione, due tendenze tra di loro
interconnesse. Le figure carismatiche fondatrici di civiltà
sono il mago, l’eroe, il Re, il profeta in un percorso che
dalla magia porta prima a credenze religiose e, infine,
nella nostra modernità, a quel processo di “illuminazio-
ne carismatica della ragione” che costituisce il punto di
arrivo delle trasformazioni del carisma e, di conseguen-
za, delle forme della società occidentale. Appare qui la grande tensione interna alla razionali-
tà umana: dal punto di vista della coerenza, è irrazionale
agire in modo efficace; dal punto di vista dell’efficacia, è
irrazionale agire con coerenza. Per di più, tale irraziona-
lità aumenta tanto più i due modelli vengono usati indi-
pendentemente: un valore assoluto cancella l’attenzione
per i mezzi; un mezzo potentissimo cancella l’attenzio-
ne al valore. L’azione umana diviene in entrambi i casi
totalmente cieca, e l’eccesso di uno dei due modelli di
razionalità porta alle medesime conseguenze: la totale
irrazionalità. Nel primo caso, predomina l’irrazionali-
tà del “devo”; nel secondo caso, quella del “posso”, due
diverse forme di totale cecità. Per riassumere. Secondo Weber, la razionalità è un
tenue lume dentro l’oscura insensatezza del mondo. Le
nostre azioni sono per lo più guidate da forze irraziona-
li che non sappiamo gestire; oppure, per lo più, da solide
consuetudini che ci nascondono i problemi con soluzio-
ni collettivamente confezionate. L’azione profondamente
umana, tipica dell’uomo maturo, prova ad usare questo
lume. Essa si muove tra Scilla e Cariddi, tra una mera
attenzione alla efficacia del mezzo e una mera osservan-
za verso i dettami che provengono dai valori, tentando di
conciliare razionalmente le due tendenze e le spinte che da
esse provengono. Dare un senso consapevolmente chiaro
alle proprie azioni implica la capacità di gestire razional-
mente responsabilità e convinzione. All’interno di questo
sfondo, appare la domanda chiave: ognuno di noi è in gra-
do di dare (razionalmente) un senso alle proprie azioni? L’azione del carisma nella storia umana è la risposta “irra-
zionale” weberiana a questa fondamentale domanda. Le società primitive fondate sulla magia e poi quelle
legate alle grandi religioni di redenzione devono le loro
caratteristiche essenziali alla specifica natura del carisma
che sta alla loro base: dal mago si passa al profeta, dalla
magia alla religione, in un passaggio che porta con sé una
prima forma di razionalizzazione. Allo stesso modo, la
nostra società attuale è ricostruibile, nei suoi tratti deci-
sivi, attraverso il modo con cui al suo interno sono anco-
ra possibili spazi per l’azione di questa qualità soggettiva
“sovrannaturale”. Essa rimane centrale perché viene indi-
viduata da Weber come l’unica capace di dare un senso al
mondo. Come abbiamo brevemente sopra visto, infatti, la
ragione non sembra in grado di rispondere alle domande
fondamentali intorno al senso. E, soprattutto, non è in
grado di farlo la ragione scientifica e tecnica. Non è da tutti riuscire a dare senso ad una realtà
che ci appare nella sua profonda irrazionalità: il cari-
sma porta, insieme al senso, ordine e giustizia, cioè
una visione morale capace di guidare l’azione collettiva. Espressioni come quelle di causa, dedizione, respon-
sabilità, fede, dovere, obbedienza, seguito, fedeltà ecc. trovano il loro senso solo all’interno di questo quadro
generale. Insisto su di un punto: Weber non concepisce
la ragione come una facoltà capace di dirci quale del-
le molteplici soluzioni storiche prodotte dal carisma al
problema del senso sia quella corretta; e neppure la ritie-
ne capace di sostituire a tali soluzioni una visione uni-
versalistica, di tipo formale, come fa, ad esempio, Kant. Semplicemente definisce le condizioni ontologiche, si
potrebbe quasi dire, entro le quali si collocano le azioni
“razionali” degli uomini: esse sono tali se possiedono un
senso assunto consapevolmente e chiaramente teso alla
realizzazione, attraverso mezzi più o meno adeguato, di
fini meritevoli di essere perseguiti, cioè più o meno coe-
renti rispetto a valori di riferimento. 2. Carisma e democrazia. Come è ampiamente noto,
il senso intenzionato ha per Weber una natura per lo più
irrazionale, e il tenue lume della ragione solo raramen-
te viene opportunamente usato. Ripercorro brevemente
pagine conosciute e concetti noti. Carisma dei valori e carisma dei fini Ciò
che è all’opera non è la forza dispiegata della tecnologia
scientifica, che dissolve la capacità della magia, o della
religione, di intervenire strumentalmente nel mondo, per
adattarlo a fini umani. Si tratta, piuttosto, della razionale
costruzione di una soggettività umana indipendente, per
il momento ancora fondata su di un’etica religiosa. Essa
impone una “vita metodica”, basata su “motivi costan-
ti” che provengono dalla dottrina. Rispetto ai primitivi
e non razionalizzati fondatori di religioni, ai maghi e ai
profeti dell’antichità, si tratta di fondatori parziali, cioè
già parzialmente razionalizzati e razionalizzanti. L’ele-
mento a mio parere decisivo è che una parte della dottri-
na che praticano è sentita come indisponibile al progetto
di Riforma, il quale si svolge all’interno di quella dimen-
sione di indisponibilità. Mantenendo intatto il quadro
dei valori di riferimento, quelli indicati dalla Bibbia per
intenderci, promuovono un nuovo sistema di fini capace
di entrare in sintonia con quei valori. Il loro è un nuo-
vo modo di vivere la stessa religione, ma così radicale
da segnare uno spartiacque profondo con il mondo tra-
dizionale di riferimento. Fondatori ed eretici, al tempo
stesso, come si diceva: l’aspetto di fondazione, questo il
punto che Weber fa emergere, è caratterizzato da una
dose di razionalità assente negli antichi fondatori cari-
smatici, che li fa essere tali in modo radicalmente nuovo. In questa stessa direzione, è interessante riflette-
re brevemente sull’esperienza dei Riformatori, come
Lutero, Zwingli, Bucero, Calvino, ecc. Sono personaggi
estremamente carismatici che, a mio parere, segnano
una tappa fondamentale del processo di razionalizza-
zione del carisma. Essi sono al tempo stesso “fondatori”
ed “eretici”. Ciò che fondano è una esperienza di rottura
che però ritiene di collocarsi, ricostituendone l’autenti-
cità, all’interno di quella stessa tradizione da cui si fuo-
riesce. Come scrive Lutero, «sarebbe ottimo se il diritto
canonico fosse distrutto dalla prima alla ultima sillaba
[…]; per quel che ci abbisogna la Bibbia mostra a suf-
ficienza come comportarsi in ogni circostanza» (Lutero
1978 [1520]: 208). In questa breve citazione sono incor-
porate le due dimensioni: quella di radicale innovazio-
ne e quella di radicale conservazione. Infatti, secondo
Weber, «non un eccesso, ma un difetto di dominio reli-
gioso sulla vita fu quello che trovarono vituperevole i
riformatori che sorsero nei Paesi allora più economica-
mente sviluppati» (Weber 1976a [1904-1905]: 111). Si tratta di un carisma che non ha ancora nulla di
democratico. Carisma dei valori e carisma dei fini Carisma dei valori e carisma dei fini zione, ma, al contrario, da quella di autenticità religio-
sa. Sempre seguendo Weber «il Dio del calvinismo non
esigeva, dai suoi, singole opere buone, ma una santità
di opere elevate a sistema […] La prassi etica dell’uomo
medio fu privata così del suo carattere non pianificato
e a-sistematico e fu trasformata in una condotta di vita
metodica e conseguente» (Ivi: 220). In questo modo, «l’a-
scesi puritana, come ogni ascesi razionale, agiva nel sen-
so di rendere capace l’uomo di mantenere e di far valere
di contro agli “affetti” i suoi “motivi constanti” […] in
modo da formare una “personalità” nel senso formale e
psicologico della parola» (Ivi: 223-224). Questa domanda è tipicamente moderna, ed è essa stessa
un prodotto tipico dell’”illuminazione carismatica della
ragione”. Nel mondo magico, religioso o dominato dal-
la metafisica, non avrebbe nessun senso, anzi non sareb-
be neppure concepibile. I processi di secolarizzazione e
di razionalizzazione portano alla luce quella dimensione
di autonomia soggettiva che magia, religione e metafisica
lasciavano per lo più nascosta, seppellita sotto la forza di
una sfera morale che si imponeva collettivamente trami-
te la sua oggettività. Il momento carismatico è quello ori-
ginario e fondativo: da lì in avanti, il carisma si esercita
all’interno di un quadro valoriale relativamente immodi-
ficabile, garantendone la permanenza e la consistenza. La
razionalità rispetto al valore delle intenzioni umane, da
questo momento in poi, si esercita nei confini di valori
indiscussi, e l’autonomia individuale viene nella sostanza
meno. Per fare un esempio, le eresie medioevali, e la forza
carismatica che in esse si attiva, non mirano a una radica-
le messa in discussione dell’ordine oggettivo della morali-
tà e dei valori condivisi, ma piuttosto rivendicano un rap-
porto di autenticità con quei valori che ritengono essersi
sbiadito, o addirittura perduto. I vescovi catari3, ma in un
certo senso anche San Francesco, per fare qualche esem-
pio, non fondano nuove religioni: pensano piuttosto che
qualcosa di fondamentale della religione condivisa sia
andato smarrito e debba essere ritrovato. È questa caratte-
ristica che li etichetta come – diversamente – eretici.l Quello che mirabilmente Weber descrive è un pro-
cesso di razionalizzazione che riguarda non tanto il rap-
porto tra mezzi e fini, bensì quello tra fini e valori. 3 Ricordo, ad esempio, i nomi di Arnaldo Oth, Guidalberto di Castres e
Benoît de Termes, che nel 1207 si scontrarono a Montreal con Domeni-
co de Guzmán in una accanita disputa teologica. Il carisma – in quan-
to risposta irrazionale alla domanda intorno al senso
– è l’espressione dello straordinario che fonda l’ordina-
rio. Implica un cambiamento interiore, il cui modello è
la μετάνοια proposta da Giovanni Battista nei Vangeli,
capace di indurre un cambiamento profondo. In questo
è l’autentica “potenza rivoluzionaria” (Cavalli 1981) della
storia. Diversamente da Durkheim, per il quale il cam-
biamento sociale è la reazione ad una crisi attraverso un
moto di effervescenza collettiva, Weber trova nella forza
della soggettività carismatica la potenza capace di pro-
durre nuovi valori, un mondo nuovo e, di conseguen-
za, nuove forme collettive, in sintonia con l’idea per cui
«solo la tensione morale suscitata dal rapporto con mete
che sono oltre il quotidiano, sopra “l’indegnità creatura-
le”, crea civiltà» (Ivi: 180). 2. Carisma e democrazia. Come è ampiamente noto,
il senso intenzionato ha per Weber una natura per lo più
irrazionale, e il tenue lume della ragione solo raramen-
te viene opportunamente usato. Ripercorro brevemente
pagine conosciute e concetti noti. Il carisma – in quan-
to risposta irrazionale alla domanda intorno al senso Se la condizione è la medesima per tutti, perché solo
pochi dotati di carisma riescono dove gli altri falliscono? 93 Carisma dei valori e carisma dei fini Però consente di identificare almeno due
diverse modalità attraverso cui il carisma può esercitar-
si: quello fondativo, che si muove senza alcun vincolo; e
quello eretico, che si muove, con diversi gradi di autono-
mia, all’interno di un quadro valoriale dato, definendo
un diverso sistema di fini perseguibili coerente con quel
quadro. La mia proposta è di chiamare il primo “carisma
dei valori” e il secondo “carisma dei fini”. L’esperienza
della Riforma sta ancora a cavallo tra le due forme, ma
segnala un’importante trasformazione epocale che va
nella progressiva affermazione della seconda. La razionalizzazione di cui sono protagonisti, para-
dossalmente, muove non da una esigenza di secolarizza- 94 Ambrogio Santambrogio normativo contiene al suo interno un sistema di valo-
ri più o meno coerente ed articolato, si può facilmente
dedurre come anche il più radicale conformismo apra
sempre e comunque alla possibilità di testimoniare quei
valori in modi diversi ed eterogenei, identificando volta
a volta sistemi relativamente coerenti di fini eterogenei. Se non esistesse questa possibilità, l’ordine normativo
di riferimento, una volta costituito, cancellerebbe total-
mente la possibilità della scelta umana: una volta che
si sia espresso, il carisma fondativo soffocherebbe com-
pletamente ogni residuo possibile di autonomia umana. Si può essere cristiani in molti modi, perché la propria
fede può essere coerentemente testimoniata lasciando un
riconoscibile spazio alle singole soggettività. Nel nostro mondo attuale, ma anche in quello di
Weber, è difficile trovare all’interno della cultura occiden-
tale fondatori di religioni, nel senso carismatico antico. Il
processo di razionalizzazione trasforma profondamente
il modello di razionalità, sia nella forma della razionalità
strumentale, cioè quella che sindaca sul rapporto tra mez-
zi e fini; sia nella forma della razionalità da valore, cioè
quella che sindaca sul rapporto tra fini e valori. i
Nel primo caso, la razionalizzazione è spinta dal
processo tecnico-scientifico; nel secondo, dal processo
di relativizzazione. I due momenti sono naturalmen-
te tra di loro connessi – poiché entrambi espressione
di una radicale secolarizzazione del mondo – ma assu-
mono aspetti diversi. Da un lato, abbiamo un aumento
di saperi oggettivi, che disincantano il nostro rapporto
con la natura. Il fulmine non è più l’espressione dell’ira
divina, ma un insieme di scariche elettriche sulle quali
l’uomo può intervenire tecnicamente. Dall’altro, abbia-
mo il venir meno dell’oggettività etica in cui gli uomi-
ni antichi vivevano. Carisma dei valori e carisma dei fini Nell’interpretazione weberiana, eroi
e profeti producono un’interpretazione mitico-religiosa
del mondo oggettiva e opaca per chi vive al suo interno. Oggi questa opacità è venuta meno, e appare la natu-
ra soggettiva della autonoma capacità umana di dare
un senso al mondo. Il carisma era nel passato la qualità
che solo alcuni, dotati di una forza misteriosa e potente,
possedevano, in grado di dare una risposta alla questio-
ne del senso. Oggi ognuno deve essere un po’ carisma-
tico per sé stesso: apparteniamo ad un mondo che non
può più contare su di una soluzione a tutti comune. Non
possediamo più una morale oggettiva, da tutti condivisa. In un mondo così fatto, la dimensione della scelta acqui-
sta uno spazio inconcepibile nel passato, e l’autonomia
soggettiva si evidenzia con maggiore estensione rispetto
agli stretti spazi in cui era tradizionalmente confinata. Quanto detto può essere utile per ragionare sul rap-
porto tra carisma e democrazia. La democrazia non è un
ordine che si costituisce carismaticamente. Diversamente
dalle religioni del passato, l’azione del carisma si eserci-
ta semmai all’interno del quadro democratico una volta
che questo si sia andato progressivamente affermando. I fondatori di democrazie – si pensi ai padri fondatori
della democrazia americana; ma anche ai nostri padri e
madri costituenti – non sono carismatici nel senso degli
antichi profeti. La democrazia porta con sé un insieme
di regole del gioco indisponibili al carisma. Queste rego-
le acquisiscono progressivamente l’aspetto formalizzato e
burocratizzato delle procedure, ma non bisogna dimen-
ticare che proprio in quelle regole si cela la natura pro-
fondamente democratica del vivere in comune: il fatto,
cioè, che una dimensione consistente di quel vivere viene
tolto dalla disponibilità dell’azione soggettiva. Anche in
democrazia esiste una moralità oggettiva, solo che non
è prodotta soggettivamente dall’azione del carisma, ma
da un processo lungo e articolato che ne fa piuttosto una
costruzione collettiva. gi
Riprendo brevemente un punto importante ai fini
della mia argomentazione. Mi sembra possibile introdur-
re una importante distinzione tra il momento fondativo
dell’ordine morale condiviso e la sua evoluzione interna,
la sua concreta storia, fatta da tutte le diverse modalità
attraverso cui quell’ordine si concretizza e si sviluppa. Carisma dei valori e carisma dei fini L’autonomia dei singoli, anche degli individui poco o per
nulla carismatici, deve essere democraticamente rispetta-
ta e salvaguardata: dentro al pluralismo dei valori demo-
cratici – garantito da valori e procedure indisponibili –
si può così esercitare la libera scelta di ognuno, che dà
più o meno liberamente un senso alle proprie azioni. Con le parole di Weber: [c]on la vittoria del razionalismo giuridico formale è
apparso in Occidente, accanto al tipo di autorità tradizio-
nale, il tipo dell’autorità legale […] non la sottomissione in
virtù della fede e della dedizione a persone dotate di cari-
sma, cioè profeti ed eroi, né in virtù della sacra tradizio-
ne e del rispetto verso un signore personale determinato
dall’ordinamento tradizionale e, eventualmente, verso i
suoi vassalli […] bensì il vincolo impersonale all’oggettivo
“dovere di ufficio” […] determinato da norme poste razio-
nalmente […] in modo così stabile e tale che la legittimità
dell’autorità si trasforma in legalità della norma generale,
ideata in conformità allo scopo, istituita e proclamata in
maniera formalmente corretta (Weber 1976a [1904-1905]:
364, corsivi miei). L’autentico “dominio carismatico” aveva bisogno
di essere testimoniato, di essere provato da una verifica
fattuale: la sua azione era qualcosa di reale, e come tale
empiricamente verificabile, anche se, naturalmente, non
in senso scientifico. Nessun leader carismatico democra-
tico è responsabile dei valori democratici nel senso cari-
smatico appena descritto: c’è qualcosa, in estrema sin-
tesi, che non dipende da lui. Egli non è responsabile del
quadro democratico, al cui interno la sua azione si inse-
risce: semmai lo è della sua qualità, del suo buon funzio-
namento e, soprattutto, dei fini che egli vuole perseguire
all’interno di quel quadro. q
q
In questa direzione, il carisma democratico è un
carisma dei fini, e non dei valori. Se ci pensiamo, non
è neppure produttore di quei valori che costituiscono il
pluralismo interno alle democrazie. La sua azione può
mirare certo a riattivare valori che si sono andati spe-
gnendo, cosa che avviene soprattutto nei momenti di
crisi. In linea di massima, però, sempre all’interno di
un quadro normativo e procedurale che non può esse-
re messo in discussione, proprio perché profondamente
interconnesso con quei valori di riferimento. Carisma dei valori e carisma dei fini Pur rimanendo in qualche modo fedele a sé stesso, man-
tenendo sempre cioè una sua riconoscibilità, qualsiasi
ordine normativo – che non coincide naturalmente con
l’ordine cognitivo, dal momento che implica un aspetto
pratico – non costituisce mai un monolite senza alter-
native. Al suo interno, si apre uno spazio – più o meno
ampio e ricco – di alternative, anche valoriali, riguar-
danti le specifiche modalità con cui si può concretamen-
te realizzare. Come sopra si diceva, esiste un numero
praticamente infinito di fini potenzialmente coerenti con
un valore di riferimento. Dal momento che un ordine Al centro, naturalmente, stanno quelle procedu-
re alla base del sistema della democrazia liberale. Ma si
tratta anche, e non meno significativamente, di quell’in-
sieme di diritti civili, politici e sociali che via via pro-
ducono una dimensione che si sottrae alla disponibilità
dell’azione di soggetti più o meno carismatici, almeno
sintantoché la loro azione si mantiene all’interno del
sistema democratico stesso. Il carisma democratico,
in sintesi, agisce all’interno di un quadro di riferimen-
to, fatto di procedure e di valori, che non può diventare
oggetto di intervento della potenza carismatica. Essa non
è più una “potenza rivoluzionaria”. Il carattere fondativo
sparisce, poiché nessuna democrazia può pensare di ave-
re un profeta come suo fondatore. La democrazia è, in
sintesi, un regime articolato di procedure e di valori che
si afferma progressivamente “anche” attraverso l’azione
di molti soggetti carismatici, ma senza che nessuno di 95 Carisma dei valori e carisma dei fini [i]l monarca cinese […] come tutti gli autentici signori cari-
smatici, era un monarca per grazia di Dio, non nel comodo
senso dei regnanti moderni che, in base a questo attributo,
pretendono di dover rispondere solo a Dio, e cioè in pra-
tica a nessuno, delle follie commesse, ma nell’antico senso
genuino del dominio carismatico. Ciò significa […] che egli
doveva legittimarsi come “Figlio del Cielo” […] attraverso
un solo modo: il benessere del popolo. Se non era in gra-
do di realizzarlo, gli mancava il carisma. (Weber 1976b
[1916]: 411) costoro possa pensarsi come capace di produrre il sen-
so che costitutivamente sta alla base dell’agire collettivo. Carisma dei valori e carisma dei fini La tendenza è
quella di isolare un tema, approfondirlo nel migliore dei
modi, lasciandolo però per lo più isolato dal contesto in
cui è inserito. Si corre così il rischio di sapere quasi tutto
di quasi niente. Non si tirano più i fili. Non si riescono
più a vedere le connessioni che esistono tra i diversi temi
che facciamo oggetto dei nostri studi. Tutto ciò è peggio-
rato dal fatto che molta di questa ricerca empirica usa
la teoria come semplice orpello iniziale, come un muto
e necessario abbellimento che non dialoga con il resto. Si tratta di una deriva empirista che trovo estremamente
pericolosa. Naturalmente, non ho nulla contro la ricerca
empirica. Le scienze sociali hanno la loro caratteristica
fondamentale nella dialettica strettissima tra idee e real-
tà, tra ciò che pensiamo del mondo e le forme che esso
assume. Proprio per rivitalizzare questa fondamentale
dialettica ritengo sia fondamentale leggere con attenzio-
ne i nostri maestri, quelli più lontani e quelli più vicini. E Luciano Cavalli è sicuramente uno di loro. Infine, Cavalli è anche però attento nel sottolineare
che «il leader di cui parlo si contrappone al leader di pura
immagine ed eterodiretto che l’età mediatica […] fareb-
be prevalere» (Cavalli 1999: 67). Il carisma non è solo
esteriorità, apparenza: la sua qualità sta nella capacità di
indicare, tra le molte possibilità, un percorso fattibile,
un’alternativa reale, coniugando responsabilità e convin-
zione. Nulla a che vedere perciò con i molti capi e capetti
che si aggirano nell’attuale panorama politico in cerca di
consenso e, soprattutto, niente a che vedere con la logi-
ca meramente mediatica che è alla base della costruzione
della loro “visibilità”. Carisma e personalizzazione della
politica non necessariamente coincidono (Calise 2007;
Marletti 2007; Bordignon 2013; Campus 2016). Per concludere. Anche in democrazia, il carisma
mantiene la sua caratteristica e intrinseca natura sogget-
tiva, espressione della capacità - e della necessità - uma-
na di dare un senso alla propria esperienza nel mondo. In democrazia, come risultato dei processi di razionaliz-
zazione della cultura occidentale, esso però subisce due
vincoli. Il primo proviene dall’alto: tutta una serie di
valori e di procedure democratiche è sottratta alla sua
disponibilità. Agisce all’interno di un quadro normati-
vo dato, che non può mettere radicalmente in discussio-
ne. Carisma dei valori e carisma dei fini Egli, ad esempio, scrive che 96 Ambrogio Santambrogio [n]egli ultimi scritti di Weber il leader carismatico della
democrazia moderna, secolarizzata, non è più, secondo il
paradigma giudaico-cristiano, l’inviato o lo strumento di
Dio. È un capo politico di grande visione e ferma convin-
zione […] È “l’uomo di fiducia delle masse”, che gli affida-
no le loro sorti […] Come esempio di questo in una moder-
na democrazia, si può forse ricordare l’ascesa alla presiden-
za di Roosevelt nel pieno della grande depressione. Subito
Roosevelt prende provvedimenti di vario tipo, a getto con-
tinuo, che sembrano dimostrare la volontà e la capacità di
affrontare la crisi (Cavalli 1999: 53). può ignorare quella “leadership diffusa”, espressione di
soggetti sociali che si sono fatti sempre più autonomi
e consapevoli rispetto alle masse, di cui parla Cavalli. Non può farlo perché il loro protagonismo è sostenuto
e incoraggiato dal quadro normativo di riferimento. La
democrazia produce partecipazione e si nutre di parteci-
pazione, nelle innumerevoli forme che essa storicamen-
te si è data e potrà darsi. Il carisma democratico è così
compiutamente un carisma dei fini e non più costitutivo
di valori. In fondo, l’unico valore che promuove, in modi
che possono essere i più diversi ed eterogenei, è il valore
più autenticamente e profondamente democratico: lo svi-
luppo dell’autonomia degli individui, il nostro personale
carisma. Appunto: Roosevelt «prende provvedimenti di vario
tipo», ma sempre all’interno del quadro di riferimento
della democrazia americana. Inoltre, sempre per Cavalli, un altro, ed egualmen-
te importante, aspetto diventa sempre più decisivo. In
democrazia, «la leadership di vertice richiede una cul-
tura democratica». Essa «è importante perché garanti-
sce la maggiore collaborazione dei cittadini e, insieme, il
loro efficace controllo sui leader di vertice», producendo
«quella leadership diffusa di ispirazione riformista e rea-
lizzatrice che tante volte si è rivelata preziose in antiche
democrazie» (Cavalli 1999: 69, corsivi miei).i 3. Conclusioni. Quello che ho appena provato a fare
è un semplice tentativo, spero almeno plausibile, in sin-
tonia con una certa idea di sociologia che ho imparato
anche da Luciano Cavalli. A questo proposito, vorrei
concludere con una breve osservazione. Leggendo la
sociologia di oggi, e non solo quella italiana, ho spesso la
spiacevole sensazione che molta parte di essa, alle volte
anche le cose migliori, siano senza respiro. Carisma dei valori e carisma dei fini Non ci può
essere autonomia soggettiva, tanto per fare un esempio,
senza libere elezioni, un sistema rappresentativo funzio-
nante, il rispetto del pluralismo, l’equilibrio della divi-
sione dei poteri, un sistema politico pluripartitico, una
società civile attiva e funzionante, ecc. ecc. All’interno
di queste regole, il politico democratico carismatico arti-
cola un sistema di fini a suo modo coerente con il qua-
dro democratico al cui interno si svolge la sua azione,
quadro che consente anche altri, e persino contrapposti,
sistemi alternativi di fini politicamente raggiungibili. Banalmente, una democrazia è compatibile con sistemi
diversissimi di tassazione; con una plurale visione del
ruolo della religione nella società; con diverse e incom-
patibili concezioni della sessualità e dei diritti che porta
con sé; con alternative concezioni del lavoro; e così via. Ed è compatibile con una pluralità di capi carismatici
diversamente orientati, cosa impossibile per l’autentico
carisma antico. Il “signore personale” potrebbe anche non esse-
re carismatico: ciò che continua a caratterizzarlo è la
dimensione personale del potere, la sua qualità soggetti-
va e non oggettiva. La natura soggettiva del potere vie-
ne progressivamente meno, invece, mano a mano che si
sviluppa una “legalità della norma generale”, sottratta
alla discrezionalità dei soggetti. Il fatto che sia “ideata in
conformità allo scopo”, cioè la natura razionale del suo
aspetto di legalità, sta a significare che la dimensione dei
valori fondativi è sottratta alla legalità, e quest’ultima
non può sindacare sulla sua legittimazione, che rimane
del tutto esterna e data. La natura della legittimazio-
ne democratica non è legale, ma neppure carismatica. Anche se molti soggetti carismatici possono aver con-
tribuito alla sua costruzione, questa qualità carismatica
si stempera progressivamente in una forma di legitti-
mazione che appare come indipendente dalle soggettivi-
tà e, soprattutto, dalle qualità carismatiche dei soggetti
che hanno contribuito alla sua costituzione. Il carisma
di Washington non è il carisma di un profeta, e la sua
qualità carismatica viene assorbita da una legittimazio-
ne che, in quanto democratica, oggettivandosi si autono-
mizza dai soggetti che hanno contribuito a produrla. Inoltre, la qualità carismatica del dominio perso-
nale poteva – e doveva – essere sottoposta alla verifica
empirica della sua efficacia: il carisma è responsabile,
completamente responsabile. Tanto per fare un esempio
significativo: La questione mi sembra puntualmente posta e
affrontata da Cavalli. Carisma dei valori e carisma dei fini Di fatto è quel processo che toglie progressivamente
al carisma la sua qualità mitico-religiosa di fondatore
di comunità, un processo al cui interno i riformato-
ri come Lutero costituiscono un importante passaggio
intermedio. Il secondo vincolo proviene dal basso: non Citation: Antonio Costabile (2023) Cari-
sma del sangue e familismo: un rap-
porto controverso. SocietàMutamen-
toPolitica 14(27): 99-104. doi: 10.36253/
smp-14339 Citation: Antonio Costabile (2023) Cari-
sma del sangue e familismo: un rap-
porto controverso. SocietàMutamen-
toPolitica 14(27): 99-104. doi: 10.36253/
smp-14339 Antonio Costabile Copyright: © 2023 Antonio Costabile. This is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. Il presente contributo analizza le possibili relazioni tra carisma del san-
gue e familismo, con alcuni specifici riferimenti alla storia italiana. Esiste,
alla luce dell’insegnamento di Max Weber e delle sollecitazioni che da esso
provengono anche attraverso i suoi studiosi, un collegamento scientificamen-
te rilevabile e sociologicamente apprezzabile tra carisma del sangue, famili-
smo sociale e familismo politico? p
La domanda è complessa perché ha molti risvolti, che possono essere
inquadrati facendo tesoro di una riflessione di Luciano Cavalli: Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. [a]bbiamo visto il concetto di carisma uscire con Weber dal chiuso della storia del
cristianesimo per diventare oggetto di analisi approfondita e, infine, uno strumen-
to fondamentale di indagine sociologica. Weber ricerca le condizioni e i modi in
cui il carisma si manifesta, i processi da esso promossi, le sue oggettivazioni e le
sue trasformazioni; studia il suo ruolo nel mutamento; si interroga sul suo avveni-
re (Cavalli 1981: 180-181). Competing Interests: The Author(s)
declare(s) no conflict of interest. 1. Partendo dai concetti di carisma e di familismo ritorniamo innanzi-
tutto a Weber, il quale, come noto, sostiene che: [i]l potere genuinamente carismatico non conosce né principi tradizionali né rego-
lamenti astratti e neppure una produzione [razionale] del diritto. Il suo diritto
[oggettivo] è l’emanazione concreta di un’esperienza vissuta, assolutamente per-
sonale, della grazia celeste e della forza divina degli eroi; esso comporta un rifiuto
del vincolo a ogni ordinamento esterno […] esso si atteggia perciò in modo rivo-
luzionario, sovvertendo ogni cosa e rompendo con qualsiasi norma tradizionale o
razionale (Weber 1981 [1922]: 222). L’autentico carisma è antitetico a qualsiasi regola preesistente, quindi
alla regola tradizionale come pure alla regola razional-legale moderna. RIFERIMENTI BIBLIOGRAFICI Bordignon F. (2013), Il partito del capo, Maggioli, Santar-
cangelo di Romagna.h Brubaker R. (2006 [1984]), The Limits of Rationality. An
Essay on the Social and Moral Thought of Max Weber,
Routledge, London and New York. g
Calise M. (2007), Il partito personale, Laterza, Roma-Bari. Campus D. (2016), Lo stile del leader, il Mulino, Bologna. 97 97 Carisma dei valori e carisma dei fini Cavalli L. (1981), «Il carisma come potenza rivoluzionar-
ia», in P. Rossi (a cura di), Max Weber e l’analisi del
mondo moderno, Einaudi, Torino, pp. 161-188.f – (1999), «Leadership personale e diffusa», in Marletti
C. (a cura di), Politica e società in Italia, V. I, Cam-
biamento politico e identità sociali, FrancoAngeli,
Milano, pp. 50-72. Lutero M. ([1520] 1978), «Alla nobiltà cristiana di nazi-
one tedesca», in Id., Scritti politici, UTET, Torino, pp. 123-224. Marletti C. (a cura di) (2007), Il leader postmoderno,
FrancoAngeli, Milano.i Santambrogio A. (2008), «Valori, fini, mezzi. Un’analisi
del concetto weberiano di razionalità», in G. Bettin
Lattes e P. Turi (a cura di), La sociologia di Luciano
Cavalli, Firenze University Press, Firenze, pp. 353-
372. Weber M. (1976a [1904-1905]), «L’etica protestante e lo
spirito del capitalismo», in Id., Sociologia delle religio-
ni, UTET, Torino, pp. 107-324. – (1976b [1916]), «Confucianesimo e taoismo», in Id.,
Sociologia delle religioni, UTET, Torino, pp. 367-582. – (1981 [1922]), Economia e società, Edizioni di Comu-
nità, Milano. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Tutta-
via, per spiegare cosa accade al tramonto della fase straordinaria guidata dal
capo carismatico, cioè al rientro nella normalità quotidiana, lo stesso Weber
segnala come nelle società del passato il più delle volte si registrava una tran-
sizione dall’eroe alla successione dinastica, in nome del carisma del sangue. Nelle società moderne, in nome dell’illuminazione carismatica della ragione,
si è registrata la transizione al primato della norma impersonale e del ruo-
lo professionale. In entrambi i casi – del carisma del sangue e del carisma SOCIETÀMUTAMENTOPOLITICA 14(27): 99-104, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14339 100 Antonio Costabile sistenza o addirittura la prevalenza dei legami familiari
e parentali nella produzione e riproduzione dei poteri e
nella selezione delle cariche. di ufficio – si tratta di forme spurie del carisma; d’altra
parte, le fasi straordinarie sono rare, mentre le fasi ordi-
narie sono comuni. Da questo fatto sorge – nei diver-
si periodi storici e nelle diverse società – il problema di
oggettivare e “normalizzare” il carisma, adattandolo
all’ambiente storico-sociale e utilizzandolo come fonte di
legittimazione di ordinamenti tradizionali ovvero statali. Ovviamente, siamo in presenza di un concetto che
racchiude fenomeni multiformi, difatti è applicabile in
contesti assai diversi, a seconda – per esempio – se tale
persistenza/primazia opera nel settore privato (nell’eco-
nomia e nelle libere professioni) e/oppure in quello pub-
blico (istituzioni, amministrazioni, reclutamento e ripro-
duzione del potere). Anche perché, soprattutto nel secon-
do caso, può dar luogo a veri e propri illeciti penali, a
gravi deformazioni degli apparati pubblici e dell’intero
processo politico democratico. Proseguiamo ancora nella lettura weberiana, secon-
do la quale il carisma del sangue si produce Mediante la concezione che il carisma sia una qualità
del sangue e che quindi inerisca al gruppo parentale e, in
particolare, ai parenti più prossimi del portatore: tale è il
carisma ereditario […] qui non vale più la credenza nelle
qualità carismatiche della persona, ma la credenza nell’ac-
quisizione legittima in virtù dell’ordine di successione (tra-
dizionalizzazione e legalizzazione) […] il carisma persona-
le può mancare del tutto […] [in quanto] non appena esiste
la credenza nel legame del carisma al vincolo di sangue, il
carisma muta completamente di significato. Mentre in ori-
gine era l’azione personale che rendeva nobili, ora l’uomo
viene legittimato soltanto dalle azioni dei suoi antenati. (Ivi: 245-248) Resta sempre vero che il secondo tipo (il familismo
in campo politico e amministrativo) può esistere sol-
tanto se è presente anche il primo (il familismo sociale
che serve a giustificarlo, in quanto genera le condizioni
socio-culturali favorevoli alla sua accettazione), mentre
non è vero il contrario. Praticamente in tutte le socie-
tà esistono aree di favoritismo familiare/parentale e di
nepotismo, ma solo in alcune si registra la corrisponden-
te diffusione del modello familista nella sfera politica. Questo dato dimostra che solo a certe precise condizioni
storiche, economiche e culturali i fenomeni dappertutto
presenti di privilegio familiare in campo privato si tra-
ducono in modelli di azione e selezione politica.i La particolarità di questa forma di carisma si inseri-
sce nell’evoluzione della società tradizionale, laddove il
legame di sangue diventa fonte permanente di autorità
politica, per farlo si istituzionalizza dando origine al pote-
re tradizionale di carattere dinastico, producendo «il rove-
sciamento del carisma genuino nel suo opposto» (ibidem). Si tratta di un campo di analisi esemplare ai fini del-
la individuazione della prospettiva originale che carat-
terizza la sociologia politica, distinguendola dalle altre
scienze sociali che pure studiano i fatti politici, dal quale
emerge immediatamente la specificità dell’angolazione
di ricerca e delle domande propriamente sociologiche
nell’indagine sulla politica. Come noto, le monarchie ereditarie esistono anco-
ra oggi in molti paesi, anche occidentali, ma risultano
svuotate del potere decisionale diretto e conservano un
ruolo di natura quasi esclusivamente simbolica-identita-
ria (come conferma, da ultimo, la forte ondata di com-
mozione popolare suscitata in GB dalla morte della regi-
na Elisabetta II). Il Familismo politico è stato molto studiato in Ita-
lia da differenti prospettive e con differenti conclusioni1,
senza però mettere finora a fuoco la possibile relazione
con il carisma del sangue, della quale – ora che abbia-
mo chiarito l’uso che intendiamo fare dei due concetti di
carisma del sangue e di familismo – possiamo occuparci. Da questo punto di vista, le monarchie parlamentari
oggi vigenti negli stati democratici sono espressione di
regimi politici del tutto diversi dagli stati assolutistici del
XVII-XVIII secolo europeo, e devono fare anche i conti
con il primato moderno del modello repubblicano. 3. 1 (Banfield 1976; Piselli Arrighi 1985; Sciolla 1997; Ginsborg 1998;
Catanzaro 1983; Fantozzi 1997; Costabile 1996; ecc.)
2 «Quando ritorna nei binari della vita di ogni giorno, allora il puro
potere del carisma vien di regola spezzato, trasportato nel campo isti-
tuzionale e piegato […] da principi strutturali completamente diversi, La società e la politica italiane negli ultimi due
secoli (quelli della graduale diffusione nell’intero terri-
torio nazionale dei modelli della modernizzazione occi-
dentale: a partire dallo stato di diritto, dal mercato eco-
nomico, dalla democrazia, fino al pieno consolidamento
di tali modelli e infine alle crisi degli ultimi decenni)
sono state contraddistinte, in diverse sfere sociali, dal-
la compresenza di modelli di azione contrastanti. Que-
sti ultimi sono stati improntati, per un verso, al carisma
dell’ordinamento impersonale; per altro verso, al cari-
sma del sangue, sia come forza legittimante di un grup-
po sia come forza legittimante del legame in quanto tale. In altre parole, l’agire familistico (e clientelare3) e l’agire
razionale-legale, sono stati più volte mescolati in manie-
ra così tanto elaborata da rendere – alla luce di quanto
emerso da decenni di ricerche empiriche e di procedi-
menti giudiziari – spesso difficile distinguere con preci-
sione tra forme di particolarismo e di universalismo, di
legalità e di illegalità, di neo-patrimonialismo oppure di
corretto uso delle prerogative legate agli uffici pubblici
ricoperti. È difatti all’interno di tali processi avanzati che il
carisma del sangue è diventato una proprietà diffusa e
manipolabile, fondata su una sacralità di base religiosa
ma poi mondanizzata, capace – lungo una serie di trapas-
si – di riassumere elementi simbolici, identitari, affettivi
e utilitaristici, validi sempre e in maniera particolare nei
periodi di più acuta sofferenza sociale. Stiamo parlan-
do di uno specifico fattore di coesione generale, di utili-
tà e di consenso, spendibile politicamente, specialmente
nelle società dove la credenza nella legalità è più debole. Una debolezza rispetto alla quale il familismo si colloca
in un rapporto ambivalente di causa-effetto: da una parte
fornendo soluzioni particolaristiche alle crisi dell’ordina-
mento impersonale; dall’altra parte utilizzando incessan-
temente tali crisi per riprodursi a suo danno, alimentando
un permanente conflitto di credenze. Carisma del sangue
e legittimità del familismo versus legalità legittima. 5. Avanzando un’ipotesi interpretativa riferita alla
storia italiana, possiamo leggere il processo in questi
termini: dall’eroe al carisma del sangue, dal carisma
del gruppo (Perulli 2008) al carisma familiare diffuso –
legato alla specificità cattolica italiana e alla civiltà par-
rocchiale, locale, familiare – al familismo sociale moder-
no come religione civile differenziata al suo interno. In primo luogo, ricordiamo un altro insegna-
mento weberiano secondo il quale le forme idealtipiche
servono senz’altro alla spiegazione dei fenomeni sociali
e alla loro comparazione; a condizione di ricordare che
in realtà non esistono mai nella forma pura riassunta e
individuata dallo studioso, che nelle sue indagini si trova
a fare i conti2 quasi sempre con «trapassi» (Weber 1981 2. Occupiamoci ora del familismo, sociale e politico. Per farlo, bisogna subito precisare che questo concetto
fa riferimento a formazioni sociali moderne e non tra-
dizionali. Viene infatti utilizzato nella ricerca scientifica
per segnalare la permanenza di relazioni di natura par-
ticolaristica, anziché universalistica, in sfere della società
moderna nelle quali – a differenza dei principi di libe-
ra concorrenza, competizione e competenza, uniti alla
imparzialità e impersonalità della norma positiva (che
dovrebbero risultare predominanti in quanto sinonimi
della modernità stessa) – si osserva, al contrario, la sus- 101 Carisma del sangue e familismo: un rapporto controverso [1922]: 31), ovvero con formazioni e fenomeni sociali nei
quali convivono caratteri riconducibili a più tipi ideali e
non a uno solo. particolare Eisenstadt con la sua teoria delle moderni-
tà multiple. In sostanza, le formazioni sociali moderne
esprimono le modalità concrete assunte in ogni paese
dalla compenetrazione delle oggettivazioni del carisma
del sangue e del carisma della ragione nel corso dei pro-
cessi di mutamento, anche alla luce delle spinte al cam-
biamento ricevute da forze endogene e/o esogene. A questo punto, il ricercatore deve cercare – serven-
dosi appunto degli idealtipi – il senso prevalente al quale
gli attori sociali ispirano le loro azioni all’interno di una
realtà pluralistica e polivalente. Se il familismo, per come lo abbiamo interpreta-
to sulla scia di una consistente letteratura sociologica,
è un fenomeno moderno attraverso il quale le socie-
tà realizzano – a certe specifiche condizioni e in ambiti
più o meno estesi – la loro transizione alla modernità e
i successivi processi di modernizzazione; il rapporto tra
familismo e carisma del sangue non deve essere ricer-
cato nel passato lontano, cioè come una manifestazione
propria di società premoderne; bensì nel passato prossi-
mo e nel presente, di conseguenza in rapporto ai proces-
si di razionalizzazione e di secolarizzazione. 4. 3 Almeno di quelle forme di clientela che conservavano e valorizzava-
no, insieme ai fattori di interesse materiale, anche dei tratti comunitari
(Eisenstadt Roniger 1992; Fantozzi 1997) oppure amalgamato e fuso con essi nelle forme più svariate» (Weber
1981 [1922]: 229) Ancora Weber: Con razionalità formale di un agire economico si deve
designare la misura del calcolo tecnicamente possibile e
realmente applicato ad esso […] Il concetto di razionalità
materiale assume significati quanto mai differenti. Esso
esprime semplicemente questo elemento comune – cioè che
l’analisi non si accontenta del fatto, constatabile in modo
(relativamente) univoco, che viene compiuto un calcolo
razionale rispetto allo scopo, con mezzi tecnici il più possi-
bile adeguati; ma fa valere invece esigenze etiche, politiche,
utilitarie, edonistiche, di ceto, di eguaglianza o di qualsi-
asi altra specie, misurando in base ad esse razionalmente
rispetto al valore o a uno scopo materiale i risultati dell’a-
gire economico, anche se questo è formalmente razionale e
calcolabile. (Weber 1981 [1922]: 81) Riassumendo, emergono così fasi successive di
oggettivazione-evoluzione del carisma di cui parliamo:
a) il carisma familiare diffuso: legato alla secolarizza-
zione ed enfatizzazione della comunità familiare come
relazione fondamentale a livello religioso e civile e come
soluzione a fratture altrimenti incomponibili. Il primato
della comunità familiare è radicato nel paese al di là dei
confini ideologici, anzi la comunità politica nazionale
e la comunità partitica sono state per molti versi proie-
zioni della comunità familiare che, in questo modo, ha
funzionato come supplenza indispensabile di legittima-
zione di fronte alla debolezza generalizzata della creden-
za della legalità e alla debolezza subculturale del Sud – si
pensi in proposito all’Italia post/unitaria o al comparag-
gio politico studiato dalla Piselli (1985) con riferimento
ai primi decenni del secondo dopoguerra nel Sud –; b)
il carisma manipolato: nei messaggi, negli appelli, nei
programmi, infine attraverso i media, sul piano concre-
to dell’agire sociale e politico-istituzionale nei periodi di
modernizzazione e democratizzazione più avanzati; c) il
carisma residuale: quando il modello familiare è radi-
calmente instabile, e i valori e vincoli di fedeltà, sacralità
sono tramontati, eppure rimane come riferimento emer-
genziale in condizioni di razionalità limitata4. 4 «Quando il carisma come potenza creatrice nel corso dell’irrigidimen-
to del potere in formazioni permanenti cede di fronte ad esse, mani-
festandosi soltanto in brevi e imprevedibili emozioni delle masse nelle
elezioni o in occasioni simili, esso resta tuttavia, anche se in senso pro-
fondamente mutato, un importantissimo elemento della struttura socia-
le» (Weber 1981 [1922]: 257) Se proviamo ad applicare il modello delle Tre Italie di
Bagnasco (1939) e Trigilia in questa direzione emergono:
il grande capitalismo familiare nel Nord-ovest, le PMI
familiare nel Nord-est, il familismo politico nel Sud. Per
altro verso, questa volta in relazione alle organizzazioni
partitiche, si possono evidenziare differenti manifesta-
zioni del familismo politico nelle diverse fasi del sistema
politico italiano: per esempio, in termini di supplenza
nei decenni della prima repubblica di fronte alla for-
za del partito politico di massa, e viceversa in termini
più espliciti e diretti, quando non addirittura sfrontati,
nei periodi difficili di transizione. C’è molto ancora da In sostanza, nella storia sociale si possono osserva-
re facilmente molteplici esempi che testimoniano, per un
verso, la radicale tensione tra carisma originario, cari-
sma del sangue e carisma della ragione. Difatti, come
abbiamo visto, il secondo e terzo tipo contraddicono
l’indiscutibile primato personale del capo e la fedeltà
completa dei seguaci alla sua rivoluzionaria visione del
mondo e missione nel mondo (“così sta scritto, ma io vi
dico”). Per l’altro verso, proprio il corso degli eventi sto-
rici ha mostrato continuamente la faticosa ricerca di for-
me di conciliazione tra queste forze carismatiche anta-
goniste; alla luce delle precondizioni religiose e culturali
ed alla situazione economica e politico-istituzionale dei
diversi paesi, nonché ai caratteri delle loro élites, come
ci ha insegnato la sociologia della modernizzazione e in 102 Antonio Costabile studiare circa le relazioni del carisma familiare diffuso
con il particolare processo di secolarizzazione realizza-
tosi nel nostro paese, che ha direttamente influenzato i
caratteri della costruzione, del consolidamento e della
democratizzazione del nostro Stato nazionale. Il carisma
familiare come giuntura compensativa, di fronte all’in-
sanabile frattura stato-chiesa, è divenuto nel tempo un
ordine cognitivo di portata generale e potenzialmente
utilizzabile in (quasi) tutti i campi dell’agire sociale –
entro limiti variabili, condizionati dagli ordinamenti di
carattere moderno e universalistico propri dei diversi
contesti politici, istituzionali ed economici nei quali la
rete familiare-parentale si trova ad operare. Ancora Weber: Si può quindi sostenere che il fondamento carismatico
del familismo sociale e politico, inteso come ordine cogni-
tivo, abbia guidato imprenditori e politici meridionali a
orientare le loro scelte – a seconda dei casi e delle opportu-
nità – a volte verso criteri di razionalità formale (per esem-
pio in alcuni decisioni innovatrici interne al mondo econo-
mico ed a quello amministrativo), più spesso verso criteri
di razionalità materiale (per esempio privilegiando in mol-
ti modi le catene particolaristiche e gli interessi immediati
a danno del dettato della norma e della volontà del legisla-
tore), generando così un singolare modello di razionalità
mista. Quest’ultimo è caratteristico della forma assunta in
queste regioni dal processo di razionalizzazione nell’agire
imprenditoriale e politico, e di conseguenza sociale, a dif-
ferenza dai modelli tipici del capitalismo d’Occidente. Questa forma di familismo carismatico ha resisti-
to, con diversa intensità, fino alla caduta della comunità
familiare tradizionale e delle ideologie collettive che ne
hanno costituito per molti aspetti l’oggettivazione nove-
centesca in Italia. Poi la società degli individui maturi
ma insicuri, le culle vuote, la scomparsa delle subcultu-
re, hanno fatto sì che il familismo sia nei fatti svuotato
dei suoi presupposti originari e di quelli secolarizzati
politicamente; ne resta soltanto l’ossatura affettivo-mate-
riale (relazioni sentimentali mescolate a interessi, profes-
sioni, imprese, sussidi). 6. Alla luce di quanto esposto possiamo formulare
un’ipotesi teorica riguardo l’esistenza di una relazione tra
carisma sangue, familismo e razionalità mista. Nel corso
del mutamento sociale, in presenza di un livello minore di
differenziazione sociale e strutturale (come nel Sud e altro-
ve), si può affermare una forma di razionalità che risulta
più collegata alla coppia weberiana razionalità materiale e
formale, che a quella più nota tra scopo e valore. 7. In conclusione, in che misura si può ancora parla-
re di carisma del sangue e familiare, laddove gli elementi
caratterizzanti delle relazioni sono delle miscele di cal-
coli, affetti personali e affari, privati, di gruppo; quindi
non solo mancano l’ordine nuovo, e straordinarietà per-
sonale ma pure è venuta meno la sacralità precedente-
mente riconosciuta al legame familiare? Sembra in effetti
che siamo di fronte ad una nuova tappa del processo di
razionalizzazione e svuotamento del carisma (in questo
caso di quello familiare), ad una variabile dell’ideologia 103 Carisma del sangue e familismo: un rapporto controverso Poggi G. (1980), La vicenda dello stato moderno, il Muli-
no, Bologna. Ancora Weber: contemporanea che ideologizza le comunità immagina-
rie in forma via via più ristretta (nazione, regione, città,
categoria, famiglia), per ovviare in qualche modo alla
caduta di tutte le altre fonti di legittimazione politica,
in condizione di incertezza radicale e di precarizzazione
generalizzata della vita individuale e collettiva. Popitz H. (1990), Fenomenologia del potere, il Mulino,
Bologna. Schluchter W. (1987), Lo sviluppo del razionalismo occi-
dentale. Un’analisi della storia sociale di Max Weber,
il Mulino, Bologna. Sciolla L. (1997), Italiani. Stereotipi di casa nostra, il
Mulino, Bologna. Citation: Mario Caciagli (2023) Storia e
scienze sociali. SocietàMutamentoPo-
litica 14(27): 105-108. doi: 10.36253/smp-
14340 Citation: Mario Caciagli (2023) Storia e
scienze sociali. SocietàMutamentoPo-
litica 14(27): 105-108. doi: 10.36253/smp-
14340 Mario Caciagli Copyright: © 2023 Mario Caciagli. This is
an open access, peer-reviewed article
published by Firenze University Press
(http://www.fupress.com/smp) and dis-
tributed under the terms of the Crea-
tive Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. 1. Luciano Cavalli e la storia1. La simbiosi tra storia e sociologia ricorre
in gran parte nell’opera di Luciano Cavalli, anche se non in tutta. Leggendo
o rileggendo i suoi testi ho cercato di enucleare vari aspetti di questa simbio-
si. Poi, sono andato a ricercarne le ragioni nella formazione dello studioso,
formazione tanto intellettuale che politica. Infine, ho individuato una frattu-
ra e l’oblio per questa simbiosi. p
q
Sociologia della storia è il titolo del primo paragrafo del medaglione che
Cavalli dedicò al suo amato Max Weber nella serie di medaglioni del volu-
me di Sociologie del nostro tempo (1973). Naturalmente vien posta in rilievo
l’attenzione del Weber al ruolo delle religioni nella storia, attenzione che cul-
mina nel rapporto fra etica e capitalismo proprio del calvinismo. In questo
Weber c’è sempre la storia sullo sfondo: storia dell’Oriente versus la storia
dell’Occidente, dove l’uomo borghese è il culmine che corrisponde alla con-
dizione umana. Allora non la storia, ma le storie di diversi popoli e diverse
civiltà, e poi la complessità del processo storico, di uomini storicamente rea-
lizzati, del ruolo delle classi sociali. In Economia e società, infine, mette in
evidenza Cavalli, il grande fattore di cambiamento nella storia è il carisma,
qui evocato per la prima volta nella sua essenza. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. q
p
p
La storia non può non ritornare nel volume del 1976, L’Italia promessa. Riflessioni sulla crisi nazionale. Nell’incipit scrive Cavalli «forse questo scritto
può esser definito come la libera riflessione di un cittadino circa la crisi tota-
le del paese […] può anche esser definito un saggio non accademico di socio-
logia della storia e della politica italiana, per la forma mentis e i riferimenti
culturali prevalenti» (Cavalli 1976: 7, corsivi miei). 1 Storia e scienze sociali era il titolo della mia relazione tenuta il 17 novembre 2022 al conve-
gno dedicato a Luciano Cavalli, svoltosi presso l’Accademia La Colombaria. Quel titolo più che
ambizioso era inappropriato come lo sarebbe per il testo che qui presento. Questo testo merita,
infatti, il titolo Un sociologo e la storia, o, ancor più precisamente, Luciano Cavalli e la storia. RIFERIMENTI BIBLIOGRAFICI Shils E. (1984), Centro e periferia, Morcelliana, Brescia. Bagnasco A. (1985), Tre Italie: la problematica territoriale
dello sviluppo italiano, il Mulino, Bologna.i Trigilia C. (2018), «La personalizzazione della leadership
politica», in D. D’Andrea e C. Trigilia (a cura di),
Max Weber oggi, il Mulino, Bologna, pp. 19-37 Banfield E. C. (1976), Le basi morali di una società arre-
trata, il Mulino, Bologna. Turi P. (2008), «Sociologia e politica nell’itinerario intel-
lettuale di Luciano Cavalli», in G. Bettin Lattes e
P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze University Press, Firenze pp. 23-189 Bobbio N. (2021), Mutamento politico e rivoluzione, Don-
zelli, Roma. Bockenforde E. W. (2006), La formazione dello Stato
come processo di secolarizzazione, Morcelliana, Bre-
scia. Viviani L. (2021), «Leadership e democrazia in Max
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società, Angeli, Milano, pp. 70-90 Calise M. (2000), Il partito personale, Laterza, Roma-Bari. Catanzaro R. (1983), «Struttura sociale, sistema politico
e azione collettiva nel Mezzogiorno», in Stato e Mer-
cato, 8: 271-315. Weber M. (1981 [1922]), Economia e società, Edizioni di
Comunità, Milano. Cavalli L. (1981), «Il carisma come potenza rivoluziona-
ria», in P. Rossi (a cura di), Max Weber e l’analisi del
mondo moderno, Einaudi, Torino, pp. 161-188 – (1982), Il capo carismatico. Per una sociologia weberia-
na della leadership, il Mulino, Bologna. – (2016), Carisma e democrazia, in R. Cipriani, Nuovo
manuale di sociologia, Maggioli, Rimini. Costabile A. (1996), Modernizzazione, famiglia e politica,
Rubbettino, Soveria Mannelli. – (2009), Legalità, manipolazione, democrazia. Line-
amenti del sistema politico meridionale, Carocci,
Roma. Eisenstadt S.N., Roniger L. (1992), lemma «Clientela»,
in Enciclopedia delle Scienze sociali, vol. II, Istituto
dell’Enciclopedia Italiana-Treccani, Roma. Fantozzi P. (1997), Comunità, società e politica nel Sud
d’Italia, Rubbettino, Soveria Mannelli. Ginsborg P. (1998), L’Italia del tempo presente. Famiglia,
Società civile, Stato, Einaudi, Torino. Izzo A. (1995), I percorsi della ragione, Carocci, Roma. Mommsen W. J. (1993), Max Weber e la politica tedesca,
il Mulino, Bologna. Perulli A. (2008), «Dal carisma personale al carisma di
gruppo. Note su Norbert Elias», in G. Bettin Lattes
e P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze University Press, Firenze, pp. 373-388. Piselli F., Arrighi G. (1985), «Parentela, clientela e comu-
nità», in P. Bevilacqua e A. Placanica (a cura di), La
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valli. RIFERIMENTI BIBLIOGRAFICI societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a SOCIETÀMUTAMENTOPOLITICA 14(27): 105-108, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14340 Nella prima pagina dell’intro-
duzione c’è un’affermazione che soddisfa pienamente la
mia curiosità conoscitiva: Non esistono confini invalicabili e domini esclusivi nel
vasto campo delle scienze sociali contemporanee. Il socio-
logo e lo storico, l’economista, l’antropologo e lo psicologo2
possono trattare la stessa materia: non di rado lo fanno. Anche in sede teorica e metodologica la distinzione non è
netta. Accade sempre più spesso che sociologi, storici e altri
studiosi abbiano una discreta conoscenza degli strumenti
concettuali e metodologici degli “altri”, e ne facciano alme-
no marginalmente uso. La differenza rilevante è, alla fine,
solo di “prospettiva”, di “taglio” e anche soltanto di “accen-
ti”. Forse si potrebbe dire che è, in ultima analisi, una dif-
ferenza di “mentalità”; in quanto ciascuno porta con s’è l’
“habitus” mentale che si è fatto frequentando certi autori,
vivendo in un certo ambiente scientifico, riflettendo soprat-
tutto su certi problemi e lavorando abitualmente con certi
strumenti» (Ibidem). Ritorno ai miei reperti. La contaminazione tra
sociologia e storia – quale strumento indispensabile per
l’approccio interdisciplinare al fenomeno studiato – è
scritto nel risvolto della sovraccoperta del volume del
1975 dal Cavalli curato nella collana di working papers
da lui stesso ideata e diretta, Il fascismo nell’analisi socio-
logica (1975). Nell’antologia vi sono testi dei suoi allievi,
nonché di sociologi, psicologi, politologi finanche di un
filosofo, José Ortega y Gasset. Quello che conta è la rot-
tura delle barriere fra le discipline. Anche se i saggi, dice
Cavalli nell’introduzione, prendono in esame il fascismo
non come storici ma come scienziati sociali. Ciò non
vieta che molti autori ricordati, in particolare Parsons e
Michels, abbiano ricercato alcune condizioni della nasci-
ta del fascismo nelle peculiarità dello sviluppo storico
tedesco e italiano. In questo lavoro si vedono certamente gli interessi
del sociologo: la struttura di classe, le classi (dalla bor-
ghesia al proletariato industriale), i dati demografici, la
distribuzione per settore degli occupati, i livelli di red-
dito, i livelli di analfabetismo, il rapporto città/campa-
gna e la grande trasformazione degli anni Sessanta. Si
vedono le chiavi di lettura: il “carattere” e le “promes-
se”, l’“ordine” e il “mutamento”, l’incapacità dei gover-
nanti di soddisfare le promesse; il tutto collocato nella
proposta di una teoria generale dello sviluppo politico. E continua Cavalli, ed è
questo il punto di merito che mi interessa: «occorre comunque rilevare, fin
dall’inizio, la libertà selettiva con cui chi scrive rievoca il passato, raffigura il
presente e progetta il futuro, in base a valori e ipotesi non sempre largamente
condivisi» (Ibidem, corsivi miei). Nella prima parte del saggio si dà ampio
spazio all’ascesa al potere del fascismo, la sua violenza, il mito della patria,
i ceti sociali coinvolti, il ruolo della burocrazia civile e militare. Sono pre-
senti: gli interessi in gioco, la responsabilità delle classi dirigenti, la sconfitta
del movimento operaio, la personalità del Duce. Per il sociologo è chiaro che SOCIETÀMUTAMENTOPOLITICA 14(27): 105-108, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14340 SOCIETÀMUTAMENTOPOLITICA 14(27): 105-108, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14340 106 Mario Caciagli Mario Caciagli di Luciano Cavalli che Paolo Turi compilò nel volume
del 2008 dedicato al maestro. Ben 166 pagine ad ono-
re dell’illustrato e dell’illustratore (Turi 2008: 23-189). Dell’itinerario intellettuale ricostruito dal Turi anche
su materiale inedito, tutto utile ai fini del mio discorso,
è più illuminante la parte dedicata alla formazione, rela-
tiva agli anni 1924-1956 (Ivi: 33-51). Accanto alla fede
antifascista coltivata in famiglia, nutrita da una salda
visione del Risorgimento, vi si legge dello studio della
storia nel corso di laurea in lettere moderne e della pre-
coce attività politica nell’allora PSIUP. Poi l’argomento
della tesi di laurea, cioè il movimento operaio a Genova
dal 1892 al 1922. Un altro prodotto di storia del PSI è l’i-
nedito Turati e il movimento socialista italiano fino alla
guerra di Libia. Su questi temi Cavalli lavorerà anche in
seguito, con studi sul movimento operaio e del lavoro,
poi delle minoranze in quel movimento e poi di sociolo-
gia elettorale e, appunto di sociologia storica (Ivi: 39, cor-
sivi miei). Poi verrà l’esperienza americana, centrale per
la formazione propriamente sociologica, sia teorica che
metodologica. E dove si farà propria la funzione “demo-
cratica” della sociologia. la democrazia sta da un’altra parte rispetto ad un regi-
me autoritario e totalitario. Per quanto riguarda la mia
prospettiva, ciò che conta è la selezione operata sui fatti
storici al fine di una lettura sociologica, l’individuazione
di nodi nella narrazione storiografica. i
Poi, quale terzo reperto, viene un intero libro, del
1974, che reca un titolo ben significativo, Sociologia della
storia italiana. 1871-1974. 2 E il politologo, aggiungo io. In proposito si veda Marco Almagisti,
Carlo Baccetti e Paolo Graziano (a cura di) (2018), Introduzione alla
politologia storica. Questioni tecniche e studi di caso, Carocci, Roma. Infine, spunta la riflessione oggi largamente condivisa,
dello “stato nazionale tardivo” con le sue tare ereditarie:
l’estraneità delle masse, la violenza invece del consen-
so, la dittatura totalitaria, il principio del capo (Musso-
lini), infine l’egemonia integralista dei cattolici (il quasi
regime della DC). Nella Sociologia della storia italiana
(Cavalli 1974) c’è molto altro che evidenzia il rapporto
fra storia e sociologia. Varrà la pena ritornare su questo
importante saggio. Il primato della politica nell’Italia del secolo XXI
(Cavalli 2001) è tutto versato sui problemi dell’oggi e
quindi il richiamo alla storia è quasi inesistente. Ma c’è
uno sviluppo storico dell’Italia nei primi quarant’anni
del secolo con un rimpianto per il Risorgimento, dovu-
to alla sua formazione familiare, ma anche, aggiungo,
per ribadire che non si fa ricerca sociologica senza uno
sfondo storico: «la catastrofica guerra del Duce ha lascia-
to il popolo italiano senza gruppi dirigenti di salda for-
mazione risorgimentale […] Duratura la differenza verso
stato e nazione […] Le secolari esperienze di un popolo
disarmato, sottomesso allo straniero e alla Chiesa» (Ivi:
4-5). È presente un lungo discorso sul ruolo della Chie-
sa (Ivi: 77-84) che partendo da lontano rivela la ferma Più avanti riporterò reperti di altre opere. Mi fermo
per aprire una parentesi che illumina la mia argomenta-
zione. Il richiamo è alla preziosa biografia intellettuale 107 Storia e scienze sociali convinzione anticlericale del nostro studioso poggia-
ta sulla prospettiva storica. Un discorso che parte dal-
la Controriforma definita «degna dell’attenzione delle
moderne scienze sociali e politiche» (Ivi: 81) per arriva-
re ad affermare poco più avanti: «Lo spirito e il metodo
di quel dominio clericale hanno avuto forza su questo
Paese […] e sembrano virtualmente riemergere all’occa-
sione: come si è potuto vedere nell’egemonia della DC»
(Ibidem). Aggiungendo: «In una valutazione sociologica
globale si deve dire che quello fu “un regime senza egua-
li per il dominio totale sull’uomo” con mezzi di indagine
e di controllo interiore che forse nessun stato occidenta-
le ha mai posseduto in seguito» (Ibidem). Tanto meglio
se «mancata formazione del cittadino, stile di governo
autoritario» (Ivi: 78) è un giudizio che si può formula-
re all’ombra di Max Weber ricordato – come nel volume
Max Weber: religione e società (Cavalli 1968) – per riba-
dire «[l’] influenza della Chiesa sul carattere naziona-
le» e per la mancata Riforma in Italia, dove eucarestia e
confessione formano individui eteronomi. restano sullo sfondo o servono soltanto di sostegno a
quelle astrazioni. Così si legge chiaramente: «Come cer-
cherò di dimostrare nella mia ricostruzione necessaria-
mente selettiva» (Ivi: 19). Si tratta, appunto, della sele-
zione che il sociologo fa del materiale storico a disposi-
zione. E più avanti viene indicata la prospettiva adottata,
alla luce cioè del paradigma che ha abbracciato: «l’autore
non rivaleggia certo con lo storico alla ricerca di nuove
fonti e di nuovi dati, ma procederà ad una ricostruzio-
ne di una vicenda nota in base alla teoria del carisma e
alla rilettura ad essi orientata dei dati e dei documenti
già acquisiti» (Ivi: 32). Nel caso di Hitler il carisma è negativo, «demonia-
co» (Ivi: 27). Ma è comunque un modello che, come tale,
può ripresentarsi in futuro; si può prevedere. Caval-
li afferma chiaramente come «la lezione di Hitler non
appartiene dunque al museo della storia» (ibidem). La
storia è derubricata a “museo” e diviene semmai “magi-
stra”, potendo essere la vicenda di Hitler una “lezione”
per i posteri. Il libro su Hitler esce, quindi, nel 1982. Non sappiamo quando Cavalli si sia soffermato in pre-
cedenza più a fondo sulla teoria del “carisma” nella sua
lunga frequentazione di Weber. Sappiamo però, gra-
zie alla puntuale bibliografia messa a punto da Umber-
ta Porta (2008) che il carisma appare per la prima volta
negli scritti del Cavalli in un articolo del febbraio 1979
per il quotidiano Il Giorno dal titolo Che cos’è il cari-
sma. Da Churchill a Mao. La teoria del carisma sarà pre-
sentata in un convegno dell’anno successivo (1980) su
Weber, poi pubblicato nel 1981 con il titolo Il carisma
come potenza rivoluzionaria in un volume curato da Pie-
tro Rossi3. Troverà sistemazione quasi definitiva nel libro
dello stesso 1981, Il capo carismatico. Per una sociologia
weberiana della leadership. 2. Il carisma demoniaco. Nel cammino intellettuale
di Cavalli c’è una svolta. In Carisma e tirannide nel seco-
lo XX. Il caso Hitler (1982), a mia opinione un capola-
voro sia perché si occupa di un mondo che io ho molto
frequentato, nella storia e nella politica, la Germania; sia
per sua intrinseca qualità. Lo Hitler del Cavalli sembra
un’opera di storia, ma non lo è. A prescindere dal titolo
– che indica come oggetto un personaggio storico – ma
quel personaggio è un “caso” che, in quanto tale, diven-
ta paradigmatico. 3 Nella bibliografia del Cavalli degli anni seguenti si trovano numero-
sissimi articoli, saggi e libri nei quali il carisma è l’oggetto privilegiato. Il testo suggerisce ben altre riflessioni
non storiografiche. i
Rileggerlo alla luce delle interpretazioni da me fatte
finora, mi sento di dire che Cavalli qui vuol fare il socio-
logo nettamente distinto dallo storico. La simbiosi pre-
sentata negli altri scritti è superata, la linea di confine è
netta. Perché quello che conta è il carisma, conosciuto da
sempre nella sua frequentazione di Max Weber, ma che
ora è il mantra per coprire la sua idea di una «demo-
crazia con capo» che coltiva da tempo e che sviluppe-
rà nell’ultima fase di studioso, incentrata, appunto, sul
paradigma del carisma. Paolo Turi fa iniziare da questi due scritti la fase
della messa a punto del paradigma, la fase dei princi-
pali contributi teorici e delle proposte di applicazione
che Cavalli esporrà per lunghi anni (Turi 2008: 99-100). Vero è che le astrazioni di cui sopra, certamente tutte
curvate per la dimostrazione della bontà del paradigma
carisma, sono ben radicate nei fatti concreti nella Ger-
mania senza i quali non starebbero in piedi. La storia
ricompare ogni tanto: il capo carismatico è «autore di
vitali sviluppi storici», scrive Cavalli e invita a «rico-
noscere il ruolo di singole individualità nella storia»
(Cavalli 1982: 9-12). Il suo libro su Hitler, Cavalli precisa nell’introdu-
zione che «non pretende di essere una biografia» (ivi:
35). Infatti, il libro è colmo di costrutti “ideali”. Ci sono
«la visione», «l’eletto», «l’estasi», «la comunità di fede»,
«i segni», «il Salvatore», «il clima di estasi collettiva»,
«i miracoli», «il signore delle masse», «la folgorazio-
ne», l’«infallibilità», i simboli, i riti, ecc. ecc. In quan-
to costruzioni ideali tutte queste sono al di là del tem-
po e dello spazio e possono valere per diversi e distin-
ti momenti storico-politici. In quel libro i fatti storici Nel suo denso saggio sul Cavalli intitolato Carisma,
tirannide e democrazia nel XX secolo, Carlo Rossetti scris-
se però giustamente: «l’analisi di Cavalli non è una pagi-
na di storia passata, superata dagli eventi. Può essere letta 108 Mario Caciagli – (a cura di) (1975), Il fascismo nell’analisi sociologica, il
Mulino, Bologna. anche come un’interpretazione di svolgimenti futuri pos-
sibili, dai quali le democrazie non sono immuni» (Rossetti
2008). Certamente, sono questi i compiti che si assegnano
gli scienziati sociali. In questo caso non solo si intende-
rebbe di prevedere, ma anche ammonire su possibili even-
ti futuri. Lo stesso Rossetti conclude però con l’ afferma-
zione: «il dato cruciale empirico che Cavalli segnala, indi-
viduato con i metodi della storiografia, pilastro della teo-
ria sociologica, è che l’autorità carismatica mette in moto
forze potentissime che tendono a sovvertire il modello di
una società razionale» (Ibidem: 250, corsivi miei).f Mastellone S. (1990), Democrazia: sociologia e storia,
Dipartimento di scienze sociali, Università di Fire-
nze, Firenze. Porta U. (2008), «Gli scritti di Luciano Cavalli», in G. Bettin Lattes e P. Turi (a cura di), La sociologia di
Luciano Cavalli, Firenze University Press, Firenze,
pp. 463-481. Rossetti C. (2008), «Carisma, tirannide e democrazia nel
XX secolo», in G. Bettin Lattes e P. Turi (a cura di),
La sociologia di Luciano Cavalli, Firenze, Firenze
University Press, pp. 229-251 L’affermazione forte, dei «metodi della storiogra-
fia» come pilastro della teoria sociologica, riporterebbe
alla mia rivisitazione illustrata in precedenza. Mi pro-
cura invece ulteriori dubbi e perplessità che non sono
in grado di risolvere in questo intervento. Dubbi che
svaniscono di fronte alla rilettura dell’intervento ad un
seminario del 1989, i cui atti vennero pubblicati l’anno
successivo da Salvo Mastellone con il titolo Democrazia:
sociologia e storia (1990). Nel suo breve intervento Caval-
li trattò a fondo il rapporto fra sociologia e democrazia,
ma niente disse della storia, nonostante essa e il suo rap-
porto con le scienze sociali fossero al centro di quasi tut-
te le relazioni. Rossi P. (a cura di) (1981), Max Weber e l’analisi del
mondo moderno, Einaudi, Torino. Turi P. (2008), «Sociologia e politica nell’itinerario intel-
lettuale di Luciano Cavalli», in G. Bettin Lattes e
P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze, Firenze University Press, pp. 23-189. Il carisma, quale chiave di lettura del potere e del
suo esercizio, diventerà centrale nella ricerca del Caval-
li. Certo non il carisma demoniaco, ma il carisma che si
addice al “capo” colui che ha guidato regimi del passato
e che dovrebbe guidare quelli democratici del presente. In particolare, e in concreto un capo servirebbe al siste-
ma italiano che ha per Cavalli il grave requisito di una
«democrazia acefala». Il giudizio sul sistema politico italiano e la soluzione
“carismatica” non andrebbero valutati tuttavia in pro-
spettiva storica? Cioè in quella prospettiva che Cavalli
aveva privilegiato nella fase precedente del suo percorso
intellettuale e scientifico? Citation: Carlo Trigilia (2023) Religione
e società: l’influenza di Weber sulla
sociologia italiana. SocietàMutamen-
toPolitica 14(27): 109-112. doi: 10.36253/
smp-14341 Carlo Trigilia Copyright: © 2023 Carlo Trigilia. This is
an open access, peer-reviewed article
published by Firenze University Press
(http://www.fupress.com/smp) and dis-
tributed under the terms of the Crea-
tive Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. 1. Mi propongo di esporre alcune considerazioni a partire da un testo
centrale nel percorso intellettuale e di ricerca di Luciano Cavalli: Max Weber:
religione e società (1968). L’importanza di questo lavoro per l’itinerario di
Cavalli è già stata sottolineata in vari interventi, specie con riferimento alla
svolta nella sua concezione della democrazia e agli sviluppi della sua socio-
logia politica. Vorrei soffermarmi qui sull’influenza che il pensiero di Weber
– letto e proposto da Cavalli – ha avuto più in generale sulla sociologia ita-
liana, sia direttamente attraverso il suo lavoro e quello dei suoi allievi, ma
anche indirettamente e meno esplicitamente contribuendo a orientare in
modo consistente e diffuso alcuni filoni di ricerca. L’Italia è uno dei paesi
dove maggiore è stata l’attenzione a Weber (Cavalli 2012). Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. Prenderò in considerazione in particolare tre aspetti: anzitutto, il con-
tributo di Religione e Società (Cavalli 1968) – in termini di storia del pen-
siero – a una più corretta e articolata interpretazione della complessa opera
di Weber; in secondo luogo, la concezione di una sociologia più vicina alla
storia, anche se distinta da essa, che viene tratteggiata da Cavalli attraver-
so la lettura di Weber, una concezione consapevolmente distante da quella
di una scienza sociale a elevato grado di generalizzazione secondo i canoni
prevalenti della metodologia neo-positivista negli anni in cui uscì Religione
e Società; e infine, l’influenza che l’interpretazione di Weber ha avuto più
specificamente su alcuni filoni di ricerca della sociologia economica e della
political economy italiana, anche se Cavalli si dedicò dopo l’approfondita let-
tura di Weber più alla sociologia politica. In relazione a quest’ultimo aspet-
to accennerò in conclusione a un tema che mi sembra rilevante. RIFERIMENTI BIBLIOGRAFICI Cavalli L. (1968), Max Weber: religione e società, il Muli-
no, Bologna. g
– (1973), Sociologie del nostro tempo, il Mulino, Bologna.l – (1976), L’Italia promessa. Riflessioni sulla crisi nazion-
ale, il Mulino, Bologna. – (1981), Il capo carismatico. Per una sociologia weberi-
ana della leadership, il Mulino, Bologna. – (1982), Carisma e tirannide nel secolo XX. Il caso Hit-
ler, il Mulino, Bologna. g
– (2001), Il primato della politica nell’Italia del secolo
XXI, il Mulino, Bologna. – (2001), Il primato della politica nell’Italia del secolo
XXI, il Mulino, Bologna. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a 1 Tratto dall’intervento Religione e società al convegno Società e democrazia nel pensiero di Luciano
Cavalli, Firenze, Accademia “La Colombaria”, 17-18 novembre 2022. Dal punto
di vista metodologico, dell’ancoraggio storico delle scienze sociali e dell’uso
della comparazione, si manifesta la vicinanza tra la sociologia politica webe-
riana di Cavalli e gli sviluppi della sociologia economica e della political eco-
nomy comparata. Le direzioni di marcia e i risultati conseguiti da un punto
di vista sostantivo, specie con riferimento ai processi di consolidamento o
destabilizzazione della democrazia contemporanea saranno però divergenti. g
Comincio dal primo aspetto che ho richiamato. Religione e Società esce
nel 1968 dopo alcuni anni di intensa preparazione. In quel momento in Italia SOCIETÀMUTAMENTOPOLITICA 14(27): 109-112, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14341 SOCIETÀMUTAMENTOPOLITICA 14(27): 109-112, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14341 110 Carlo Trigilia Weber è ancora molto poco conosciuto. La sociologia sta
facendo i primi passi in termini di riconoscimento come
disciplina accademica. Tra i principali lavori dedicati a
Weber ve ne sono due: Lo storicismo tedesco contempo-
raneo di Pietro Rossi, apparso nel 1956 e in nuova edi-
zione nel 1971; Max Weber e il destino della Ragione di
Franco Ferrarotti, pubblicato nel 1965. Il volume di Ros-
si, un pioniere degli studi su Weber in Italia, contiene
un’ampia parte dedicata ai saggi metodologici di Weber
(Rossi tradurrà e introdurrà poi per Einaudi, nel 1958, i
principali saggi metodologici con il titolo Il metodo delle
scienze storico-sociali). L’altro contributo – quello di Fer-
rarotti – dedica anch’esso molto spazio alla concezione
weberiana della sociologia, ma discute il contributo di
Weber soprattutto alla luce del dibattito culturale dell’e-
poca, e in particolare rispetto al marxismo. Manca dun-
que in Italia, ancora alla fine degli anni ‘60, un lavoro
sistematico capace anche di approfondire il legame tra
le posizioni metodologiche e il contributo sostantivo di
Weber: cioè la sua interpretazione delle origini del capi-
talismo moderno nell’ambito della specifica civiltà occi-
dentale. Una presentazione sistematica viene offerta da
Max Weber: religione e società (Cavalli 1968). Si tratta, a
mio avviso, di un lavoro di particolare importanza, non
solo perché prende in considerazione l’intera produzione
di Weber, ma perché offre al contempo un’interpretazio-
ne solida ed efficace delle sue idee sulle origini del capi-
talismo moderno nel più ampio quadro dello sviluppo
della civiltà occidentale. dizione religiosa, ma sono connessi ad altre variabili più
specifiche e autonome, come in particolare la forza o la
debolezza dei grandi imperi che condizionano i carat-
teri delle città. Queste ultime assumono un ruolo cru-
ciale: sono il luogo nel quale nell’esperienza occidentale
prenderanno forma le diverse componenti del motore
del capitalismo moderno. Cavalli mette anche in evi-
denza come in questo quadro più ampio si ridimensio-
ni il ruolo inizialmente assegnato da Weber all’idea di
predestinazione nella formazione e diffusione dell’etica
economica a favore della maggiore importanza attribui-
ta alle sette protestanti. Queste ultime si basavano su un
meccanismo organizzativo capace di formare e soste-
nere comportamenti imprenditoriali congruenti con lo
sviluppo del capitalismo moderno perché eticamente
vincolati. Allo stesso tempo, l’appartenenza alle sette
protestanti generava fiducia, era una garanzia di affida-
bilità, riduceva il dualismo etico e favoriva le transazio-
ni economiche. 2. Cavalli è il primo nel contesto italiano a chiari-
re e mettere in evidenza questa chiave interpretativa
più complessa del pensiero di Weber. Il rilievo del suo
contributo emerge anche da uno sguardo a livello inter-
nazionale. I due principali testi di riferimento sull’opera
di Weber all’epoca erano The structure of social action
di Talcott Parsons (1937) e Max Weber: An Intellectual
Portrait di Reinhard Bendix (1962). Cavalli prende le
distanze da entrambi per fondati motivi. Da Parsons,
per sottrarre la lettura di Weber al tentativo di farne
un tassello della sua costruzione di una teoria socio-
logica a elevata generalizzazione che contrastava con
l’ancoraggio storico del lavoro del sociologo tedesco. Da Bendix Cavalli si differenzia in un certo senso per
il motivo opposto, perché questo autore dà molto spazio
sul piano descrittivo e dell’analisi storica alle ricerche
di Weber, ma non offre una chiave interpretativa più
generale e compiuta della sua interpretazione del capi-
talismo occidentale. Cavalli si avvicina invece a questo
obiettivo anche perché si vale ampiamente, nella sua let-
tura, di un testo che fino ad allora era stato trascurato:
Storia economica (1993), che raccoglie le lezioni tenute
da Weber all’università di Monaco poco prima della
sua scomparsa. È da ricordare, in proposito, che que-
sto testo sarà poi base di uno dei contributi più noti di
interpretazione di Weber: l’articolo di Randall Collins
apparso nel 1980 nell’American Sociological Review con
il titolo Weber’s Last Theory of Capitalism: A Systema-
tization. La lettura di Collins è molto vicina a quella di
Cavalli apparsa più di dieci anni prima. Vale la pena
anche notare che la scoperta del capitale sociale riman-
dava anche piuttosto chiaramente al ruolo assegnato alle
sette nello sviluppo del capitalismo da Weber. gua inglese, certo più difficile da ottenere per un libro
di sociologia italiano alla fine degli anni ‘60. gi
g
Tuttavia, l’importanza del contributo di Cavalli per
far conoscere una versione meno semplicistica dell’ope-
ra di Weber è stata certo rilevante in Italia, e lo è stata
inizialmente più sul terreno della sociologia economica
in relazione allo studio del capitalismo e delle sue varie
forme, ma anche allo studio di classi e ceti. Mi soffer-
merò ora brevemente su quest’aspetto, scusandomi per
alcuni riferimenti personali. L’influenza weberiana sulla
sociologia economica è comprensibile alla luce dell’inte-
resse del sociologo tedesco per il capitalismo, per le sue
origini ma anche per i suoi sviluppi. Essa si può cogliere
a vari livelli. Anzitutto, nei testi di riferimento teorico e
negli strumenti di formazione per gli studenti, poi nel-
le problematiche di ricerca sulla società italiana e infine
nell’approccio più generale della sociologia economica
come political economy comparata. p
y
p
Per quel che riguarda il primo punto, oltre allo stes-
so contributo di Religione e Società (Cavalli L. 1968),
sono da ricordare alcuni testi che hanno una duplice
funzione: di presentazione dei riferimenti teorici del-
la sociologia economica e di strumenti per la didattica. Mi riferisco in particolare all’antologia curata da Ales-
sandro Cavalli, apparsa nel 1972 e poi ripubblicata dal
Mulino in più edizioni e al volume di saggi di Alberto
Martinelli, uscito più tardi nel 1986. Entrambi que-
sti lavori mostrano fin dal titolo identico – Economia
e Società – una spiccata impronta weberiana. A questi
testi aggiungerei poi la traduzione della Storia Econo-
mica – della cui importanza anche per l’interpretazione
di Cavalli ho già detto – pubblicata nel 1993 da Don-
zelli con una mia introduzione chiaramente influenzata
dalla lettura e dall’uso che ne aveva fatto Cavalli, oltre
che Randall Collins. Si trattò di una decisione non faci-
le per l’editore, data la mole e la complessità dell’opera,
che avevano già scoraggiato precedenti tentativi. Infi-
ne, mi permetto di menzionare anche il mio manuale
di sociologia economica (Trigilia 1998). Evidentemen-
te sulla possibile influenza del lavoro di Cavalli a livello
internazionale ha pesato la mancata traduzione in lin- Non posso, ovviamente, motivare qui in modo det-
tagliato questo giudizio. Mi limiterò a segnalare sche-
maticamente alcuni elementi essenziali su cui si fonda. Anzitutto, Cavalli prende una chiara posizione critica
nei riguardi della tesi che attribuisce a Weber un’in-
terpretazione basata sul ruolo chiave della religione per
spiegare il capitalismo moderno in Occidente. Com’è
noto, questa interpretazione era al centro delle con-
troversie e delle critiche suscitate dalla cosiddetta “tesi
Weber”. Insistendo sull’importanza dell’interpretazione
“plurifattoriale” di Weber, Cavalli rimette la religione
protestante (in particolare il calvinismo) al suo posto,
molto rilevante ma non unico ed esclusivo. Il prote-
stantesimo serve a spiegare la formazione dello spirito
del capitalismo: una particolare etica economica che è
componente essenziale dello sviluppo del capitalismo in
Occidente, ma che non ne è l’unica causa. Per compren-
dere questo fenomeno più ampio e complesso è neces-
sario guardare non soltanto al percorso evolutivo della
religione in Occidente, ma anche a tre specifici fattori
istituzionali che emergono meglio dalle grandi ricerche
comparate: le città, la scienza razionale e lo stato razio-
nale. Tali fattori risentono certo, a loro volta, della tra- 111 Religione e società: l’influenza di Weber sulla sociologia italiana primi passi nel clima di mobilitazione sociale degli anni
‘70. L’esempio più tipico per cogliere questo fenomeno è
costituito dalle ricerche sullo sviluppo di piccola impre-
sa di cui ha già parlato Arnaldo Bagnasco che ne è stato
protagonista. Per parte mia vorrei aggiungere che pro-
prio la ricerca empirica sulle regioni della Terza Italia
mise ben presto in luce come non potesse interpretarsi
il fiorire delle piccole imprese solo come conseguenza
del forte conflitto industriale manifestatosi nelle grandi
aziende delle grandi città industriali nei primi anni ‘70. E che quindi una lettura del fenomeno influenzata dal
marxismo e basata sull’idea del decentramento produt-
tivo fosse palesemente insufficiente. In realtà, lo sviluppo
di piccola impresa concentrato in alcune aree del paese
sollecitò una spiegazione istituzionale plurifattoriale cer-
to influenzata dalla lezione di Weber. Bisognava infatti
guardare alla rete di città, al saper fare diffuso, al ruolo
della famiglia, delle associazioni, delle subculture politi-
che. E bisognava guardare al particolare capitale sociale
capace di favorire fiducia e sostenere l’intensa coope-
razione produttiva tra le piccole imprese. In particolare, si pre-
sentano comparazioni tra sistemi pluralisti e neo-corpo-
rativi, si analizza la varietà dei capitalismi distinguendo
tra “capitalismo anglo-sassone” e “capitalismo renano”
caratterizzati da diversi assetti istituzionali. E se ne met-
tono in luce le conseguenze non solo sulla performance
economica ma anche sulle disuguaglianze sociali. È da
notare la sintonia da un punto di vista metodologico con
l’approccio weberiano, quindi la convergenza su questo
terreno con gli sviluppi del lavoro di Cavalli che lega
saldamente la sociologia alla storia. Allo stesso tempo si
può però cogliere la divergenza tra la sociologia politica
di Cavalli e la sociologia economica dei capitalismi sul
piano sostantivo. Com’è noto, Cavalli mette in eviden-
za il ruolo – a suo avviso fondamentale – per assicurare
governabilità e stabilità delle istituzioni democratiche di
quelle forme di democrazia plebiscitaria che assicura-
no una leadership personale forte, cioè con le sue paro-
le «capi devoti agli interessi di fondo di un paese, e di
una civiltà, e muniti dei poteri necessari per governare
effettivamente» (Cavalli 1981: 256). Egli vede i segni più
consistenti di questa leadership nelle democrazie anglo-
sassoni e in quella francese, contrapposte alle «democra-
zie acefale» (tra cui l’Italia). Dall’altra parte, la sociologia
economica e la political economy contrappongono a que-
sta prospettiva weberiana un’altra visione che si potrebbe
definire invece “kelseniana”; nel senso che dà rilievo non
solo alla governabilità, ma anche alla capacità dell’asset-
to istituzionale di favorire l’integrazione sociale e uno
sviluppo inclusivo attraverso la rappresentanza degli
interessi più deboli, il compromesso sociale e politico, la
concertazione degli interessi, l’istituzionalizzazione del
conflitto. Insomma, quella che si potrebbe definire una
“democrazia negoziale” basata su un’efficace condivi-
sione del potere politico piuttosto che una “democrazia
maggioritaria e plebiscitaria” che punta alla verticalizza-
zione del potere attraverso la personalizzazione della lea-
dership e le sue qualità carismatiche. ship e soprattutto segnalano che la personalizzazione
non favorisca specie negli anni più recenti – come nelle
attese della visione weberiana – una maggiore capacità
di risposta delle democrazie maggioritarie ai processi di
destabilizzazione sociale e alle sfide per le stesse istitu-
zioni democratiche che sono cresciute. Vorrei concludere su questo confronto e sui proble-
mi di ricerca che restano aperti con una nota personale. Ho avuto la possibilità di discuterne con Luciano Cavalli
in occasione del convegno Max Weber oggi tenutosi nel
2015 presso il Dipartimento di Scienze Politiche e Socia-
li. In quella circostanza, commentando con attenzione
la mia relazione, Cavalli mi confermò le sue convinzio-
ni sul ruolo dei leader carismatici e la democrazia ple-
biscitaria weberiana. Per parte mia, cercai di spiegare
ulteriormente i dubbi e gli interrogativi, credo con non
molto successo. Ma voglio concludere ricordando che
l’incontro con Weber che ebbi prima come studente alle
lezioni di Cavalli, e poi da giovane ricercatore al quale
Cavalli chiese di preparare dei seminari sulla metodo-
logia weberiana e sulle origini del capitalismo, hanno
segnato a fondo il mio percorso di ricerca. È un testo con
una chiara impronta weberiana dal punto di vista del
metodo, che àncora la sociologia alla storia, valorizza le
macro-comparazioni come generatrici di ipotesi causa-
li e sostiene una visione istituzionalista dell’economia e
dello sviluppo come fuoco per delimitare i confini e lo
spazio della sociologia economica. Ricordo ancora un aspetto delle problematiche di
ricerca: quello relativo al Mezzogiorno. Ben presto l’a-
nalisi dello sviluppo della Terza Italia finì infatti per
sollecitare interrogativi sul mancato sviluppo autono-
mo del Sud. E anche qui favorì la messa a punto di una
spiegazione istituzionale basata sul contesto endogeno
in contrasto con interpretazioni di taglio economico –
allora ma ancora oggi prevalenti – o a semplicistiche ma
ricorrenti spiegazioni neomarxiste, come quelle ispira-
te dall’idea di mero sfruttamento delle regioni arretra-
te da parte di quelle avanzate (teorie della dipendenza). Insomma, si cominciò a guardare anche qui al ruolo
delle città, che in chiave weberiana apparivano più come
“città di consumatori” che “di produttori”, prevalenti
invece nel centro-nord. Si pose anche più attenzione al
“capitale sociale”, qui invece sfavorevole allo sviluppo
perché le relazioni fiduciarie sono più impiegate nella
cattura di benefici particolaristici da parte della politica
o alla presa della criminalità. E naturalmente ci si chiese
il perché, facendo largo uso dell’analisi comparata come
generatrice di ipotesi e spiegazioni causali, e costruendo
modelli di sviluppo territoriale con delle coordinate spa-
ziali e temporali ben delimitate (nel linguaggio weberia-
no dei “tipi ideali”). 3. Venendo ora alle problematiche di ricerca che
hanno visto impegnata la nascente sociologia economi-
ca italiana, vorrei ricordare come l’influenza di Weber
abbia contribuito a contrastare prima e a ridimensio-
nare dopo il forte ancoraggio al marxismo – o meglio
a varie forme di neo-marxismo – che ne orientarono i Questo mi porta all’ultimo punto che vorrei tratta-
re, quello relativo alla partecipazione di una parte della
nuova sociologia economica italiana all’esperienza della
rivista Stato e Mercato. La rivista nasce nel 1981 e si basa 112 Carlo Trigilia Carlo Trigilia sulla collaborazione tra studiosi italiani e stranieri, pre-
valentemente americani. Questi ultimi contribuiscono a
orientare la rivista verso la political economy comparata,
cioè un approccio che si propone di analizzare come l’e-
conomia sia influenzata dall’assetto politico-istituzionale
dei diversi paesi. Vengono costruiti tipi ideali empiri-
camente fondati dei diversi modelli di capitalismo ed è
ampiamente utilizzata la macro-comparazione per la for-
mulazione di spiegazioni causali. In particolare, si pre-
sentano comparazioni tra sistemi pluralisti e neo-corpo-
rativi, si analizza la varietà dei capitalismi distinguendo
tra “capitalismo anglo-sassone” e “capitalismo renano”
caratterizzati da diversi assetti istituzionali. E se ne met-
tono in luce le conseguenze non solo sulla performance
economica ma anche sulle disuguaglianze sociali. È da
notare la sintonia da un punto di vista metodologico con
l’approccio weberiano, quindi la convergenza su questo
terreno con gli sviluppi del lavoro di Cavalli che lega
saldamente la sociologia alla storia. Allo stesso tempo si
può però cogliere la divergenza tra la sociologia politica
di Cavalli e la sociologia economica dei capitalismi sul
piano sostantivo. Com’è noto, Cavalli mette in eviden-
za il ruolo – a suo avviso fondamentale – per assicurare
governabilità e stabilità delle istituzioni democratiche di
quelle forme di democrazia plebiscitaria che assicura-
no una leadership personale forte, cioè con le sue paro-
le «capi devoti agli interessi di fondo di un paese, e di
una civiltà, e muniti dei poteri necessari per governare
effettivamente» (Cavalli 1981: 256). Egli vede i segni più
consistenti di questa leadership nelle democrazie anglo-
sassoni e in quella francese, contrapposte alle «democra-
zie acefale» (tra cui l’Italia). Dall’altra parte, la sociologia
economica e la political economy contrappongono a que-
sta prospettiva weberiana un’altra visione che si potrebbe
definire invece “kelseniana”; nel senso che dà rilievo non
solo alla governabilità, ma anche alla capacità dell’asset-
to istituzionale di favorire l’integrazione sociale e uno
sviluppo inclusivo attraverso la rappresentanza degli
interessi più deboli, il compromesso sociale e politico, la
concertazione degli interessi, l’istituzionalizzazione del
conflitto. Insomma, quella che si potrebbe definire una
“democrazia negoziale” basata su un’efficace condivi-
sione del potere politico piuttosto che una “democrazia
maggioritaria e plebiscitaria” che punta alla verticalizza-
zione del potere attraverso la personalizzazione della lea-
dership e le sue qualità carismatiche. Il confronto su queste diverse visioni della democra- sulla collaborazione tra studiosi italiani e stranieri, pre-
valentemente americani. Questi ultimi contribuiscono a
orientare la rivista verso la political economy comparata,
cioè un approccio che si propone di analizzare come l’e-
conomia sia influenzata dall’assetto politico-istituzionale
dei diversi paesi. Vengono costruiti tipi ideali empiri-
camente fondati dei diversi modelli di capitalismo ed è
ampiamente utilizzata la macro-comparazione per la for-
mulazione di spiegazioni causali. Citation: Rossana Trifiletti (2023) Cavalli,
Weber e la formazione dei sociologi
sui testi classici. SocietàMutamento-
Politica 14(27): 113-121. doi: 10.36253/
smp-14342 Rossana Trifiletti Copyright: © 2023 Rossana Trifiletti. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Premessa. Avevo già tentato di tracciare un bilancio dell’influenza che
Luciano Cavalli ha esercitato mediante la sua assidua frequentazione dei clas-
sici della sociologia su tutti i suoi allievi – anche, chi più chi meno - quelli
allontanatisi in seguito dai suoi temi, ma spesso andati poi in cerca di altri
classici altrettanto pregnanti (Trifiletti 2008) anche in altre discipline. Sono
ormai trascorsi più di 50 anni da un fatidico seminario su Max Weber reli-
gione e società (Cavalli L. 1968), tenutosi l’anno immediatamente successivo
alla uscita di quel libro e un anno prima della pubblicazione de Il mutamen-
to sociale (Cavalli L. 1970). Il nostro percorso di allievi di allora nella disci-
plina sociologica oggi è forse alla fine ma devo constatare che alcuni aspetti
di questo legame a quei due lavori e, per loro tramite a Weber, non li avevo
ancora capiti fino in fondo e me ne sono dovuta rendere conto ancora succes-
sivamente a quel primo bilancio. Forse i debiti, specie quelli di questa natu-
ra, non si capiscono mai del tutto, e solo un poco per volta. E si è rafforzata
nel tempo soprattutto l’impressione, anche un po’ inattesa, che molti aspetti
di questa influenza di Cavalli siano stati verosimilmente altrettanto impor-
tanti per colleghi che hanno condiviso allora quella esperienza o anche che
siano stati coinvolti successivamente su questi stessi temi cruciali, pur appro-
dando poi a percorsi diversissimi, anche in altre scienze sociali. Mi sembra,
oggi, che questo discenda dal fatto che i classici in sociologia sono davvero
costitutivi della disciplina più che in altri settori (Alexander 1981; Cavalli A. 2022) e questo ce lo aveva mostrato nella didattica quotidiana ma anche con
riferimento alla sua pratica di ricerca genovese Luciano Cavalli proprio in
quel primo seminario, col suo modo di approcciarsi a Weber e di utilizzare i
suoi strumenti per la conoscenza empirica della sua e della nostra contempo-
raneità. RIFERIMENTI BIBLIOGRAFICI Bendix R. (1962), Max Weber: An Intellectual Portrait,
Methuen, Londra. Cavalli A. (1972), Economia e società, il Mulino, Bologna. – (2012), Max Weber e la sociologia italiana, in Momenti
di storia del pensiero sociologico, Ledizioni, Milano. Cavalli L. (1968), Max Weber religione e società, Il Muli-
no, Bologna. g
– (1981), Il capo carismatico, il Mulino, Bologna. Collins R. (1980), «Weber’s Last Theory of Capitalism: A
Systematization», in American Sociological Review,
vol. 45, n.6. Ferrarotti F. (1965), Max Weber e il destino della ragione,
Laterza, Bari. Martinelli A. (1986), Economia e società: Marx, Weber,
Schumpeter, Polanyi, Parsons e Smelser, Edizioni di
Comunità, Milano. Parsons T. (1937), The structure of social action,
McGrawHill, New York. Rossi P. (1971 [1956]), Lo storicismo tedesco contempora-
neo, Einaudi, Milano. Trigilia C. (1998), Sociologia economica. Stato, mercato e
società nel capitalismo moderno, il Mulino, Bologna. Il confronto su queste diverse visioni della democra-
zia resta aperto, anche perché la ricerca comparata sulle
democrazie avanzate mostra dei risultati che da un lato
confermano la tendenza crescente alla personalizzazione
politica, ma dall’altro lato indicano come essa si accom-
pagni a un sempre più rapido “consumo” delle leader- – (2007), «Introduzione», in M. Weber, La storia econom-
ica, Donzelli Editore, Roma, pp. III-LVI. ica, Donzelli Editore, Roma, pp. III-LVI. Weber M. (1958), Il metodo delle scienze storico-sociali,
Einaudi, Milano. – (1993), Storia Economica, Donzelli Editore, Roma. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 113-121, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14342 Un
po’ come nel racconto di Edward Shils riportato da Scaff
che, quando leggeva Weber, a tratti si sentiva mancare
il fiato intravedendo nessi causali a cui non avrebbe mai
pensato e doveva alzarsi e fare due passi per riprendersi
(Scaff 2011: 255). E bisogna non sottovalutare il fatto –
altro veicolo di fascinazione per noi, sia allievi che gio-
vani assistenti – che negli anni ’60 le ricerche empiriche
italiane importanti della prima generazione dei sociologi
si potevano ancora elencare in poche righe (cfr. Cavalli
A. 2021: 28). quelle circostanze abbiamo respirato. Certamente non era
quella che gli avrebbe poi assegnato la scuola sociologi-
ca empirista, nella formulazione più chiara di Boudon,
secondo cui classico della sociologia si può ritenere solo
un autore che abbia “contribuito a fare della discipli-
na una scienza a tutto tondo, una scienza come le altre,
finalizzata essenzialmente a spiegare fenomeni sociali
enigmatici a partire da teorie che obbediscono a criteri ai
quali è asservita qualsiasi disciplina scientifica degna di
questo nome” (Boudon 2002 [1998]: 7, corsivo mio). Un
simile taglio scientista avrebbe portato a mettere al cen-
tro di quel seminario piuttosto il Weber della Wissen-
schaftslehre che non era, tuttavia, così cruciale per Caval-
li e forse, secondo la sua lettura di allora, nemmeno per
Weber stesso. Eppure, certo non si sottovalutavano nel
corso i grandi passi di fondazione nella lettura di insieme
di Weber compiuti in Italia soprattutto da Pietro Rossi a
partire dal metodo (Rossi 1954; 1956; cfr. Marra 2009). A uno dei suoi libri più famosi nella weberologia italia-
na (Rossi 1981), anni dopo, avrebbe del resto collaborato
anche Luciano Cavalli. Tuttavia, in quel famoso semina-
rio la metodologia di Weber entrò per noi, ma a latere,
per iniziativa autonoma di un allievo, magari uno assai
brillante. Ci bastavano, in fondo, per entrare nel metodo
comparativo di Weber la comprensione del multifattoria-
lismo e della funzione degli idealtipi, senza entrare nelle
sottigliezze del bilanciamento fra Rickert e Dilthey. Semmai in quegli anni Cavalli per l’insegnamento
più generale del metodo in sociologia si appoggiava più
volentieri alle esperienze di campo (Cavalli L. Un aspetto importante di questa necessaria riconsiderazione di quella
esperienza deriva sicuramente dai grandi progressi che si sono nel frattempo
realizzati nell’analisi critica del pensiero di Weber. Anche per Cavalli, allo-
ra, come sosterrà Robert Bellah diversi anni dopo, la sociologia delle grandi
religioni di salvazione che ripercorremmo in quel seminario, ma soprattutto
la Vorbemerkung (Weber 1965 [1920]) e la Zwischenbetrachtung (Weber 1989
[1915-16]) successive che collegavano quegli studi al resto dell’opera, erano da
considerarsi i testi chiave del sistema teorico weberiano (Bellah 1999: 279; cfr. Marra 2022: 353), certo non un aspetto secondario o accessorio ai temi poli-
tici o economici. Veniva così anticipata in quegli anni nella lettura italiana di Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 113-121, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14342 114 Rossana Trifiletti Weber una svolta che nel dibattito internazionale anglo-
sassone, ancora influenzato dalle traduzioni di Gerth e
Mills (1949) e di Parsons si avrà solo a partire dal 1975
(Schluchter 2021: 14; Silla 2016) soprattutto con la Weber
renaissance, una rilettura, finalmente, dell’insieme della
sua opera, focalizzata appunto soprattutto sulla socio-
logia delle religioni nella versione di Tenbruck (1975) e,
attraverso di essa, sul processo di razionalizzazione in
quella di Schluchter (1987 [1979])1. Erano effettivamente
questi i due caposaldi anche della lettura weberiana di
Cavalli di allora che, pur utilizzandola largamente, non
mi sembra che considerasse Economia e Società il capo-
lavoro compiuto di Weber, come era normale a quell’e-
poca2, mentre, come è stato ben dimostrato in seguito,
il testo trasportava molte ingenuità editoriali di Marian-
ne e dello stesso Winckelmann (Schluchter 2021; Adair-
Toteff 2011). E così ci appariva Cavalli a quell’epoca, da
studenti: forte delle sue ricerche di campo genovesi ma
allo stesso tempo weberiano3 appassionato di storia e di
comparatistica quanto Weber stesso; un filone dei suoi
interessi che avrebbe sviluppato di lì a poco (Cavalli L. 1974; 1976). Era anche un caso - agli occhi semplificato-
ri di giovani studenti - di apparente somiglianza fra due
personalità con la loro idea coinvolgente dello studio, ma
soprattutto un caso di endless fascination di Cavalli attra-
verso Max Weber (Scaff 2011: 250; Adair-Toteff 2014). 3 Anche secondo Alessandro Cavalli, Luciano Cavalli va forse conside-
rato l’unico autentico weberiano fra i sociologi italiani insieme a Sandro
Segre (Cavalli A. 2006). 1 Con una possibile ricaduta delle nuove attenzioni del dibattito statuni-
tense sulla stessa sociologia tedesca che veniva da una parentesi di oblio
di Weber nel dopoguerra (Connell 1997: 1540), a far data in realtà dal
1964 col famoso Convegno di sociologia di Heidelberg a 100 anni dalla
nascita di Weber, dove, appunto, erano presenti oltre a Marcuse e Ador-
no anche Parsons e Bendix e un giovane Pietro Rossi (Schmitt 2006).
2 Basti citare il giudizio di un classico - lontanissimo dai weberolo-
gi riconosciuti - Howard Becker, su Economia e Società, un libro a
suo dire “quasi sovrumano”. E questo ancor prima della traduzione di
Gerth e Mills e subito dopo la traduzione di Parsons dell’Etica (Becker
1934: 403, n. 10).
3 Anche secondo Alessandro Cavalli, Luciano Cavalli va forse conside-
rato l’unico autentico weberiano fra i sociologi italiani insieme a Sandro
Segre (Cavalli A. 2006). 1 Con una possibile ricaduta delle nuove attenzioni del dibattito statuni-
tense sulla stessa sociologia tedesca che veniva da una parentesi di oblio
di Weber nel dopoguerra (Connell 1997: 1540), a far data in realtà dal
1964 col famoso Convegno di sociologia di Heidelberg a 100 anni dalla
nascita di Weber, dove, appunto, erano presenti oltre a Marcuse e Ador-
no anche Parsons e Bendix e un giovane Pietro Rossi (Schmitt 2006). 2 Basti citare il giudizio di un classico - lontanissimo dai weberolo-
gi riconosciuti - Howard Becker, su Economia e Società, un libro a
suo dire “quasi sovrumano”. E questo ancor prima della traduzione di
Gerth e Mills e subito dopo la traduzione di Parsons dell’Etica (Becker
1934: 403, n. 10). In questo modo quel seminario ci ha fatto addentrare
in mondi come la Cina e l’India, paesi affascinanti di
mistero che la nostra cultura scolastica aveva semmai
appena sfiorato, ricostruendo un affresco complesso
dove le concatenazioni causali si moltiplicavano, erano
biunivoche e inattese come in una analisi figurazionale
(Knöbl 2015: 543) – anche se allora non sapevamo cosa
fosse. E che l’argomentazione di Weber si facesse strada
introducendo altri e diversi nessi causali che complica-
vano via via il ragionamento era – se possibile – anche
più affascinante dal punto di vista della funzione di tou-
chstone: era la metodologia applicata, la dimostrazio-
ne pratica di come l’insight epocale dell’Etica, nella sua
natura idealtipica, si articolava e si complicava se messo
a confronto con aspetti multiformi della realtà storica,
culturale, istituzionale di altre civiltà, pur restando del
tutto plausibile la sostanza della prima intuizione. sociologia americana esattamente il difetto di non essere
seriamente storica e comparativa, come l’uso degli ideal-
tipi weberiani avrebbe reso possibile (Becker 1934). Del resto anche Weber è stato, a suo modo, un
metodologo controvoglia come si è sempre più chiarito
negli anni e come molti avrebbero sottolineato in segui-
to (Veca 1981: 4-5; Oakes 1982; Scaff 2016 [2015]: 56):
come è noto gli scritti metodologici di Weber sono occa-
sionali, incompiuti e asistematici (Cavalli A. 1981: 27), da
appassionato delle scienze di realtà e da vero empirista
che considerava il metodo migliore quello che di volta in
volta era servito a risolvere concreti problemi di ricerca,
senza alcuna ambizione a porsi come metodologo puro,
arrivando piuttosto a parlare di “pestilenza metodologi-
ca” a proposito del Methodenstreit suo contemporaneo
(Roversi 1980: IX): Le scienze sono state fondate, e il loro metodo viene ulte-
riormente sviluppato, soltanto ponendo in rilievo e risol-
vendo problemi sostanziali; e mai finora hanno contribuito
in maniera decisiva a tale scopo le pure considerazioni del-
la conoscenza e di metodologia (Weber 1958 [1906]: 148). 1961, parti
II e III), facendo riferimento soprattutto ai Lynd, a Tho-
mas e al primo Merton di Teoria e struttura sociale che,
a somiglianza di Boudon, teorizzava effettivamente la
separazione analitica necessaria fra la storia – rigorosa-
mente non scientifica – della sociologia e dei suoi mae-
stri e una teoria scientifica che si sarebbe prima o poi
sviluppata permettendo il funzionamento della scien-
za ordinaria e la cumulazione delle conoscenze (1971
[1949]: 50) dimenticando i suoi classici. Ma Cavalli, pur
apprezzando di Merton la capacità di temperare l’empi-
rismo radicale con la teoria di medio raggio e la consa-
pevolezza delle conseguenze inintenzionali dell’azione,
nello sviluppo dell’unica teoria scientifica cumulativa
non ci credeva già più molto (Cavalli L. 1962b: XII-XII)
e criticava anche esplicitamente Merton per i rischi di
astoricismo che potevano sempre aprire una possibilità
di “contrabbandare senza più discussione tutti i tentativi
scientistici” (Cavalli L. 1963: 222): come da allora in poi
si sarebbe verificato sempre più nella sociologia america-
na mainstream. Colpisce in questo senso la quasi perfet-
ta assonanza con un padre indiscusso della contrapposta
sociologia qualitativa statunitense che rimproverava alla 2. La funzione dei classici in sociologia e il mutamen-
to sociale. Vale la pena allora di chiedersi meglio quale
fosse esattamente l’idea della funzione dei classici che in 115 Cavalli, Weber e la formazione dei sociologi sui testi classici dialogo implicito con i suoi Zeitgenossen (Lepsius 2016)
e il suo possibile pubblico, come bisognerebbe sem-
pre fare andando oltre il testo (cfr. Skinner 1969: 47). Questo era senz’altro vero e particolarmente suggestivo
quando, dopo la rivelazione del nesso causale dell’Etica
protestante con l’agire economico, ci si addentrava nel-
le molte variazioni multicausali e biunivoche che Weber
identificava via via nelle altre religioni di salvazione,
nei distinti livelli della condotta di vita, dell’organizza-
zione societaria, della cultura e delle visioni del mondo
(D’Andrea 2018), nei loro possibili nessi causali con l’e-
conomia, le istituzioni economiche, la storia economica
(cfr. Schluchter 2020, parte seconda). Oltretutto Cavalli
era immune dai molti errori di traduzione che sono stati
documentati negli anni (Bianco 2006), perché per i ter-
mini più pregnanti si basava sempre sull’originale tede-
sco, certo quello dei Gesammelte Aufsätze zur Religions-
soziologie nell’edizione del 1966 di cui disponeva all’epo-
ca. 5 Un giudizio anche un poco esagerato, si veda la sua critica alla autore-
ferenzialità delle diverse tendenze tecniche via via di moda per misurare
le covariazioni (Stinchcombe 1984: 55). 4 Nonostante le cautele al riguardo espresse dallo stesso Weber nella
Vorbemerkung. L’a-
more dei classici allora sembra poter diventare una via
temperata fra questi due estremi e se ne identificano le
funzioni positive. Secondo Alexander (1990 [1987]: 75)
nelle scienze sociali “sovradeterminate dalla teoria e sot-
todeterminate dai fatti”, e a maggior ragione, quindi, in
sociologia, lo studio dei classici ha una funzione solidifi-
cante, semplifica la complessità: permette di non dovere
sempre riandare ai presupposti ultimi del discorso e di
dare per scontate “le assunzioni di sfondo più generali
che rimangono implicite e relativamente invisibili nelle
scienze naturali” (ibidem: 70) finché vige il paradigma. Poiché la crisi di paradigma è, invece, inevitabile e ende-
mica nelle scienze sociali, il riferimento ai classici per-
metterebbe poi di giungere a “covering laws relativamen-
te predittive […] all’interno di diversi e concorrenziali
punti di vista” (ibidem: 71). cata di John Madge (1966 [1962]), una raccolta, appun-
to, non tanto di principi metodologici astratti, quanto di
esperienze di ricerca “classiche” analizzate criticamente e
che già, prima che Merton la teorizzasse (Merton e Bar-
ber (2002 [1992]), erano ricche di casi di serendipity. E
non a caso in quel testo ai Lynd si affiancavano, Thomas,
Mayo, Foote Whyte e Zorbaugh, che abbiamo amato
allora come eroi sul campo e maestri fondatori di socio-
logie speciali. Allo stesso modo la sua specifica atten-
zione, tutt’altro che comune per l’epoca, per la tecnica
dello studio di caso (Cavalli L. 1962a) testimonia della
sua apertura verso metodi più qualitativi, che aveva del
resto già messo in pratica nelle ricerche genovesi (Cavalli
L. 1961) e avrebbe ampiamente utilizzato in seguito, con
una particolare attenzione per il metodo biografico6. Ma
anche allora, nel Mutamento Sociale che in un certo sen-
so abbiamo visto comporsi in quegli anni, si approccia-
va a ciascun autore di quelli che ci ha insegnato a fre-
quentare, con un forte interesse biografico e una chiara
intenzione storicizzante che leggesse il contesto in modo
non riduttivo e non figé in senso deterministico. E certo
non è un caso che il manuale rivelasse la sua imposta-
zione weberiana fino dal sottotitolo: Sette ricerche sulla
civiltà occidentale. Ma forse il Mutamento Sociale, era
anche un po’, come allora non avevo capito, un tentativo
di “rimettere in piedi” un Parsons capovolto, completan-
do la Struttura dell’azione sociale per l’aspetto del muta-
mento, che come Cavalli sottolineava, nell’impostazione
del primo testo di Parsons “non è ancora approfondito
ma sembra chiaro fin d’ora che sarà difficile spiegarlo”
(Cavalli L. 1964: 57; cfr. 64 ss. 80 ss.). Dimenticare i classici ha comportato secondo altri
(Cavalli A. 2022) anche un eccesso di frammentazione
nelle sociologie speciali. La confusione fra storia e siste-
matica che Merton criticava non sarebbe, allora, solo
inevitabile ma addirittura positiva, per Alexander la
separazione non può esistere (1990 [1987]: 32 trad. mia), i
sociologi debbono barcamenarsi tra scienze sociali e stu-
di umanistici, appunto, con la storia in particolar modo
(cfr. Cavalli A. 1998). Del resto, anche chi si è applicato
a documentare le esagerazioni nella valutazione e le tra-
sformazioni nel tempo della selezione dei maestri classi-
ci ai fini della formulazione del canone sociologico del
mondo anglosassone, conclude che comunque Marx,
Durkheim e Weber saranno sempre presenti nella sto-
ria della disciplina, anche se, magari, in proporzioni più
realistiche (Connell 1997). Guardando da oggi al Mutamento sociale, certo,
mancavano allora molti classici che ci sono sembrati
fondamentali in seguito: sicuramente Simmel, Schütz,
Bourdieu, Goffman, Elias, ma forse, col senno di poi
aggiungerei anche, Boltanski, Latour, e sicuramente
Burawoy per il suo appello alla public sociology, proposta
che sicuramente avrebbe riscosso la simpatia di Luciano
Cavalli. In passato ero stata affascinata dalla proposta di let-
tura di Arthur Stinchcombe della funzione dei classici
in sociologia, per spiegarmi l’atmosfera straordinaria di
quel lontano seminario (Trifiletti 2008): nei suoi termini
la funzione di touchstone dei classici sarebbe non tanto
quella, impropria, di derivare ipotesi ancora attuali dai
loro scritti, ma piuttosto quella di fornire uno standard
intellettuale elevato ed esemplare cui misurarsi o ambi-
re di misurarsi, una pietra di paragone che tenderebbe
ad avere una specifica ricaduta preferenziale “di svilup-
po” sulla formazione dei giovani sociologi (Stinchcombe
1968). Tanto più che qualche anno dopo Stinchcombe
avrebbe precisato la funzione di touchstone come molto
vicina all’idea del paradigma scientifico di Kuhn (Stin-
chcombe 1982), come se la funzione dei classici fosse un
po’ quella di mostrare in concreto i massimi livelli a cui
è possibile portare in sociologia la scienza normale. Di
recente ho scoperto che lo stesso Merton ha insegnato
per 10 anni un corso su Tarde, Cooley, Pareto, Weber e
Durkheim, rifiutandosi, effettivamente, di usare la let-
teratura critica su di essi, tuttavia indagando a fondo le
loro strategie di ricerca (Swedberg 2020). Invece Cavalli,
allora, prendeva molto seriamente la funzione di esplo-
razione del paradigma della sociologia, documentando-
ci la vastità dell’erudizione transdisciplinare di Weber
messa al servizio degli studi religiosi, come la massima
possibile per la sua epoca4, guardando a fondo anche il E, come ci suggerisce lo stesso Stinchcombe (1982:
5), abbiamo qui una riprova che “la scienza di buon
livello [first class] funziona con standards anche estetici
quanto con standards logici ed empirici”.f Tuttavia, Jeffrey Alexander nella sua difesa del ruo-
lo dei classici ha criticato la posizione di Stinchcombe
considerandolo una sorta di positivista mascherato5 che
sottovaluterebbe il contesto non empirico in cui lavora-
no sempre i sociologi teorici, che “non lavorano solo coi
fatti applicando la ragione. Lavorano con teorie, le teorie
dei loro padri, cercando di elaborarle e specificarle per
spiegare serie di fatti nuovi e inattesi” (Alexander 1981:
290) entro tradizioni di scuole consolidate. In realtà Ale-
xander va molto più in là di questo: la centralità dei clas- 116 Rossana Trifiletti sici e delle tradizioni di scuola per la sociologia risulte-
rebbe poco condivisa e forse un poco sospetta non solo
ai positivisti radicali – alla ricerca della scientificità più
dura – ma anche ai convinti umanisti à la Skinner. 6 Rimando al contributo di Paolo Turi in questo stesso volume. Molti di questi contributi, però, sono carat-
terizzati dal vizio psicologistico di far derivare i tratti
dell’opera dalle circostanze della vita (Wallach Bologh
1987; Whimster 1999) e qualcuno rasenta i rischi peg-
giori della psicobiografia (Mitzman 1970; Radkau 2005;
2008); spesso la “rivelazione” passa oggi per una riva-
lutazione della biografia di Marianne (1995 [1984]) e
delle sue allusioni allo stato di salute del marito (Meu-
rer 2010; Antonelli 2020; Nocenzi, Deriu 2020), ma mi
sento di dire che c’era già tutto ciò che riguarda davve-
ro l’insieme della teoria weberiana nel grande affresco
della Zwischenbetrachtung. Come dice Robert Bellah è il
testo dei testi, “il testo chiave di tutto il corpus weberia-
no” (1999: 179 trad. mia); ma più in generale la sociolo-
gia delle religioni resta sempre “il più ambizioso studio
comparativo” di tutta la sociologia. Nella Zwischenbe-
trachtung Weber è particolarmente attento alla sfera
emotiva mentre descrive le complesse interdipendenze e
tensioni fra le sfere della vita (Cavalli A. 2020: 660-661;
Scaff 1987: 743) e le considera tutte egualmente rilevan-
ti, arte ed erotica comprese. Ma non bisogna dimenticare
che considera questo un effetto della razionalizzazione
esterna che la religione opera in Occidente rafforzando
paradossalmente la Eigengesetzlichkeit di ogni sfera (cfr. Marra 2020: 132). psicologia e la psicanalisi. E Luciano Cavalli, pur nel suo
modo prudente, era sicuramente un trespasser (cfr. Hir-
schman 1981), curioso di storia ma anche di psicologia,
di psicanalisi, di biografia, di letteratura… Un esempio
affascinante ma anche dannatamente esplicativo di cosa
dovesse essere una buona sociologia capace di traversa-
re continuamente i confini delle discipline: proprio come
sostiene oggi Alessandro Cavalli che abbiamo disimpa-
rato a fare più di recente in sociologia, chiudendoci nei
recinti ristretti delle sociologie speciali. Ma Il mutamen-
to sociale era anche, un tentativo abbastanza nuovo per
l’epoca, un ponte gettato tra classici consolidati e perso-
naggi contemporanei che ben rendevano il colore della
sua sociologia che, appunto, nella sua formazione ameri-
cana sapeva distinguere fra cattivi e buoni maestri, met-
tendo volutamente Robert Lynd e Charles Wright Mills
sullo stesso piano di Marx, Durkheim, Mosca e Weber. 3. Il lascito di Weber o del Weber di allora? 7 Si vedano l’Antikritisches Schlusswort 1910 che Cavalli conosceva
benissimo (1968: 42) come le precedenti risposte ai critici comparse
sull’Archiv egualmente utilizzate in Max Weber religione e società.
8 Anche se Baumgarten (1964) ha sottolineato l’importanza delle
aggiunte successive alla Zwischenbetrachtung che ora si potrebbero
ricostruire sistematicamente. Chi fra i suoi allievi ha iniziato poi dei percorsi
verso questi o altri autori seguendo le proprie preferenze
usava comunque il metodo che ci ha insegnato lui: non
solo serietà e fedeltà nel leggere i classici sulla base di un
esteso lavoro testuale e della conoscenza filologica della
critica relativa, ma osare, come loro, varcando i confini
disciplinari, cercando nuovi ambiti influenzati da saperi
dissimili, dove le diverse discipline sociali si incontrano
e si intrecciano A ben guardare, anche nel Mutamen-
to sociale con Durkheim si incontravano la statistica
sociale e l’antropologia, con Mosca la scienza politica,
con Marx e Weber l’economia e la storia, con Parsons la È anche vero che negli anni ’70 il desiderio di una
sociologia sostanziosamente scientifica era più vivo e
urgente di adesso, ma Luciano Cavalli non è mai stato
aridamente positivista e scientista: la sua precoce cri-
tica della Natural Science Sociology testimonia della
sua conoscenza di prima mano delle origini devian-
ti dell’empirismo astratto nella sociologia americana
(Cavalli L. 1963), che era stata tuttavia, per altre impor-
tanti direzioni di ricerca, la base della sua formazione e
di cui andava giustamente orgoglioso. Certo allora stu-
diavamo il manuale di William Goode e Paul Hatt di cui
Cavalli aveva curato con Anna Servettaz la traduzione
per Il Mulino, ma di cui pure sottolineava ampiamente
i limiti nell’introduzione (Cavalli L. 1962b: XIV-XVI);
questo approccio, comunque, veniva temperato nella
nostra formazione dalla grande antologia oggi dimenti- 117 Cavalli, Weber e la formazione dei sociologi sui testi classici Weber tra il 1915 e il 1920, e per una pubblicazione a cui
teneva molto. In questo il nostro Weber di allora, bor-
ghese tutto di un pezzo, risulta però assai dissimile dal
Weber “riscoperto” di recente e in qualche modo ufficia-
lizzato in Italia nel grande convegno del 2020 Con Max
Weber a 100 anni dalla morte, come persona profon-
damente mutata negli ultimi anni della sua vita, aven-
do riscoperto e rivalutato amore, erotismo, arte e forse
persino una contenuta gioia di vivere (Green 1986; Scaff
1989: 102-112; Whimster, Heuer 1999; Cotesta 2019; Pal-
ma 2020; Rusconi 2020; Marra 2020). In realtà sapeva-
mo già quasi tutto dallo straordinario numero di biogra-
fie di Weber accumulatesi nei decenni (Schwinn 2020:
353-354). In Max Weber religione e società la musica
armonica e sinfonica razionale compariva all’improvviso
come raggiungimento del razionalismo occidentale solo
nella Vorbemerkung, che però Cavalli scindeva in due
distinte trattazioni, all’inizio e nelle conclusioni, sepa-
rando giustamente le sue funzioni introduttive e quelle
di punto di arrivo di tutto lo sviluppo teorico; eppu-
re per degli studenti ai primi passi in sociologia sentire
arrivare il risultato della Sociologia della musica, con “la
nostra orchestra col quartetto ad archi posto a nucleo
centrale e colla sua organizzazione dell’insieme degli
strumenti a fiato, il basso di accompagnamento […] le
nostre sonate, sinfonie ed opere” (Weber 1965 [1920]:
65), più che un effetto di serendipity era una vera e pro-
pria rivelazione, quanto l’uso borghese del pianoforte. di Marianne (Weber 2012 [1918-20]; Roth 2010). Ma la
sostanza più interessante di quello che scoprono i com-
mentatori di oggi che recuperano quella parte nascosta
della vita di Weber, c’era già tutta. Con nessi, già molto
chiari negli anni ’60, che ci portavano dalla Zwischenbe-
trachtung, alla Einleitung, alle conferenze del 1917 e del
1919 e alla Vorbemerkung, sottolineando aspetti cruciali
di unitarietà dell’opera. Per esempio, a proposito della Zwischenbetrachtung
Cavalli sottolineava che il destino del puritano, idealiz-
zato fino a quel momento, sarebbe stato nel processo di
razionalizzazione (e nella gabbia di acciaio)9 quello di
essere vincolato a una “chiarezza senza amore” (Caval-
li L. 1968: 288) nella condotta di vita, pur se questo era
indicato di passata in un paragrafo chiaramente presen-
tato come “accessorio ai nostri temi”, significativamen-
te intitolato Altre sfere. Forse questo tratto di una certa
pudicizia è di nuovo abbastanza tipico di Luciano Caval-
li anche se, più avanti negli anni, sono sicura che cono-
sceva bene almeno l’inizio della svolta di valutazione
della biografia di Weber: ho visto sia il Mitzman (1970)
che il Green (1974) tra i suoi libri.f Forse, alla luce della critica successiva, un’altra sot-
tovalutazione di allora riguarda l’unitarietà delle due
conferenze sul Beruf, anche per effetto della stermina-
ta passione di Cavalli per Politik als Beruf, ma i legami
delle due conferenze con la Zwischenbetrachtung e le due
etiche erano già molto chiari. E forse,
a un punto di svolta come quello odierno negli studi
weberiani, di recente segnalato da Wolfgang Schluchter,
in cui la conclusione del pluridecennale cantiere della
Gesamtausgabe ci potrebbe consegnare un Weber mol-
to diverso da quello che abbiamo ingessato nelle vecchie
traduzioni su cui ci siamo formati, vale la pena di fare
il punto sul Weber “di quegli anni” da confrontare col
Weber di oggi se non addirittura con quello possibile -
secondo il suggerimento dello stesso Schluchter - di una
nuova generazione di studiosi weberiani che ci si può
aspettare si formerà da ora in poi sui testi profondamen-
te mutati della Gesamtausgabe (Müller, Steffen 2021; cfr. Müller 2020; Rossi 2022). Tuttavia nel seminario prima
ricordato ci si occupava in specifico dei saggi di socio-
logia della religione nella versione comparsa sull’Archiv
für Soziologie und Sozialpsychologie e poi nella revisione
uscita postuma dei Gesammelte Aufsätze zur Religionss-
soziologie del 1920-21: Weber ribadiva e articolava la tesi
dell’Etica precisando con puntiglio7 l’intreccio comples-
so tra immagini del mondo (Vassalle 2012; D’Andrea
2018) e circostanze materiali, istituzionali e sociali delle
grandi religioni di salvazione. Quindi – con l’eccezione
dell’Etica, i cui aggiustamenti sono già stati magistral-
mente documentati (Weber 2002 [1904-05]), il nostro di
allora non è tutto sommato un ambito di grandissimi
spostamenti8 dovuti agli approfondimenti della Gesam-
tausgabe: sono per lo più testi rivisti direttamente da Mancava certo, ai tempi della nostra iniziazione, il
Weber esteta e che si definiva “divenuto Simmelesco”
e lieto della primavera in una lettera del 1908 (Weber
1908[1990]: 500) e forse in particolare il Weber di socio-
logia della musica, saggio che appunto è chiaramente il
portato della sua relazione matura con Mina Tobler e
che forse è stato l’aspetto più inatteso per Weber stesso,
giunto a compimento perfetto della tesi nur im Okzident. Ai tratti già noti dell’interesse di Weber per la cerchia
contro-culturale ante litteram che si riuniva a Heidel-
berg intorno a Otto Gross e per suoi viaggi presso la
comunità quasi hippy di Monte Verità vicino ad Asco-
na, mancavano solo le lettere della maturità, quelle che
hanno scandalizzato Jaspers rivelando il suo tradimento 118 Rossana Trifiletti secondo gran parte della critica, nel saggio si tratta, alla
fine, più dei fondamenti razionali che di quelli sociali
della musica, come suonerebbe il sottotitolo (Del Grosso
Destrieri 1982; Konoval 2019; contra Kaesler 2004 [1998]:
225-228). Forse allora, la vera differenza tra il Weber di allo-
ra e il Weber di oggi è che allora non valutavamo la sua
uscita dalla depressione come una vera cesura sul piano
personale, ma piuttosto come una resurrezione: la sua
figura di studioso restava del tutto coerente e conferma-
va con le altre religioni punto per punto la tesi dell’Etica
in un quadro allargato mondiale; oggi siamo più inclini
a cercare degli spostamenti di posizione, assai verosimi-
li dopo un tale percorso nella malattia, anche se biso-
gnerebbe evitare possibilmente gli eccessi della psico-
biografia (Green 1986; Cotesta 2019; Demm 2017). Allo
stesso modo non mi sembra proprio necessario metter-
si a ipotizzare tendenze sadico masochiste nel rapporto
di Weber con Frieda Jaffé, sulla base di qualche scherzo
nelle lettere, comprese quelle a Marianne (Radkau 2008). Viene da pensare che forse avesse persino ragione Caval-
li a sorvolare sugli aspetti erotizzanti della Zwischenbe-
trachtung che si collegavano con la biografia; sono sicura
che avrebbe detto sbrigativamente a lezione “queste cose
non ci interessano”, liquidando storie che alla fine sanno
anche un poco di inutile pettegolezzo. Ma soprattutto lo erano molti anni prima della con-
clusione della Gesamtausgabe e del bilancio odierno che
può tracciare Wolfgang Schluchter nel suo ultimo libro,
essendo l’unico rimasto in vita degli illustri curatori,
quello che la ha seguita per tutti i 4/5 decenni necessari
e che è stato suggestivamente descritto come “il grande
maestro nel gioco delle perle di vetro della tassonomi-
ca e dell’esegetica, che ha scavato i testi di Max Weber
per trarne le più raffinate implicazioni. Nessuno, vivo
o morto, ha analizzato l’insieme completo dell’opera
di Weber più a lungo e con effetti più profondi [con la]
tenacia […] e la determinazione di arrivare a una sintesi
teorica del corpus weberiano frammentario e incompiu-
to” (Oakes 2021: 195). Un altro tratto del corpus webe-
riano che Cavalli ha sempre avuto come sfondo del suo
lavoro, esattamente come Schluchter, insieme al pathos
della sobrietà e dell’onestà intellettuale (cfr. Schluchter
2004: XVIII). In un certo senso tutti e due hanno lavo-
rato non “su” ma “Mit Max Weber” nello studio e nella
condotta di vita (Schluchter 2020). Peccato non poter più
sapere cosa Luciano Cavalli avrebbe pensato di questa
estrema sintesi di Schluchter: posso azzardarmi a imma-
ginare che la avrebbe trovata un po’ troppo filosofica per
lui (cfr. 9 Utilizzo l’espressione anche se è stato ben dimostrato che deriva dalla
traduzione impropria di Parsons – forse la traduzione “bella e infedele”
più longeva e influente della storia – mentre stahlhartes Gehäuse indica
piuttosto un involucro, una conchiglia, dura come l’acciaio, comunque
non una iron cage (Baehr 2001; Douglass 2016). RIFERIMENTI BIBLIOGRAFICI con Alessandro Cavalli», in Rassegna Italiana di
Sociologia, 61(3): 651-670. Adair-Toteff C. (2011) «My ‘Sociology’: Wolf-
gang Schluchter on the History of Max Weber’s
Wirtschaft und Gesellschaft», in Journal of Classical
Sociology, 11(4): 495-505.f Cavalli A. (2021), «Passaggi generazionali: la sociolo-
gia in Italia nella seconda metà del Novecento», in
Quaderni di Sociologia, 65(85): 27-34. Quaderni di Sociologia, 65(85): 27-34. Cavalli A. (2022) «La sociologia nello spazio pubblico»,
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Alexander, J.C. (1990 [1987]), Sociologia e discorso: la
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Ascona. Piuttosto, col senno di poi, all’epoca abbiamo sicura-
mente sottovalutato la sua sociologia della musica come
un cruciale nur im Okzident che deve essere sembrato
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some Comparison with the Work of Foucault», in
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ologist, 17(2): 2-11.h Whimster, S. Citation: Marco Bontempi (2023) L’analisi
del mutamento sociale nella socio-
logia di Luciano Cavalli. SocietàMu-
tamentoPolitica 14(27): 123-128. doi:
10.36253/smp-14343 Citation: Marco Bontempi (2023) L’analisi
del mutamento sociale nella socio-
logia di Luciano Cavalli. SocietàMu-
tamentoPolitica 14(27): 123-128. doi:
10.36253/smp-14343 Citation: Marco Bontempi (2023) L’analisi
del mutamento sociale nella socio-
logia di Luciano Cavalli. SocietàMu-
tamentoPolitica 14(27): 123-128. doi:
10.36253/smp-14343 Marco Bontempi Copyright: © 2023 Marco Bontempi. This
is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. Il tema del mutamento sociale è uno degli assi portanti degli interessi e
della produzione scientifica di Luciano Cavalli (Turi 2008: 27), ed è possibi-
le individuare concetti e metodi che tematizzano questo interesse dai suoi
primi lavori fino agli ultimi scritti sulla leadership. In questo contributo mi
propongo di delineare alcuni degli elementi, a mio parere significativi, che
emergono da un’analisi di tre volumi che Cavalli ha specificamente dedicato
al mutamento sociale e che ha pubblicato tra il 1968 e il 1974. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. 1. Nel 1968 Cavalli pubblica Max Weber: religione e società, una ricerca,
a tratti enciclopedica, che immerge e conduce il lettore in una ricostruzione
brillante della sociologia weberiana delle religioni, con un accentuato interesse
per l’analisi sulle relazioni tra idee religiose ed ethos economico-professionale
svolta in L’Etica protestante e lo spirito del capitalismo. Cavalli si impegna in
un corpo a corpo con i temi weberiani, illustrandone e discutendone i tratti
e le critiche, in primis in relazione all’intreccio tra idee ascetiche del prote-
stantesimo puritano, agire dei fedeli e condizioni di possibilità dello sviluppo
inintenzionale, e talvolta intenzionale, di un ethos dell’agire economico capi-
talisticamente orientato. Con una conoscenza e passione che ne attestano una
personale partecipazione, Cavalli mostra come la dottrina di Giovanni Calvi-
no, solo in parte considerata da Weber, possa avere elementi rilevanti più di
quanto Weber non considerasse, per svolgere un ruolo decisivo nella forma-
zione dell’ethos capitalistico. RIFERIMENTI BIBLIOGRAFICI (1999) (ed.), Max Weber and the Culture of
Anarchy, Palgrave Macmillan, London. g
Stinchcombe A.L. (1984), «The Origins of Sociology as a
Discipline», in Acta Sociologica, 27(1): 51-61. Whimster S. e Heuer G. (1999), «Otto Gross and Else
Jaffé and Max Weber, in Love and Eroticism», in
Theory, Culture and Society, 15(1): 3-4. Swedberg R. (2022), «Robert K. Merton’s Approach to
Teaching the Classics in Sociology», in The Ameri-
can Sociologist, 53(1): 107-128. Tenbruck F.H. (1975), «Das Werk Max Webers», in
Kőlner Zeitschrift für Soziologie und Sozialpsycholo-
gie, 27(3): 663-702.i Trifiletti R. (2008) «Da Weber a Simmel e oltre? Note
sull’uso dei classici in sociologia», in G. Bettin Lattes
e P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze University Press, Firenze, pp. 425-435. Vassalle A. (2012), «Costruzione di mondi. Note sul con-
cetto weberiano di Weltbild», in Annali del Diparti-
mento di Filosofia, 18: 127-149. i
Veca S. (1981), «Il metodo e le condizioni dell’oggettivi-
tà», in P. Rossi (ed.), Max Weber e l’analisi del mon-
do moderno, Einaudi, Torino, pp. 3-26. Wallach Bologh R. (1987), «Max Weber on Erotic Love. A Feminist Inquiry», in S. Whimster and S. Lash
(eds), Max Weber, Rationality and Modernity, Allen
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fia, il Mulino, Bologna. i
Weber Max (1958 [1906]), «Studi critici intorno alla logi-
ca delle scienze della cultura», in Id., Il metodo delle
scienze storico-sociali, Mondadori, Milano. Weber Max (1990 [1908]), «Brief an Marianne Weber 2
April 1908», in Max Weber Gesamtausgabe (MWG)
II/5, Mohr Siebeck, Tübingen, p. 500. Weber Max (1989 [1915-1916]) Zwischenbetrachtung,
in Die Wirtschaftsethik der Weltreligionen. Ver-
gleichende religionssoziologische Versuche, in Max
Weber Gesamtausgabe (MWG) I/19, H. Schmidt-
Glinzer and P. Kolonko (eds.), Mohr Siebeck, Tübin-
gen, pp. 479-522. Weber Max (1965 [1920], Introduzione a L’etica protes-
tante e lo spirito del capitalismo, Firenze, Sansoni. Weber Max (1999), «Letters from Ascona», in S. Whim-
ster (ed.), Max Weber and the Culture of Anarchy,
Palgrave Macmillan, London, pp. 129-160. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 123-128, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14343 Cavalli si impegna a più riprese nella ricostru-
zione del contesto storico, sia della Ginevra di Calvino, che della Rivoluzio-
ne inglese, illuminando aspetti solo in apparenza minori, ma significativi per
una comprensione più articolata del tema di ricerca weberiano. Un accento
particolare è posto, ad esempio, sulla rilevanza delle istituzioni ecclesiastiche
calviniste per la formazione di una personalità autodiretta e rivolta all’interes-
se della comunità. Come è noto, Weber tratterà di questo aspetto solo nel sag-
gio su Le Sette protestanti e lo spirito del capitalismo, mentre Cavalli mostra,
in modo tutto sommato convincente, l’opportunità di considerarlo già nella
discussione sull’Etica e di valorizzare le idee calviniste, in confronto ad altri
teologi protestanti, maggiormente di quanto Weber non faccia. Competing Interests: The Author(s)
declare(s) no conflict of interest. L’interesse per Calvino è, tuttavia, genuinamente sociologico, in pri-
mo luogo perché, attraverso l’indagine su questa figura, viene posta speciale
attenzione ad un tema che – con declinazioni differenti – occuperà la pro- SOCIETÀMUTAMENTOPOLITICA 14(27): 123-128, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14343 124 Marco Bontempi Marco Bontempi duzione scientifica cavalliana in maniera costante: la for-
mazione di una personalità attraverso la costruzione di
un assetto istituzionale fondato sui valori di promozione
della libertà individuale, della responsabilità e del servi-
zio alla comunità. Su questo aspetto, non è il luteranesi-
mo ad essere una sorgente significativa, anzi è semmai
una condizione di quella «dominazione burocratica» che
già Weber segnalava. È invece «nei paesi anglosassoni
e calvinisti [che] l’etica e gli istituti della responsabilità
avevano formato degli uomini e dei ceti capaci di con-
trollare la burocrazia» (Cavalli 1968: 179). Le idee di
Calvino porterebbero infatti fino alle piene conseguen-
ze quel processo di razionalizzazione della condotta di
vita che invece resterebbe incompiuto nel luteranesimo,
inculcando nei tedeschi il sentimento di autorità e una
concezione metafisica dello Stato. tica il piano biografico dell’impegno politico di Weber,
secondo l’idea che i concetti della sociologia weberiana
possano trovare ulteriore chiarificazione rispetto al testo
se letti alla luce del suo impegno politico nella Germania
dell’epoca. Quanto la vita dell’Autore possa essere utiliz-
zata come una risorsa esegetica della sua produzione è
un tema di lunga discussione che non è certo il caso di
trattare qui. Certo è che i concetti della sociologia webe-
riana, fortemente orientati a concettualizzare non stati
di fatto, ma “condizioni di possibilità”, e per giunta non
sempre impiegati da Weber con il medesimo significato,
si prestano facilmente a questo tipo di operazione esege-
tica. Mi pare che la lettura cavalliana operi connettendo
due livelli più di quanto non li connetta Weber: la “sco-
perta” weberiana della relazione tra ordinamento istitu-
zionale calvinista e personalità autodiretta, responsabile
e orientata all’interesse della comunità, viene assunta
da Cavalli come il nucleo identificativo dell’Occidente
moderno, nel quale libertà individuale e interesse del-
la collettività trovano un equilibrio peculiare. Questo
nucleo etico-istituzionale è in Cavalli una risorsa per
l’azione politica, la quale diviene capace di fornire un
orientamento al mutamento politico-sociale nella misura
in cui lo guida alla realizzazione di condizioni istituzio-
nali dello sviluppo di valori e soggettività etico-politiche
coerenti con questo “nucleo occidentale”, per quanto
ormai completamente secolarizzato. È uno spostamen-
to rilevante rispetto all’Etica, che per essere qualificato
come weberiano richiede di convocare e interpretare gli
interessi politici del Weber intellettuale del suo tempo
come parte delle sue analisi sociologiche. i
L’assetto istituzionale delle sette protestanti è analiz-
zato da Cavalli non solo nella chiave weberiana del feno-
meno storico-sociale dello sviluppo dell’individuo capi-
talistico, ma come una possibilità efficace nel favorire
la formazione e la selezione di una specifica personalità
nei propri membri e soprattutto nei propri capi. È infatti
nella setta, osserva Cavalli, che si può trovare [l]a soluzione del problema storico del rapporto individuo-
gruppo che non comporta né alienazione per l’individuo,
da un lato, né anarchia sociale, dall’altro, perché questa
soluzione si fonda, com’è implicito nel concetto stesso di
“setta”, sulla partecipazione: che assicura a ciascuno il libe-
ro svolgimento di se stesso entro il rispetto dei fini e delle
regole del gruppo, interiorizzati e manifestantisi anche e
prima di tutto attraverso un continuo autocontrollo; e che
garantisce il gruppo e la più vasta società dall’anarchia,
perché comporta responsabilità comune e del controllo di
tutti e di ciascuno su tutti e su ciascuno, come controllo in
primo luogo morale (Ivi: 204). La seconda ragione concerne il ruolo del leader cari-
smatico in questi processi di orientamento del muta-
mento sociale. In questo volume si trovano a più riprese
elementi di una chiara anticipazione delle idee posteriori
sulla centralità della leadership carismatica. Ci mostra come questa
figura sorga nel milieu del protestantesimo, in particola-
re in Inghilterra, e si connoti per quell’istanza di prima-
to della libertà personale e della libera coscienza proprie
dell’ethos dell’intellettuale occidentale moderno. Questa
figura del dissenter compare anche negli altri volumi,
come un omaggio alle figure che non temono di assume-
re posizioni politicamente radicali, anche se per queste
possono trovarsi in posizioni marginali. 2. La pubblicazione de Il mutamento sociale (1970)
segna un punto di maturazione intellettuale significativo
per l’elaborazione della connessione tra teoria e analisi
del mutamento. Lungi dall’essere una semplice rassegna
di autori di studi sul mutamento sociale, la selezione, la
composizione e soprattutto l’esposizione del lavoro degli
autori scelti, che Cavalli chiama «i miei autori» – espri-
mendo così una relazione più intima di quella puramente
funzionale ai propri interessi di ricerca – sono condotti
secondo un metodo di ricerca teorica strutturato intor-
no a interrogativi eminentemente politici, concernenti 3
aspetti e cioè: il posizionamento del mondo occidenta-
le «tra gli altri “mondi”», come le sue «interne contrad-
dizioni e i suoi conflitti si rapportino alla sua posizione
mondiale» e quali direzioni di cambiamento e quali forze
«si possano indicare come […] possibili e desiderabili dal
punto di vista di un “occidentalista”» (Ivi: VII). In questa architettura, da un lato, Durkheim è valo-
rizzato come analista e come critico della società occi-
dentale, capace di analisi e anche di proposte di orien-
tamento del mutamento sociale, sempre così distanti
dalle marxiane, e indifferenti a queste, da dare, osserva
Cavalli, «per più versi la bizzarra impressione di essere
vissuto e di aver scritto prima di Marx» (ivi: XI). Sebbe-
ne impegnato, come Marx, in una teoria generale della
società, Durkheim condivide con Weber, stanti grandis-
sime differenze, una notevole distanza dalla riduzione
marxiana della religione a epifenomeno sovrastruttura-
le e una risonanza di fondo sull’interesse per l’influen-
za delle idee religiose nella plasmazione e nei mutamen-
ti delle forme della vita sociale. In questa prospettiva, a
Cavalli interessa mostrare come le tesi durkheimiane,
che fondano, parallelamente a Weber, la sociologia come
disciplina scientifica, illustrino al lettore critiche, anche
forti, al capitalismo e ai suoi conflitti interni, ma sempre
con un’attenzione alla libertà individuale tale da consi-
derarne la riduzione un pericolo e mai un mezzo per la
soluzione delle degenerazioni del capitalismo. È interes-
sante come sull’emersione del capo carismatico già in
questo lavoro Cavalli mostri un interesse pratico-politico
che nei testi sociologici weberiani non è altrettanto niti-
do, stanti le diverse condizioni dalle quali possono sor-
gere figure profetiche, e per di più non necessariamente
secolarizzate. In altre parole, è precipuamente cavalliana,
direi, l’idea che meriti l’impegno di una vita la ricerca
delle condizioni di formazione di una personalità occi-
dentale, nel suo senso, attraverso la costruzione di istitu-
zioni politiche – e quindi di classi politiche – orientate a
valori da una leadership carismatica. Insomma, è questo
“il modo” cavalliano di essere weberiano. È forte per lui l’attrazione di assumere questo insie-
me di istituzioni e personalità non solo come un «pro-
dotto storico», ma come uno scopo per l’azione politica
nell’epoca contemporanea. Così intendeva Weber, scri-
ve Cavalli, quando «voleva rifare la società tedesca del
dopoguerra in base al principio della honesty e all’isti-
tuzione generalizzata dell’associazione esclusiva», ma
questo genere di tentativi richiedono in via prelimina-
re «una radicale distruzione di principi e istituzioni, e
l’attuazione di nuove condizioni materiali e spirituali –
ossia quel genere di cambiamento che in altre pagine egli
sembra affidare come compito storico ai grandi leaders
carismatici» (Ivi: 205). Emerge, in questa ricerca, anche un’altra interessante
figura della modernità, questa volta affatto carismatica: il
dissenter. L’Autore guarda con evidente simpatia all’indi-
viduo, meglio all’intellettuale critico, controcorrente, ma Questo passo è, a mio parere, interessante, almeno
per due ragioni. La prima concerne l’esegesi weberiana. Cavalli segue qui Weber, ma introduce nell’interpreta-
zione delle connessioni concettuali weberiane dell’E- 125 L’analisi del mutamento sociale nella sociologia di Luciano Cavalli cazioni pratico-politiche, come «ideologia dei proletariati
interni ed esterni» (Ivi: XIV) all’Occidente, e in que-
sto come minaccia per la sopravvivenza dell’Occidente
medesimo. Su questo piano, la “differenza specifica” tra
Marx e i marxisti è per Cavalli decisiva: del primo molte
intuizioni vanno nella direzione che poi Weber ha colto
con maggiore acutezza e sviluppato con efficacia, molte
altre no, ma la potenza teoretica di Marx per lo studio
del mutamento sociale è fuori discussione. Il marxismo
invece è visto in primo luogo nel suo significato di azio-
ne politica, come una minaccia operata da forze anti-
occidentali al futuro della civiltà occidentale. interessato all’interesse collettivo. Il lavoro di Parsons,
dunque, rappresenta «un invito all’inerzia politica» e
nella sua «scarsa propensione per la critica della socie-
tà e il deficiente interesse per il problema della libertà»
(ibidem) Cavalli vede un auto-intrappolamento di Par-
sons nella propria teoria e una conseguente mancanza
di realismo. In questa chiave è evidente quanto la rice-
zione parsonsiana di Weber possa risultare a Cavalli, già
alla fine degli anni ’60, assai discutibile, perché parziale
e distorta, com’è oggi universalmente noto, ma non di
meno per il tentativo, a suo dire persino «illegittimo», di
«far “confluire” Pareto, Durkheim e Weber negli schemi
di una teoria generale» (Ivi: IX). [u]n modello di analisi di una società come sistema di isti-
tuzioni che, benché artigianalmente elaborato e, come ho
detto, non sostenuto teoricamente, resta ancora suggesti-
vo. Occorreranno uomini come Mills perché la sociologia
riprenda a battere questa via fruttuosa (con gli strumenti
di una teoria e di una metodologia assai più efficaci); in
Italia, nessuno ha più tentato, nemmeno in questo dopo-
guerra, un esame siffatto della società contemporanea (Ivi:
263). Alcuni anni dopo, nel 1974, Cavalli pubblica Socio-
logia della storia italiana (1871-1974), proprio come un
primo tentativo di analisi politico-istituzionale del muta-
mento sociale in Italia. L’inclusione di Lynd e di Mills esprime bene, a mio
parere, diversi elementi degli interessi intellettuali di
Cavalli. In primo luogo, l’attrazione per la figura del dis-
senter, lo studioso controcorrente, acribico appassionato
e creativo. Si tratta di un profilo di intellettuale che cer-
tamente si attaglia bene anche allo stesso Luciano Caval-
li, critico talvolta anche radicale dell’ordine sociale, ma
allo stesso tempo lontanissimo dalla contestazione main-
stream degli anni ’60-’70. In secondo luogo, l’attenzione
al potere e specialmente alle classi dirigenti come atto-
ri capaci di orientare o frenare il mutamento, ma anche
alle pratiche manipolatorie del potere. Cavalli guarda
con simpatia al “quasi-marxismo” di Lynd, che descrive
come «l’analista spietato del moderno capitalismo ame-
ricano, di cui mostra, forse per primo e con ineguaglia-
ta acutezza, le conseguenze deteriori sull’individuo e
la società» e vede in Mills «la forma più “moderna” del
dissenso entro la società occidentale», un illuminista che
«con i migliori strumenti della grande sociologia euro-
pea, si dedica all’analisi critica del potere di minoranza
e della manipolazione, in America e in URSS, erigendo-
si infine ad aperto oppositore della politica dei blocchi»
(Ivi: XIII). In que-
sto, il lavoro di Parsons è trovato distante dalle istanze
degli autori europei, in primis Weber, ma anche Marx. È
interessante, ad esempio, l’osservazione che quanto più,
nella teoria di Parsons, si accentuano gli aspetti di “mec-
canismo sociale”, tanto più si tende a giustificare «un’e-
tica simile a quella implicita nelle teorizzazioni di alcu-
ni antropologi funzionalisti: tutto ciò che è funzionale
è buono, tutto ciò che non è funzionale è cattivo […]
Anche la critica della società viene ad avere come pre-
cipuo riferimento i valori vigenti [e] mancherà sempre
di una vera ragione morale, almeno dal punto di vista
di uno spettatore convinto della pluralità e irrazionalità
dei sistemi di valore» (Ivi: 519-520). Il lavoro di Parsons,
dunque, rappresenta «un invito all’inerzia politica» e
nella sua «scarsa propensione per la critica della socie-
tà e il deficiente interesse per il problema della libertà»
(ibidem) Cavalli vede un auto-intrappolamento di Par-
sons nella propria teoria e una conseguente mancanza
di realismo. In questa chiave è evidente quanto la rice-
zione parsonsiana di Weber possa risultare a Cavalli, già
alla fine degli anni ’60, assai discutibile, perché parziale
e distorta, com’è oggi universalmente noto, ma non di
meno per il tentativo, a suo dire persino «illegittimo», di
«far “confluire” Pareto, Durkheim e Weber negli schemi
di una teoria generale» (Ivi: IX). tanto il mutamento strutturale, quanto le motivazioni
alla critica e le condizioni di possibilità dell’attivazio-
ne politico-sociale dei processi di mutamento. In que-
sto, il lavoro di Parsons è trovato distante dalle istanze
degli autori europei, in primis Weber, ma anche Marx. È
interessante, ad esempio, l’osservazione che quanto più,
nella teoria di Parsons, si accentuano gli aspetti di “mec-
canismo sociale”, tanto più si tende a giustificare «un’e-
tica simile a quella implicita nelle teorizzazioni di alcu-
ni antropologi funzionalisti: tutto ciò che è funzionale
è buono, tutto ciò che non è funzionale è cattivo […]
Anche la critica della società viene ad avere come pre-
cipuo riferimento i valori vigenti [e] mancherà sempre
di una vera ragione morale, almeno dal punto di vista
di uno spettatore convinto della pluralità e irrazionalità
dei sistemi di valore» (Ivi: 519-520). Pur organizzando il volume in un ordine cronolo-
gico, l’architettura di questa ricerca si sviluppa su più
piani che attraversano in modi e gradi diversi tutti gli
autori. Al fondamento di questo impianto viene posta
quella parte della sociologia weberiana che offre – nella
sua prospettiva – gli strumenti metodologici per «“indi-
viduare” l’Occidente […] come una civiltà caratterizzata
alti e peculiari raggiungimenti di libertà» che nel con-
testo della guerra fredda lo impegnano in «una lotta di
civiltà e potere» contro i paesi comunisti «dell’Oriente
europeo» (Ivi: XIII). Mentre a Weber è fatto svolgere il
ruolo del fondatore di una comprensione occidentale
dell’Occidente, Marx è posto nel ruolo dell’interlocuto-
re di Weber in primis, e però in una posizione asimme-
trica rispetto a lui e alle sue idee, ma più in generale è
interlocutore per ciascuno degli altri autori trattati e più
di quanto non lo siano tra loro. Marx è un interlocuto-
re la cui analisi «dell’Occidente contemporaneo non è
soltanto acutissima, ma influentissima, anche se le idee
di Marx più influenti ed efficaci praticamente, in sen-
so eversivo, antioccidentale, sono state spesso le meno
acute, o comunque, le meno valide scientificamente»,
nell’economia dell’analisi cavalliana, tanto meno valide,
quanto più distanti dalle idee di Weber (Ibidem). La cri-
tica di Marx al capitalismo è vista anche nelle sue impli- La trattazione del lavoro di Parsons è inserita in
questa architettura in modo lucido e originale. In con-
trotendenza rispetto alle semplificazioni che ne facevano
una teoria generale della società, Cavalli osserva che Parsons resta dell’opinione che una teoria completa del
cambiamento sociale sia impossibile; e si limita a conside-
rare quei tipi di cambiamento che hanno oggi rilevanza e
che si prestano “ad essere trattati efficacemente con le cate-
gorie del sistema”. Il risultato è ovviamente quello di trat-
tare sempre il cambiamento entro il sistema; tuttavia, il
fatto è spesso occultato dall’uso del termine “cambiamento
strutturale”, che è quanto mai equivoco (Ivi: 526). Nella ricostruzione della teoria parsonsiana, emer-
ge una prospettiva teorica efficace nel fornire una spie-
gazione della società contemporanea altra e perfino in
antitesi a quella marxiana. Coerentemente con i propri
criteri di analisi, a Cavalli interessa verificare quali ele-
menti possano concettualizzare e anche spiegare non 126 Marco Bontempi Marco Bontempi tanto il mutamento strutturale, quanto le motivazioni
alla critica e le condizioni di possibilità dell’attivazio-
ne politico-sociale dei processi di mutamento. L’analisi del mutamento sociale nella sociologia di Luciano Cavalli Pur nelle sue artico-
lazioni interne, la borghesia costituisce un’unità cultura-
le, plasmata lungo un processo plurisecolare, ed è «la più
grande e matura parte di questa classe che ha fatto l’uni-
tà» (Ivi: 19) dello Stato italiano. Ma questa unità, realiz-
zata sul piano politico-militare, è rimasta un’aspirazio-
ne, non si è mai compiuta per la resistenza della classe
lavoratrice. Una classe creata dal capitalismo moderno e
proprio per questo non solo strutturalmente in conflit-
to con la borghesia, ma «esclusa dalla partecipazione alla
cultura umanistica, e quindi alla storia, alla tradizione
essenziale, ai valori occidentali» (Ivi: 20). Un’estraneità
quella della borghesia primariamente culturale, dunque,
e poi anche economico-politica che spiegherebbe la resi-
stenza della classe lavoratrice all’integrazione nello Stato
nazionale, nonostante gli sforzi della classe politica bor-
ghese. A fronte di questa estraneità sta la seconda alteri-
tà istituzionale, la chiesa cattolica, nemica dell’unità fin
dal primo momento che oppone resistenza allo Stato. La
storia d’Italia viene così punteggiata dagli intrecci di lot- In questa prospettiva, l’Italia della metà degli anni
’70 appare essere il frutto maturo di questo lungo arco
temporale nel quale è stato continuamente rinviato il
lavoro di costruzione di un’unità nazionale fondata su
valori condivisi. La lotta contro l’avversario, di volta in
volta definita come decisiva per la sopravvivenza, ha
visto le gerarchie cattoliche impegnarsi prima contro la
classe risorgimentale, poi contro i marxisti e a loro vol-
ta questi ultimi impegnarsi continuativamente contro la
borghesia e la chiesa. Di volta in volta, dal fascismo al
dopoguerra, altre da quelle risorgimentali sono state le
“promesse”, cioè le giustificazioni dell’integrazione poli-
tica, fino a quella del secondo dopoguerra «individuali-
stica […] dell’arricchimento e del godimento individua-
le». L’esito è che [i]l popolo italiano non è più un’entità politica […] non è
più un popolo, ma un insieme di individui e gruppi ammi-
nistrati da un corpo di amministratori omogeneo ed ete-
rodipendente, che deve in primo luogo manipolare la for-
mazione e i comportamenti degli amministrati ai fini del
consenso e del conformismo. Questi amministratori, d’al-
tronde, proprio perché privi della tensione ideale che si
ricongiunge ad una “promessa” e che fa capaci di “servire”
e di “sacrificarsi”, sono essenzialmente attenti ai problemi
propri e del loro gruppo. Ecco quello che io chiamo il qua-
si-Regime. […] La caduta del centrismo ne è solo la dimo-
strazione più evidente (Ivi: 25). L’analisi del mutamento sociale nella sociologia di Luciano Cavalli rali”, le meno “italiane”» (Ivi: xvii), cioè la chiesa cattoli-
ca, la DC e il PCI1. rali”, le meno “italiane”» (Ivi: xvii), cioè la chiesa cattoli-
ca, la DC e il PCI1. te tra queste tre istituzioni che, di volta in volta, allean-
dosi ora con l’una ora con l’altra conseguono il risultato
di bloccare il processo di formazione di una coscienza
nazionale – una «noità», nelle sue parole – come, secon-
do Cavalli, era stato interpretato dalla classe politica
risorgimentale. In breve, la storia dell’Italia unita «è la
storia di una unità nazionale tentata tardi e mai compiu-
tamente attuata, indebolita e minacciata dalla secessione
religiosa, e, soprattutto, di classe, e dei tentativi sempre
rinnovati di attuare, secondo “modelli” e con “promesse”
diverse, questa unità da parte di chi aveva il potere e del-
le forze sociali che lo sostenevano» (Ivi: 22). 3. Con Sociologia della storia italiana (1961-1974)
(1974) Cavalli tenta un’applicazione di molti dei con-
cetti elaborati e discussi in Il mutamento sociale (1970)
sviluppando un’analisi del mutamento politico-istitu-
zionale, in primis delle tensioni e lotte che attraversano
l’élite del potere. Centrale in questo lavoro è il concetto
di istituzione come sistemi integrati di gruppi formal-
mente organizzati «con norme che ne regolano la strut-
turazione e il funzionamento in ogni aspetto, interno e
nell’ambiente sociale in cui si collocano, e con una giu-
stificazione ideologica, che sotto un certo profilo è pure
una “promessa”» (Cavalli 1974: 15) cioè «il senso speci-
fico che viene conferito all’unità politica di una socie-
tà» (Ivi: 11). Sono istituzioni lo Stato, i partiti, la chie-
sa cattolica. Sono i conflitti tra loro che caratterizzano,
secondo Cavalli, la storia dell’Italia unita, ostacolandone
lo sviluppo come nazione. L’analisi decostruisce l’in-
terpretazione marxista delle lotte tra le classi, mostran-
do, weberianamente, l’articolazione della stratificazione
interna alla borghesia e le diversità di aspirazioni e di
motivazioni politiche tra proletariato e piccola-media
borghesia. La borghesia nel suo insieme è la protagoni-
sta di questa ricerca, Cavalli ne tratteggia i valori, defi-
nendoli il prodotto non di un modo di produzione, ma
della «lunga storia occidentale. Così come, del resto, lo
Stato nazionale è il prodotto di una lunga elaborazione
collettiva, che rapporta in modo originale l’individuo e
la comunità, in base a quei valori». 1 All’incirca il 65-70% dell’elettorato dell’epoca. Il tema del potere manipolativo – e con esso
la lotta contro le oligarchie – è un fil rouge che attra-
versa le analisi di molti degli autori discussi da Cavalli,
da Marx a Mills, passando per Weber e la formulazione
comunitaria del potere manipolativo fatta da Lynd. La scelta di includere Gaetano Mosca in una ricer-
ca come questa sarà apparsa sorprendente a più di uno
dei lettori, ma illustra bene alcune istanze metodo-
logiche e concettuali di questa prospettiva. Lettore ed
illustratore appassionato dei lavori di Mosca, Caval-
li lo convoca per la convergenza con il proprio punto
vista «circa le minoranze elette, la classe media, l’unità
occidentale» (Ivi: XI). Il lavoro di Mosca, che ha aper-
to «la strada agli altri elitisti fino a Dahrendorf» (ivi:
258), gli consente di tematizzare il mutamento sociale
secondo una chiave elitista che sposta in misura mol-
to rilevante il fuoco di analisi dai processi macrosociali
discussi negli autori precedenti ad una specifica mino-
ranza organizzata, la classe politica e alle sue dinami-
che di trasformazione che riflettono e traducono nell’a-
zione istituzionale il mutamento sociale, economico e
culturale che si verifica nella società. Mosca consente
a Cavalli di introdurre nella sua ricerca un approccio
metodologico e concettuale che interpreta in chiave
politico-istituzionale il mutamento sociale e riconosce
un ruolo fondamentale alla classe politica, fa questo
avendo a riferimento proprio il mutamento sociale in
Italia. Senza trascurare di rilevarne i limiti, Cavalli leg-
ge La teorica dei governi anche come Il cuore di questo lavoro si viene dunque strutturan-
do progressivamente attraverso la discussione di mol-
tissimi temi e problemi teorici e politici fino a prendere
forma come una meta-prospettiva di ricerca teorica che
individua nella leadership e nelle pratiche manipolatorie
della classe politica, il fattore di primaria importanza del
mutamento istituzionale. In questa chiave, Cavalli asse-
gna alla leadership un compito costruttivo, ma solo dopo
che la critica più radicale dei movimenti abbia operato in
senso distruttivo «in tutte le istituzioni ove le oligarchie
del passato hanno il potere [senza risparmiare] quelle
che sono ad un tempo le più importanti e le meno “libe- 127 L’analisi del mutamento sociale nella sociologia di Luciano Cavalli L’analisi del mutamento sociale nella sociologia di Luciano Cavalli È in questa lucida consapevolezza dello stato di cri-
si profondissima dell’Italia della metà degli anni ’70 che
l’interesse sulla personalità carismatica e le sue istitu-
zioni, già presente da tempo nelle sue ricerche, prende a
focalizzarsi nell’approfondimento teorico e storico-empi-
rico delle condizioni di possibilità del carisma nella lea-
dership politica delle democrazie occidentali. Al di là dei
suoi contenuti, questa scelta può essere letta come il ten-
tativo di alzare uno sguardo di prospettiva proprio dove
e quando sembra che non ci siano e ci possano essere le
condizioni di un cambiamento del genere e impegnarsi,
per la propria parte, a lavorare perché possano formarsi. 128 Marco Bontempi 4. Le riflessioni sviluppate fin qui intendono essere
solo degli abbozzi della peculiare ricezione dei classi-
ci e della creatività intellettuale con le quali Cavalli ha
saputo comporre temi, concetti e prospettive differenti
in relazioni di attrazione e distinzione, facendoli propri
come risorse alternative e complementari di interroga-
zione intellettuale del presente e sul presente, guidato da
un interesse genuinamente politico per la partecipazione
politico-intellettuale ai processi di mutamento sociale. Fedele a questa impostazione, la sociologia cavallia-
na ci affida una testimonianza della necessità per i socio-
logi di pensare il presente dal suo interno, intrecciando
in forma situata e personale spessore teorico, analisi
empirica e impegno politico. Al di là dei singoli temi o
concetti, il cuore del lascito cavalliano mi pare consiste-
re in questa capacità di fedeltà al rigore dell’analisi e alla
propria posizione nel mondo, perché, come ha scritto
in Il mutamento sociale «ciascun uomo fa la sua ricer-
ca, anche quando gli intenti dichiarati sono i medesimi;
e ovviamente non può essere che così ed è bene che sia
così: che una ricerca sia una ricerca di un uomo, soprat-
tutto quando abbia l’ampiezza di scopo conoscitivo e il
significato etico-politico che necessariamente investe
questa ricerca» (Cavalli 1970: VIII). Citation: Andrea Millefiorini (2023) Un’al-
tra importante strada aperta da Lucia-
no Cavalli: lo studio del pensiero politi-
co nei classici della letteratura. Socie-
tàMutamentoPolitica 14(27): 129-137. doi:
10.36253/smp-14344 Andrea Millefiorini Copyright: © 2023 Andrea Millefiorini. This is an open access, peer-reviewed
article published by Firenze Univer-
sity Press (http://www.fupress.com/
smp) and 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. 1. Premessa. Ripensando al percorso attraverso il quale è avvenuta la mia
formazione scientifica, la figura di Luciano Cavalli, sebbene non sempre “in
presenza” – come si direbbe oggi – ha detenuto un ruolo che, al tirar delle
somme, si è rivelato di prim’ordine. Prima ancora di superare il concorso per il dottorato di ricerca in Socio-
logia politica della Facoltà di Scienze politiche “Cesare Alfieri” di Firenze,
nella quale Cavalli ha insegnato e dove ha fondato quel dottorato, il mio
maestro Luciano Pellicani mi aveva assegnato una lista di testi imprescindi-
bili da studiare per prepararmi al concorso. In questa lista figuravano tutte
le principali opere sino ad allora pubblicate da Cavalli, a cominciare (vado
a memoria) da Il capo carismatico, per proseguire poi con opere altrettanto
rilevanti come Il presidente americano, Carisma e tirannide nel Secolo XX,
Governo del leader e regime dei partiti, più almeno altre tre o quattro opere
altrettanto significative che completavano la voce “Cavalli” nella lista prepa-
ratami da Pellicani. I due Luciani, tra l’altro, erano molto amici e tra loro
c’è sempre stata una reciproca e profonda stima. Cavalli era più anziano di
Pellicani, tant’è che fu proprio lui che nella commissione di concorso a pro-
fessore ordinario del Luciano più piccolo volle fortemente la sua abilitazione,
riuscendo a convincere tutto il resto della Commissione. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. Quanto invece al mio concorso, quello per l’ammissione al dottorato,
la lista preparatami da Pellicani si rivelò provvidenziale. Alla prima prova,
lo scritto (allora c’era anche quello, diversamente dalle selezioni ai dottorati
come vengono organizzate oggi) una candidata fu chiamata ad estrarre una
delle buste in cui erano contenuti gli argomenti. RIFERIMENTI BIBLIOGRAFICI Cavalli L. (1970), Il mutamento sociale. Sette ricerche sulla
civiltà occidentale, il Mulino, Bologna. g
– (1968), Max Weber: religione e società, il Mulino, Bolo-
gna. – (1974), Sociologia della storia italiana (1961-1974), il
Mulino, Bologna. Turi P. (2008), «Sociologia e politica nell’itinerario intel-
lettuale di Luciano Cavalli», in G. Bettin Lattes e P. Turi (a cura di), La sociologia di Luciano Cavalli,
Firenze University Press, Firenze, pp. 23-189 societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Certo, viene manipolata di
volta in volta dal leader di turno, ma è proprio attraverso
questa dinamica che Cavalli può mostrare come la logica
della teatralità si esalti: «Si fanno decisive le capacità tea-
trali dei principali protagonisti […] Trionfa la recitazio-
ne, con il suo carico machiavellico di menzogna, simula-
zione, dissimulazione. Che, in verità, è il filo rosso della
politica e, in specie, dei suoi drammi» (Cavalli 2006: 22).i Non essendo qui possibile, con un singolo contri-
buto, affrontare la vasta opera che distinse Cavalli nella
storia del pensiero sociologico italiano, vorrei approfitta-
re di questo spazio su SMP per proporre la lettura e il
mio personale commento ad una delle sue ultime opere,
Giulio Cesare, Coriolano e il teatro della Repubblica. Una
lettura politica di Shakespeare, pubblicata per Rubbettino
nel 2006. Ecco quindi, inevitabile, anche una dose di finzio-
ne che il leader deve mettere in atto nei confronti della
massa, e quindi un contrasto “lacerante” che inevitabil-
mente scaturisce tra ciò che il protagonista “deve” essere
e ciò che il protagonista “vuole” essere. E guai a cambiare
la “faccia” che si mette (direbbe Goffman) in un deter-
minato contesto. Questa deve restare sempre la stessa,
pena la perdita di credibilità e di fiducia che si è riusciti
a conquistare nel pubblico. La capacità di Cesare rispet-
to ai suoi avversari fu infatti proprio quella di mante-
nere la sua immagine pubblica «alta e ferma», coerente,
«costante» (Ivi: 35), in netto contrasto con l’inconsisten-
za dell’immagine del personale di governo della vecchia
repubblica. Ne segue che «nella visione shakespeariana
della politica, incapacità e rifiuto di recitare sono prova
sicura di inettitudine alla leadership» (Ivi: 147). Trovo questo lavoro di Cavalli estremamente utile
per due motivi: il primo perché valorizza uno Shake-
speare “politico” che da sempre meriterebbe di essere
approfondito meglio di quanto la critica letteraria e la
letteratura sociologica non abbiano fatto sino ad oggi. Ne uscirebbe, e in parte questo avviene senz’altro nel
lavoro di Cavalli, un drammaturgo che fu al contempo
anche un pensatore politico. Il secondo motivo sta nel
fatto che da questa opera di Cavalli la politica esce con
una lettura ad ampio spettro, grazie alla quale emergono
lati non sempre sufficientemente trattati, come ad esem-
pio uno dei temi che danno il titolo stesso alla ricerca: la
politica come teatro. Uscì il tema della leadership
carismatica, e a quel punto per me il problema divenne non più cosa scrivere
ma quanto scrivere: tra quantità e qualità, come noto, vi è quasi sempre il
rischio che la prima vada a detrimento della seconda. Riuscii a mantenere un
giusto equilibrio tra le due e la commissione apprezzò. Cominciava per me
un periodo, quello del dottorato di ricerca a Firenze, che ricorderò sempre
con grande nostalgia. Perché quel dottorato, allora coordinato dal prof. Gian-
franco Bettin, allievo di Cavalli, ebbe il pregio di mantenere salde le radici
teoriche e scientifiche della tradizione della sociologia politica italiana, alla
quale Luciano Cavalli appartiene da protagonista, e di darci al contempo SOCIETÀMUTAMENTOPOLITICA 14(27): 129-137, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14344 130 Andrea Millefiorini fischiare o addirittura sbeffeggiare. E c’è infine un pal-
coscenico. i una preparazione che non perse di vista gli orientamenti
metodologici che andavano affermandosi dalla fine degli
anni Novanta. Ebbene, tutte queste figure e queste cose sono pre-
senti, con millimetrica coincidenza, anche nell’arena
politica. I diversi modi attraverso i quali queste persone,
queste cose e queste tecniche si combinano tra loro, dan-
no luogo a diversi scenari politici. Nell’ambito di questi
ultimi, la teatralità della politica si estrinseca in diverse
dimensioni e su diversi livelli. Cerchiamo, seguendo il
testo di Cavalli, di enuclearne i principali. Ricordo una giornata nell’aula “Alberto Spreafico” del
Dipartimento di Scienza politica e Sociologia, nella qua-
le si tenevano le lezioni del dottorato, alla quale partecipò
il Professor Cavalli, per ascoltare alcuni di noi dottorandi
esporre lo stato della propria ricerca. La sua figura destava
grande rispetto e deferenza: era seduto al centro, tra i suoi
colleghi e di fronte a noi studenti emanando proprio quel
carisma del quale aveva nei suoi studi spiegato e interpre-
tato così bene le dinamiche e gli effetti. La sua voce era
ferma e densa, il suo sguardo acceso. Per ognuno di noi
ebbe parole tanto franche quanto utili e chiare. Si perce-
piva benissimo la sua passione che a un tempo era di stu-
dioso e di docente, che manteneva allora pur essendo già
pervenuto da qualche anno al pensionamento. Uno dei temi più ricorrenti in quest’opera è quel-
lo del teatro politico di massa. In entrambe le tragedie
shakespeariane trattate dall’Autore, il Giulio Cesare e il
Coriolano, infatti, la plebe è sempre presente in modo
non esclusivamente passivo. Pensare che, grazie a quello che è
stato probabilmente il più grande drammaturgo dell’era
moderna, la politica sia stata spiegata anche ricorrendo
alla metafora del teatro, è qualcosa che conferisce ancora
più incisività e valore a tale spiegazione. Del resto, non
si tratta nemmeno di una semplice metafora. La politica
non è “come” il teatro, la politica “è” anche teatro. Questo ovviamente non significa che un attore
politico sia condannato ad indossare sempre la stessa
maschera. L’importante è che non la cambi all’interno
di uno specifico contesto. Egli stesso potrà però cam-
biare quel contesto, con nuovi scenari, nuovi personag-
gi e quindi nuove maschere. Come seppe fare Antonio:
«L’Antonio shakespeariano può provare il suo genio del
teatro politico, ristrutturando la scena per poter eserci-
tare la massima suggestione sulla massa. Davvero egli è
ora autore, regista e primo attore» (Ivi: 69). 2. La politica come teatro. Il teatro è fatto di atto-
ri, registi, maschere, costumi, scenografi, sceneggiatori,
autori tragici, drammatici e comici e altrettanti attori
tragici, drammatici e comici. C’è un copione, ci sono
delle tecniche di recitazione, ci sono dei ruoli e del-
le interpretazioni, che possono essere buone o cattive. C’è poi, non ultimo, un pubblico che può applaudire, Anche nel Coriolano troviamo diversi riferimenti al
teatro della politica. Il protagonista subisce spesso l’in-
fluenza e il condizionamento della madre, Veturia (nel
testo Cavalli la chiama erroneamente Volumnia, che era 131 Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura invece la moglie di Coriolano), la quale lo istruisce all’ar-
te della simulazione e dissimulazione. Arte sulla quale,
nel XVII secolo, il cardinale Mazzarino ebbe a scrivere
interessanti pagine nel suo Breviario dei politici 1989). Roma dei primissimi anni della repubblica, esisteva già
una salda cultura politica che aveva in odio l’oppressione
e il dominio di un autocrate, o quello di una classe su
un’altra. E la conseguenza dell’opzione di Coriolano fu,
lungi dal garantire una ricomposizione del tessuto socia-
le romano, esattamente il contrario, cioè un nuovo con-
flitto civile. E il capro espiatorio di questa crisi, alla fine,
fu proprio Coriolano, condannato all’esilio.i g
p
Ma l’aspetto certamente più rilevante della figu-
ra di Coriolano concerne la sua insopprimibile volontà
di non separare l’uomo dall’attore, di non scindere la
sua interiorità dalla sua esteriorità, il suo credo dal suo
linguaggio. Ebbene, proprio questa qualità – qualità,
ovviamente, per l’etica dell’homo civicus, non certo per
quella dell’homo politicus – lo porterà alla rovina poli-
tica: «Incapace di abiezione, pronuncia sul Foro soltanto
poche parole di concordia e pace, che i tribuni contro-
battono con rinnovate accuse di tradimento e tirannide. Coriolano irato manda al diavolo tribuni e popolo. L’e-
roe precipita ormai in un rovinoso destino» (Ivi: 138). L’ordine è quindi figlio di un vincolo politico che
si fonda, certo, anche sul monopolio della forza, ma
senza che quest’ultimo possa essere considerata con-
dizione necessaria e sufficiente per il mantenimento
dell’ordine stesso. Esiste un rapporto di reciprocità, in
quel vincolo, per il quale il potere politico deve pog-
giare necessariamente anche su una base di riconosci-
mento e di legittimazione, senza la quale «subentrano
disordine diffuso e, tosto, disgregazione» (Cavalli 2006:
142). Cavalli sottolinea infine come anche la saldezza
dell’ordine gerarchico che connota la catena del coman-
do politico, dal vertice fino agli esecutori, costituisca
un elemento a garanzia del mantenimento dell’ordine. Se quella catena si spezzasse, anche in questo caso la
comunità sarebbe esposta al rischio del disordine e del-
la disgregazione. Elemento, quest’ultimo, spesso trala-
sciato dalle analisi della scienza politica, e che tuttavia
Cavalli mette giustamente in rilievo in quanto anch’es-
so elemento fondamentale per il funzionamento della
macchina del potere politico. 3. Il problema dell’ordine. Cavalli chiarisce sin dall’i-
nizio del suo lavoro che l’oggetto principale del Giu-
lio Cesare shakespeariano è a suo avviso «la questione
dell’ordine in una società-stato che attraversa un critico
mutamento imposto dalla dinamica del processo sto-
rico» (Ivi: 18). Come egli spiega molto bene, dentro il
problema dell’ordine sono contenute infatti tutte le prin-
cipali questioni relative, più in generale, alla categoria
della politica: la struttura del potere, le figure dell’auto-
rità, l’apparato simbolico, il conflitto. La lettura dei fat-
ti e delle azioni che si susseguono nella scena del Giulio
Cesare debbono quindi essere interpretate non tanto e
non solo in rapporto al problema del potere, ma anche e
soprattutto in relazione al problema dell’ordine. 4. Ma è soprattutto nella ben nota orazione funebre di
Antonio per Giulio Cesare che è possibile riscontrare in 132 Andrea Millefiorini modo più chiaro la capacità di un politico di definire la
situazione: sfortunati saranno coloro che sono governati da essa, ed
essa stessa sarà presto o tardi scalzata da un’altra politica
in grado di farlo. Antonio non è prigioniero di un personale mondo ideale,
come Bruto. Appare intento a cogliere gli umori della gen-
te che lo circonda e ad adeguarvisi e, quando occorra, a
modificarli, con duttilità, astuzia, pazienza, avendo sempre
di vista lo scopo ultimo: stabilire presso il popolo romano
la sua definizione della “realtà”, che deve psicologicamente
determinare l’azione contri i congiurati. Egli trova subito il
tono giusto, familiare e rispettoso a un tempo, che gli per-
mette di stabilire un buon rapporto con la massa. Per gua-
dagnarsela, si richiama a concreti eventi, naturalmente da
lui ridefiniti, rivolgendosi assai più al sentimento che alla
ragione, con immagini che operano in modo diretto sulla
fantasia e l’emotività popolari – le modalità che già Le Bon
gli avrebbe raccomandato (Ivi: 66-67). Nelle tragedie shakespeariane il capo politico, sia
esso un monarca, un generale, un qualsivoglia leader
di qualsivoglia gruppo o comunità, viene sempre trat-
teggiato ricorrendo all’immagine di un uomo, al fondo,
solo con sé stesso. E così è, né potrebbe essere diversa-
mente. Riccardo Muti ebbe a dire una volta, a proposito
del ruolo del direttore d’orchestra: «Il podio è un’isola di
solitudine», dove il podio è il palchetto sul quale il diret-
tore sale per dirigere l’orchestra. Non si potrebbe trovare
metafora più adatta per descrivere il ruolo di guida di un
leader. E Cavalli sottolinea giustamente questo elemento
tipico della leadership. Due personaggi dei quali l’Autore mette in eviden-
za “in negativo”, nel senso di assenza di qualità di lea-
der, le caratteristiche personali, sono Bruto e Coriolano. Cioè dire, come non deve essere un leader e cosa non
deve fare un leader. Privo di intuito politico, scarso nei
giudizi sulle situazioni e sugli uomini, troppo rigido nei
principi al punto da essere troppo ideologico e quin-
di privo di realismo, Bruto fa scelte sbagliate e prende
quindi decisioni rovinose. Così come privo di realismo
fu Coriolano. La politica come definizione della realtà. Uno dei
pregi principali dell’analisi di Cavalli è l’aver messo in
evidenza, e spiegato molto bene, come Shakespeare inse-
risca nella dinamica e nella successione dei fatti narrati
una funzione essenziale della politica: la definizione del-
la situazione. Ben prima di Thomas e Znaniecki, dun-
que (i quali tra l’altro evidenziarono questa dimensione
non nella politica ma nell’ambito dell’interazione socia-
le), Shakespeare attribuisce, sebbene certo in modo non
esplicito ma comunque ben ravvisabile nel suo testo,
questa dimensione così importante e imprescindibile
della politica e ancor più di un leader politico. Cesare, nella sua persona, incarna l’ordine. E l’ol-
traggio alla sua persona perpetrato dai congiurati si tra-
durrà immediatamente in oltraggio all’ordine. Quell’or-
dine che l’ultima classe politica della repubblica non era
più in grado di garantire e, soprattutto, di impersonare. «Bruto è povero di intelligenza politica, come d’altron-
de gli altri membri della congiura di cui è il capo. Non
si sono resi conto del fatto che Cesare ormai impersona
l’ordine, lo Stato, e quindi la sua uccisione provocherà
verosimilmente il caos» (Ivi: 30). E tuttavia, il problema dell’ordine non si risolve
attraverso il dominio. È, questo, un altro fondamentale
contributo che ci viene dalla scienza politica shakespea-
riana (Krippendorff 2005). Ciò è quanto si palesa molto
bene nel Coriolano. Il generale romano, cresciuto nell’e-
ducazione patrizia, e che aveva personalmente in spregio
la plebe, eroe sul campo di battaglia grazie alle sue virtù
di soldato e di ufficiale, credeva che il problema dell’or-
dine a Roma potesse essere risolto attraverso il puro
dominio sulle masse. E la sua sconfitta politica, pari
per entità alla sua vittoria militare, è la prova che, nella Quando un gruppo politico intraprende un percorso
per arrivare al potere, deve essere portatore di una lettu-
ra della realtà e quindi di una sua definizione. Dopo l’as-
sassinio di Cesare la repubblica si trovava, politicamen-
te, sotto un cumulo di macerie, tra le quali i vari gruppi
politici cercavano di prevalere uno sull’altro. E ciascuno
cercava di far prevalere la propria definizione della real-
tà presso il popolo. Nessuno riuscì a prevalere e si andò
alla guerra civile. Il quale tuttavia prese decisioni che si rive-
larono sbagliate non per lo scarso intuito o per giudizi
errati su persone o cose, ma per la totale assenza, nel
suo bagaglio personale, di un altro, fondamentale attri-
buto di un vero leader politico: il senso storico. Non
aver saputo cogliere che la storia stava virando verso
una ascesa della plebe, e il voler essere rimasto fermo su
posizioni oramai indifendibili costò a Coriolano l’accusa
di traditore e la pena dell’esilio. Se quindi le fazioni repubblicane avevano cercato,
prima del discorso di Antonio, di diffondere la loro ver-
sione dell’assassinio di Cesare, versione che era fino ad
allora in qualche modo riuscita a far breccia nelle plebe,
con l’orazione di Antonio tutto viene rovesciato: adesso
Bruto e Cassio non sono più i salvatori di Roma, i cam-
pioni delle virtù repubblicane. Essi diventano, dopo quel
discorso, i traditori di Roma e i responsabili del disordi-
ne nel quale la città era precipitata. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. 133 Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura patie della plebe, divenendo così autore, regista e primo
attore. Ma non solo. Scrive Cavalli: Il progressivo affievolirsi dell’autorità delle istituzio-
ni è inversamente proporzionale alla crescente attrazio-
ne, da parte delle masse, verso figure più o meno cari-
smatiche: «La massa popolare di Roma richiede il potere
personalizzato, contro le illusioni repubblicane dei con-
giurati» (Ivi: 23). [q]uesto essenziale racconto della seduzione di massa
condotta da Antonio, e dei suoi rivoluzionari effetti, ha
lasciato in ombra alcuni meccanismi sociologici che han-
no trasmesso la nuova, motivante definizione della realtà,
suscitando conformi comportamenti di massa. Lo studioso
di questi fenomeni sa bene che, a questi fini, il meneur ha
bisogno di intermediari tra la massa, che possono addirit-
tura assolvere la funzione di leadership vicaria. E lo sape-
va bene Shakespeare, per l’acuta osservazione della vita e
la profonda riflessione sulla storia. Nel Richard III rappre-
senta con grande evidenza una folla manipolata per mezzo
degli uomini di Buckingham infiltratisi in essa, per insce-
nare una chiamata popolare di Riccardo sul trono d’Inghil-
terra. Tra la folla disorganizzata del Julius Caesar si hanno
corrispondenti fenomeni spontanei (Ivi: 73-74). Se però non sussiste un leader in grado di governare
le masse, in grado di dirigerle anche “contro” le passio-
ni e gli istinti che circolano di esse, queste ultime posso-
no diventare una forza “primigenia” distruttiva, che può
cadere preda di demagoghi, di imbonitori e di incanta-
tori. Ed è proprio questa la situazione che venne a pre-
sentarsi dopo la morte di Cesare, e tutta la drammatici-
tà e il pericolo che questo stato di cose portava con sé ci
viene descritto da Cavalli, come meglio non si potrebbe,
in occasione delle esequie solenni e dei discorsi fune-
bri in ricordo di Cesare tenuti prima da Bruto e poi da
Marco Antonio. Bruto e Marco Antonio impersonano i
due esempi opposti di come, rispettivamente, “non va” o
“va” gestito il potere sulle masse in momenti come que-
sti. Già subito dopo l’assassinio del dittatore da parte
dei congiurati repubblicani, capeggiati da Bruto e Cas-
sio, Marco Antonio, che non faceva parte del complotto
e che era sempre stato un generale fedele a Cesare, col-
se quella occasione per iniziare il corso della sua ascesa
politica. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Lo fece strappando subito a Bruto la promessa
di poter pronunciare anch’egli un elogio funebre in occa-
sione delle solenni celebrazioni. Errore fatale di Bruto, il
quale, non conoscendo minimamente la legge che secoli
più tardi avrebbe formulato Machiavelli, regalò in questo
modo al suo nemico quell’ultimo respiro che lo tenne in
vita e che gli permise, grazie proprio a quel discorso, di
ribaltare completamente a suo favore una situazione che
sembrava invece saldamente in mano ai congiurati. Mar-
co Antonio, intervenuto a parlare dopo Bruto, riesce, con
il suo famoso discorso, a screditare e mettere in cattiva
luce l’operato dei congiurati, risvegliando nel popolo sen-
timenti morali di offesa all’onore e al prestigio di Roma. In ciò Antonio riuscì utilizzando sapientemente gli stru-
menti della retorica, della demagogia e, non ultimi,
dell’arte teatrale, «che si manifestano nell’uso della parola
magica e dei sussidiari strumenti retorici – il testamento
[di Cesare, N.d.A.], la veste, le spoglie di Cesare» (Ivi: 61). Non solo in questo riuscì Antonio, ma anche nel ricon-
giungere il popolo al suo iniziale affetto verso Cesare
e nel saper esprimere gratitudine e riconoscenza per il
grande leader. Tutto ciò significò, al contempo, il volger-
si dei sentimenti verso i congiurati, dall’iniziale consenso
alla successiva condanna e risentimento verso coloro che
ormai, dopo il discorso di Antonio, apparivano come dei
vili e dei traditori. Antonio riesce in tal modo a “ristrut-
t
l
”
l
l t
f
l
i Se Antonio mostra palesemente doti di leadership
superiori a quelle di Bruto, ciò non toglie che esse sia-
no accompagnate, nel suo caso, anche da una massiccia
dose di demagogia. Sta in effetti proprio qui una delle
questioni che con più frequenza Cavalli presenta al let-
tore come “opacità” che non di rado emerge dalla figura
di un leader. Una leadership pienamente e funzional-
mente rispondente ai bisogni per cui è chiamata a ope-
rare dovrebbe invece essere in grado di prescindere dal
momento demagogico. Esattamente come seppe fare
Giulio Cesare. Sotto questo aspetto potremmo mettere a confron-
to la figura di Cesare, guida politica del suo popolo, con
quella di Alcibiade, ateniese che anch’egli, per un cer-
to periodo, si trovò a guidare i suoi cittadini. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Un primo elemento che emerge dalla trattazione di
Cavalli riguarda il leader come uomo in grado di tener
testa e di gestire situazioni in rapido cambiamento. Un
leader riesce a rimanere al centro della scena politi-
ca anche in circostanze che cambiano rapidamente. È
anche in questo modo che egli riesce a mantenere salda
la presa sulle moltitudini, sul suo gruppo, sui più stretti
collaboratori. Dando risposte e rassicurando, dando una
lettura convincente della situazione e fornendo quindi
indicazioni chiare per l’azione successiva. Collegato a
questo aspetto del mutevole corso degli eventi vi è poi
un secondo, altrettanto essenziale fattore necessario e,
diremmo, indispensabile a colui che ambisca a ergersi a
leader di un gruppo o di una intera comunità politica:
un leader deve essere «rapido a cogliere l’occasione favo-
revole, nella estrema mutevolezza degli uomini e delle
circostanze» (Ivi: 38). Qui Cavalli coglie un punto che
costituisce a nostro avviso uno dei cardini della figura di
un capo politico ma anche, più estensivamente, di tutta
la cultura dell’azione politica in quanto tale. Se la poli-
tica non è in grado di sfruttare a suo favore un’occasio-
ne, di anticipare i tempi, di saper cogliere al volo un’op-
portunità o di saperla volgere a suo favore, ebbene se la
politica non sa far valere questa tecnica indispensabile, Altra prerogativa fondamentale della leadership, come
Cavalli evince chiaramente dal testo shakespeariano, è la
capacità di comporre i conflitti interni e di saper difende-
re il gruppo, o la comunità, da minacce esterne. In questo
senso, la capacità di sintesi e di composizione dei diversi
interessi presenti all’interno del gruppo o della comunità
si rivela lo strumento migliore per evitare conflitti inter-
ni. Sempre, naturalmente, se tale capacità è accompagnata
dal carisma, solo in grado di sapere e potere applicare le
decisioni anche, se necessario, imponendole. Comune alle due vicende storiche, come più volte
ricorda l’Autore, sta la precarietà e la fragilità delle isti-
tuzioni politiche precedentemente costituitesi. Per insor-
gente “senilità” di esse, nel caso di Cesare, o per il loro
ancora troppo flebile sviluppo, nel caso di Coriolano. Ed
è proprio in situazioni di questo genere che la questione
della leadership può diventare decisiva, in senso risoluti-
vo o, viceversa, involutivo rispetto al problema dell’ordi-
ne sociale. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Mentre
il primo esercitò la sua leadership senza dover arrivare
a toccare, o peggio ancora a manipolare, le corde sen-
timentali, passionali o umorali dei suoi concittadini, il
secondo, come ricorda molto bene Bertrand de Jouvenel
(1997), fece della sua abilità nell’orientare le opinioni del
popolo la sua fortuna. Fortuna che tuttavia, come sap-
piamo grazie a Tucidide (2003), non durò a lungo, e si
volse tempo dopo in fallimento. E tuttavia ben pochi
sono i leader che riescono a fare a meno del momento
demagogico. Spesso, per un leader, il popolo non è un
valore in sé, ma solo uno strumento di lotta politica tra
capi, scrive Cavalli, sicché il popolo stesso «deve essere
indotto a credersi sempre il vero protagonista, che pen-
sa, decide, agisce per autonoma deliberazione. Questa
adulazione “populistica” consente di farne ciò che si
vuole» (Cavalli 2006: 95). Ed è esattamente contro tale aspetto, tipico dei tri-
buni sin dagli inizi della Repubblica, che si scagliò l’altro
protagonista dell’opera di Cavalli, Coriolano. Quest’ul-
timo teneva in spregio sia i tribuni, manipolatori del 134 Andrea Millefiorini popolo, sia il popolo stesso, considerato come un gregge
che segue docilmente i comandi del suo pastore. Anche nel Coriolano, tuttavia, il tema di cui stiamo
parlando non è assente. In una prospettiva e sotto una
filigrana però in parte diversa. In questo caso Coriolano
rappresenta sì, eccome, l’esempio dell’etica della respon-
sabilità. Il suo gesto militare ne è la logica conseguenza. Tuttavia Shakespeare – la cui grandezza è stata anche
nel permettere allo spettatore di poter scomporre le
azioni dei suoi protagonisti in diverse angolature, sotto
diverse prospettive, potendo coglierne così anche l’in-
trinseca contraddittorietà e ambiguità – nel presentare
Coriolano come il campione di un’aristocrazia guerrie-
ra – «amor patrio, orgoglio di casta, franchezza, lealtà,
disprezzo del denaro, rifiuto di ogni cedimento e com-
promesso, […] fortezza in ogni circostanza» (Ivi: 112)
–, ci mostra quanto queste virtù, se non accompagnate
anche da scaltrezza, lucidità, rapidità di analisi e di deci-
sione, rischino di rendere l’etica della responsabilità un
vuoto involucro, non in grado di assolvere alle funzioni
per cui un politico è chiamato. E Coriolano, che non è
soltanto una storia teatrale ma una persona e una vicen-
da realmente esistita, ne è uno degli esempi più chiari. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Anche qui, lo Shakespeare politico ci mostra il lato opa-
co di un’etica che se “stirata” fino a tenderla oltre i suoi
limiti, si ritorce contro colui che, pur in buona fede, ne
applica acriticamente i dettami, producendo effetti con-
trari a quelli auspicati. «La via dell’inferno è lastricata
di buone intenzioni», recita un noto detto. Ebbene, la
grandezza di Shakespeare sta nel mostrarci come a volte
la “via dell’inferno” possa essere, paradossalmente, per-
corsa non solo dall’etica della convinzione – concetto,
questo, ampiamente riscontrabile nella storia del Nove-
cento – ma anche dalla stessa etica della responsabilità,
se non accompagnata dal giusto distacco e da quel pizzi-
co di sano disincanto, necessari entrambi per mantenere
abbastanza vivo il chiarore di quel «crepuscolo delle pos-
sibilità» alla luce del quale John Locke ci ha spiegato che
vengono prese le decisioni più importanti. Puntualmente anche Coriolano, come Alcibiade, fal-
lirà. Fallirà non perché si oppose ai tribuni demagoghi,
ma perché anch’egli, come Alcibiade, in fondo al cuore
non aveva alcuna stima per il popolo. Giulio Cesare non
fallì mai, politicamente. Dovette essere ucciso fisicamente
per essere tolto di mezzo. E Giulio Cesare aveva una pro-
fondo rispetto per il popolo. Infatti non lo manipolava. 5. Cultura politica, ideologia ed etica della responsa-
bilità. Shakespeare sembra aver anticipato Weber su una
tematica del calibro di quella dell’etica della responsabi-
lità, come ben mette in evidenza Cavalli. La figura sulla
quale in maggior misura è possibile impostare uno stu-
dio sul rapporto tra etica della responsabilità ed etica
della convinzione, nelle due tragedie, è quella di Bruto. Egli pare assumere, ante litteram, la posa dell’homo ide-
ologicus di novecentesca memoria. L’ideale repubblicano
assume ormai, in lui, più un tratto ideologico che non
una cultura politica, di per sé stessa flessibile e quindi
aperta. In altri termini Bruto non riesce a contestualiz-
zare i pur fondamentali valori e simboli della Repub-
blica, mettendoli in relazione alla grave e irreversibile
congiuntura sociale e politica di quegli anni. In breve:
Bruto restò fedele all’etica della convinzione a detri-
mento dell’etica della responsabilità. E ne pagò un prez-
zo molto alto. Questo è infatti ciò che prima o poi acca-
de – come traspare chiaramente dal pensiero di Caval-
li – ai politici che antepongono acriticamente la prima
alla seconda. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. La congiura fu infatti, come tutte le con-
giure, affare di una ristretta oligarchia, sebbene, a fatto
avvenuto, il Senato manifestò una certa condiscenden-
za verso quel tragico gesto. Questa oligarchia era ormai
investita da odio verso colui che aveva dimostrato sul
campo le doti di leader in grado di mantenere salda
l’integrità di una città e di una società intera, a dispet-
to dei pur frequenti tentativi di destabilizzazione poli-
tica che si susseguivano ormai da tempo. Sicché, vuote
e vane risulteranno, nell’orazione funebre, le parole di
Bruto che, rivolgendosi all’uditorio, domanda retorica-
mente «chi è tanto vile da non amare la Patria?», quasi
una excusatio non petita per il gesto compiuto. Senon-
ché, la risposta della plebe, con quel «Viva Bruto! Viva
Bruto!» gridato dalla folla a squarciagola, non gli fa
minimamente sospettare che in quel grido vi era tutto il
bisogno, tutta la necessità, da parte del popolo di Roma,
di trovare una figura in grado di rassicurarlo, di ridargli
quella fiducia che da tempo le istituzioni repubblicane,
per come si erano andate indebolendo, non erano più
in grado di assicurare ai cittadini. Proprio ciò che aveva
fatto, e stava facendo, Giulio Cesare. Sicché, se l’etica della responsabilità poggia soltanto
su rigidi valori, essa non diviene altro che pura etica del-
la convinzione, esattamente come accaduto a Coriolano. La linea che divide l’una dall’altra è dunque molto
sottile, difficile a volte anche da distinguere. E un vero
leader, un vero statista, si può riconoscere anche dalla
capacità di riconoscere quella linea di confine sulla qua-
le termina l’etica della responsabilità e inizia l’etica della
convinzione. La flessibilità, l’apertura mentale, la dispo-
nibilità al compromesso costituiscono, a questo riguar-
do, elementi essenziali al di qua di quella immaginaria
linea di confine tra le due etiche. 6. La questione del sistema politico. Diversi riferi-
menti nel testo di Cavalli rimandano alla questione della 135 Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura zionale. Senonché, il tentativo di Cesare fu stroncato dalla
ben nota congiura di Bruto. E l’errore di Bruto fu proprio
un errore «di giudizio storico-politico. La personalizza-
zione del potere, infatti, corrispondeva alle necessità del
tempo. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Se il potere personale è associato alla dittatura, alla
monarchia o all’impero, il potere oligarchico è associato,
per lo meno nella storia di Roma, in genere alla Repub-
blica, o al massimo all’ultima fase del periodo monar-
chico, quando sebbene un Re cingesse sul suo capo una
corona, di fatto alcune famiglie patrizie erano in grado
di essere determinanti per la sua ascesa e mantenimen-
to al trono. Tant’è che negli ultimi anni della monarchia
il potere dell’oligarchia patrizia divenne talmente pre-
ponderante che si rese necessario quel cambio di assetto
politico che portò all’approdo repubblicano. Per Shake-
speare, sembra che tra potere personale e potere oligar-
chico, e potere oligarchico che tende anche alla demo-
crazia (degli antichi), sia senza alcun dubbio da preferire
il primo rispetto al secondo. Ciò emerge chiaramente,
osserva Cavalli, soprattutto dal Coriolano, la cui disami-
na, da parte di Shakespeare, «ha anche un evidente valo-
re critico verso la democrazia degli antichi» (Ivi: 127). Cavalli ha il merito di porre la questione del cesari-
smo mettendone in luce la sua specificità, se confrontato
con altre esperienze, compresa quella imperiale, succes-
sivamente affermatasi proprio a Roma. E infatti – come
scrive – il cesarismo «è oggi riconosciuto dagli studi
politici come un fenomeno a sé» (Ivi: 51), ben distin-
to da altre forme di potere personale, come la tirannia,
l’autoritarismo, il bonapartismo, la monarchia assoluta o
altre fattispecie. Con ciò, Cavalli non esclude che anche
il cesarismo rientri in quel tipo di assetti istituzionali
monocratici nei quali Shakespeare vedeva una garanzia
di pace e di ordine all’interno di uno Stato. Ma un conto
è riconoscere questo giudizio in Shakespeare, conside-
rando il periodo storico in cui egli visse, altro è ritenerlo
valido ai nostri giorni, cosa che Cavalli si guarda infatti
molto bene dal fare: «Il tallone d’Achille del cesarismo
come sistema politico [sta nel fatto che] il potere è stret-
tamente personale. Non è prevista ordinata successione
in termini costituzionali […] ed anche da questo fatto
conseguiranno l’assassinio e la guerra civile» (Ivi: 27). 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. […] Uccidendo Cesare, pilastro centrale dell’or-
dine-ponte, Bruto precipita Roma nel caos della violenza
anarchica, cui farà seguito la più selvaggia guerra civile»
(Ivi: 79). La storia proseguì poi il suo corso seguendo le
sue vie imprevedibili e imperscrutabili. Vie che portarono
al nuovo assetto uscito dopo la lunga fase che portò infi-
ne al prevalere di Augusto. Fu, quello disegnato dal pri-
mo imperatore romano, il sistema politico migliore per le
condizioni, i mezzi e gli strumenti che quella élite politica
aveva a disposizione? Non spetta a noi rispondere a questa
domanda. Certo è che il corso della storia, qualora Cesare
fosse rimasto in vita, sarebbe stato probabilmente diverso. Migliore? Peggiore? Anche qui, non spetta ad un sociolo-
go dare giudizi. Gli spetta tuttavia rilevare tale condizio-
ne. Così come sempre al sociologo spetta ricordare come
«nella storia di Roma, come nella tragedia [di Shakespe-
are] confliggono l’oligarchia della tradizione ed il potere
personale sorto dalla guerra civile» (Ivi: 93). personalizzazione del potere, con altrettante riflessioni
che l’autore permette di fare anche in merito al miglior
governo e al sistema politico non solo ai tempi di Cesare
e di Coriolano, ma anche ai nostri giorni. L’idea weberiana che in tempi di crisi delle istituzio-
ni sia molto frequente il fenomeno della personalizzazio-
ne del potere viene sostanzialmente ripresa da Cavalli,
che la declina qui, e le dà ulteriore conferma, collocan-
dola storicamente ai tempi di Roma.i La figura di Giulio Cesare, da sempre, ha prodotto
due opposte letture della vicenda che lo vide protago-
nista di uno dei periodi della storia umana più densi di
simboli e di significati. La prima, favorevole, lo consi-
dera colui che, grazie al suo carisma e alle sue capacità
politiche e militari, evitò che Roma arrestasse il processo
di sviluppo, di crescita e di influenza politica che stava
progressivamente acquisendo nel Mediterraneo e che le
permise di diventare la culla della civiltà occidentale. La
seconda vede invece nella dittatura di Cesare la sostan-
ziale fine della cultura politica e giuridica nata sotto la
Repubblica, e quindi la svolta verso un tipo di regime
sostanzialmente dittatoriale, mascherato successivamen-
te dalle riforme augustee, riforme che tuttavia non ne
cambiarono la sostanza. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Potremmo suddividere in tre grandi gruppi le tecni-
che in oggetto: quelle manipolatorie, quelle della retorica
e quelle tattico-strategiche. Tra le tecniche tattico-strategiche che vengono
menzionate, una in particolare merita qui considerazio-
ne. In sostanza, si tratta della antica questione del che
fare e come comportarsi con il nemico una volta che
questi sia sull’orlo della sconfitta, o sia stato definitiva-
mente sconfitto. Tra le prime incontriamo la lusinga. Bruto “lusinga”
Giulio Cesare interpretando positivamente il suo sogno
di Calpurnia, nel quale egli muore. E lo fa con lo sco-
po di convincerlo a recarsi al Senato, dove tutto era già
pronto per il suo assassinio. Dopo quel sogno, infatti,
Cesare ebbe un presentimento che lo induceva a non
andare in Senato. Cesare non era certamente un politico
facilmente adulabile, ma la grandezza di Shakespeare sta
nel sottolineare come, nel suo caso, la lusinga non avve-
nisse su cose reali o su tratti reali della sua persona, ma,
ancora più profondamente, sul suo inconscio, sul suo
Sé più profondo. Del resto, non fu proprio Shakespeare
a pronunciare la famosa frase «Siamo fatti della stessa
sostanza con cui son fatti i sogni»? Nel primo caso (il nemico messo all’angolo), Sun
Tzu insegna, ne L’arte della guerra (2003), che al nemi-
co va sempre lasciata la possibilità di fuggire. In questo
modo si può ottenere una vittoria senza gravi perdite o
ulteriori spargimenti di sangue. Diversamente, al nemi-
co non resterebbe che una istintiva reazione aggressiva,
come una bestia feroce ferita, tentando il tutto per tutto
non avendo nulla da perdere.i Nel secondo caso (il nemico sconfitto), Machiavel-
li, ne Il Principe, raccomanda di annientare del tutto il
nemico dopo la sua sconfitta, senza lasciargli la benché
minima possibilità di potersi riprendere. Diversamen-
te, quel nemico non farà altro che attendere il momen-
to propizio per vendicare il suo odio contro il vincitore,
potendo magari anche riuscire a ribaltare completamen-
te l’esito della iniziale sconfitta in una vittoria. A sua volta, Bruto viene manipolato da Cassio, che
riesce a convincerlo ad ordire la congiura contra Cesa-
re. Cassio è quindi la vera mente e il vero artefice delle
idi di marzo. E riuscì nel suo scopo grazie all’arte della
manipolazione. Capacità della quale le personalità ego-
centrate e narcisistiche sono dotate in misura enorme:
«Cassio espropria Bruto dell’autonomo giudizio. 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Si conferma così una lettura di Shakespeare politico,
presente anche in altri autori, che ci descrive il grande
drammaturgo inglese convergere con il clima storico
nel quale visse, e che trovò successivamente in Hobbes
il suo più autorevole teorico: l’assolutismo del potere
come strumento che, in quell’epoca, si affermava come
migliore strumento non solo per il problema dell’ordi-
ne, ma anche per quella secolare e titanica sfida che fu
la costruzione dello Stato-Nazione. Cavalli, chiaramen-
te, storicizza a sua volta la posizione di Shakespeare,
mostrandone appunto i limiti se comparata con le con-
dizioni sociali, economiche e istituzionali di oggi. Una cosa, tuttavia, è certa: Cavalli, grazie a Shake-
speare, lascia chiaramente intendere che le istituzioni
repubblicane, o meglio “quelle” istituzioni repubblicane,
avevano ormai perso ogni capacità di poter incidere sui
profondi processi di mutamento che stavano investendo
da tempo la società romana. In questo senso, il cesarismo
aveva iniziato a svolgere una funzione che, piaccia o no,
stava traghettando Roma verso un nuovo assetto istitu- 136 Andrea Millefiorini stringe le mani insanguinate dei congiurati, nominando-
li uno a uno. Ma poi, rivolto al corpo giacente di Cesare,
il suo dolente affetto prorompe: “Pardon me, Jiulius! Here
was thou bayed, brave heart” [Perdonami Giulio! Qui sei
stato tratto in inganno, cuore coraggioso]. E, sviluppando
quest’altra metafora, ridefinisce cripticamente l’assassinio
in modo opposto al rito dei congiurati, che sembrano non
avvertire la minaccia. L’immagine del regale cervo, che del
mondo era “the heart”, il centro vitale, ed è stato tratto in
agguato e trucidato dai cacciatori, contraddice drastica-
mente il modello del tirannicidio sacrificale proposto dai
congiurati, facendo dell’uccisione di Cesare un crimine di
significato universale da pagare con le vite di tutti coloro
che hanno partecipato alla congiura (Ivi: 42-43). 7. Tecniche del potere. Vi è infine un ultimo, non
meno interessante tema che emerge con un suo profi-
lo ben definito dalle pagine del libro. Si tratta di quella
che molti “manuali” sulla gestione e l’esercizio del pote-
re trattano come materia da offrire a lettori dotati di un
certo appetito per il dominio e per l’influenza sugli altri. Nel nostro caso lo stile non è ovviamente quello, ma
dalle pagine di Cavalli si possono trarre alcune consi-
derazioni di rilievo in conseguenza dei ritratti dei diver-
si personaggi che calcano il palcoscenico delle tragedie
shakespeariane. Antonio ottiene – fatale errore di Bruto – di poter recita-
re l’elogio funebre sul cadavere di Cesare. Allora Antonio 5. La questione del leader. E veniamo così ad uno dei
contenuti centrali dell’opera. Gli
impone la sua immagine di Bruto che mira ad uccidere
Cesare, come d’altronde l’immagine demonizzata di un
Cesare tiranno che deve essere assassinato. Quasi un’o-
perazione ipnotica. Degna dell’attenzione di un Le Bon»
(Ivi: 81-82). In questa “operazione ipnotica” l’arte mani-
polatoria di Cassio nei confronti di Bruto prende forma
attraverso lo strumento della “vilificazione”, come la
definisce Cavalli, cioè nel dipingere come vile l’immagi-
ne e la persona di Cesare. Altro strumento utilizzato da
Cassio è la sua “demonizzazione”, fenomeno in questo
caso ben noto a psicologi, sociologi e politologi. Ebbene, Bruto commette esattamente l’errore di cui
parla Machiavelli. Egli «commette l’errore strategico
decisivo, che rovescia il corso degli eventi contro i con-
giurati: lascia che Antonio viva, nonostante l’avviso con-
trario di Cassio. Anzi, gli promette – sempre a dispetto
di Cassio – che potrà farne l’elogio funebre» (Ivi: 85). 8. Un’opera seminale per uno stile di ricerca. Con
il suo Giulio Cesare, Coriolano e il Teatro della Repub-
blica Luciano Cavalli ha offerto un grande servigio
alla sociologia politica italiana. Egli ha infatti ripreso
e ridato valore e significato ad un filone di studi, quel-
lo del pensiero politico presente nelle opere letterarie,
non sempre considerato degno di attenzione da parte
della ricerca scientifica. Pochissimi erano stati infatti,
sino alla pubblicazione dell’opera di Cavalli, i prece-
denti simili. Come esempio di tecniche del secondo tipo, quelle
che fanno affidamento sulla retorica, della quale Roma
fu maestra, Cavalli ci propone ovviamente il ben noto
elogio funebre di Antonio: Antonio ottiene – fatale errore di Bruto – di poter recita-
re l’elogio funebre sul cadavere di Cesare. Allora Antonio 137 Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura Un’altra importante strada aperta da Luciano Cavalli: lo studio del pensiero politico nei classici della letteratura I risultati ottenuti da Cavalli con questa ricerca sono
di indiscutibile valore sia sul piano dell’esegesi del pen-
siero politico shakespeariano, sia su quello della compa-
razione e della messa alla prova di quel pensiero con le
più consolidate teorie sociologico-politiche sul potere e
sulla politica. Un’opera insomma che non dovrebbe mancare nella
libreria di ogni scrupoloso studioso di sociologia politica. 1 Testo virgolettato delle parole di Don Abbondio in Alessandro Manzoni (1985), I promessi sposi,
Mondadori, Milano, p.55.
2 Va ricordato che a partire dall’anno accademico 1968-69 inizia il processo di riforma dell’ordina-
mento didattico della Facoltà di Scienze Politiche di Firenze che proprio per iniziativa di Cavalli e
Sartori porterà ad una importante valorizzazione dell’insegnamento delle scienze sociali. RIFERIMENTI BIBLIOGRAFICI Cavalli L. (2006), Giulio Cesare, Coriolano e il teatro della
Repubblica. Una lettura politica di Shakespeare, Rub-
bettino, Roma. De Jouvenel B. (1997), «Saggezza e azione: lo pseudo-
Alcibiade», in Id., La teoria pura della politica (intro-
duzione di Mario Stoppino), Giuffré, Milano, pp. 23-28. Krippendorff E. (2005), Shakespeare politico. Drammi
storici, drammi romani, tragedie, Fazi, Roma. Mazzarino G. (1989), Breviario dei politici, Macchia G. (a
cura di), Rizzoli, Milano. Sun Tzu (2003), L’arte della guerra, Mondadori, Milano. Tucidide (2003), La guerra del Peloponneso (introduzione
e traduzione di Savino E.), Garzanti, Milano. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Citation: Franca Bonichi (2023) Il “rea-
lismo radicale” di Luciano Cavalli. Appunti per una riflessione. Socie-
tàMutamentoPolitica 14(27): 139-148. doi:
10.36253/smp-14345 Citation: Franca Bonichi (2023) Il “rea-
lismo radicale” di Luciano Cavalli. Appunti per una riflessione. Socie-
tàMutamentoPolitica 14(27): 139-148. doi:
10.36253/smp-14345 Franca Bonichi Copyright: © 2023 Franca Bonichi. This is
an open access, peer-reviewed article
published by Firenze University Press
(http://www.fupress.com/smp) and dis-
tributed under the terms of the Crea-
tive Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. Quelli che fanno il bene,
lo fanno all’ingrosso: quando
hanno provato quella soddisfazione,
n’hanno abbastanza, e non
si vogliono seccare a star dietro
a tutte le conseguenze; ma coloro
che hanno quel gusto di fare male,
ci mettono più diligenza, ci stanno
dietro fino alla fine, non prendon
mai requie, perché hanno quel canchero
che li rode [Don Abbondio]1. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. Competing Interests: The Author(s)
declare(s) no conflict of interest. 1. Introduzione. Mi pare si possa facilmente convenire che un approccio
realistico ai fenomeni della politica costituisca una sorta di “filo rosso”, indi-
viduabile in tutto il percorso intellettuale di Luciano Cavalli; uno sguardo
critico che, a partire dalle prime opere, si precisa e si formalizza negli anni,
fino a raggiungere l’articolazione e la complessità di un vero e proprio para-
digma teorico. g
La proposta di una sociologia critica, mi si perdoni questo riferimento
autobiografico, non poteva che conquistare quanti tra noi, nel lontano anno
accademico 1968-69, avevano scelto di iscriversi a Scienze Politiche proprio
allo scopo di poter disporre di strumenti concettuali più specifici e “moder-
ni” per analizzare e possibilmente per cambiare la società in cui vivevamo2. Le lezioni di Cavalli in effetti ci mettono in contatto non solo con impor-
tanti, e allora ancora poco conosciuti sociologi americani, come Parsons, ma
soprattutto con sociologi radical come Mills e i coniugi Lynd che Cavalli ave-
va conosciuto solo pochi anni prima alla Columbia University. SOCIETÀMUTAMENTOPOLITICA 14(27): 139-148, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14345 Proprio per questo suo ruolo istituziona-
le, in coerenza con una tradizione di pensiero cui par-
tecipano autori come Tucidide e Machiavelli, anche per
Cavalli l’interesse della Stato finisce pertanto con l’iden-
tificarsi col bene comune e quindi a costituire “la stella
polare” dell’azione politica. L’impostazione realista di Cavalli si completa poi
con una concezione coerentemente elitista del potere che
individua nell’agire strategico della funzione di leader-
ship e nell’incapacità politica della massa i suoi capisaldi. Una funzione complessa, quella di leadership (cui Caval-
li, come ben sappiamo, ha dedicato la maggior parte del-
la sua attività di studio e di ricerca), che nella storia «si
manifesta al meglio quando sia concentrata in una sola
persona». Affermazione cui fa riscontro la convinzio-
ne, si potrebbe affermare quasi un topos della tradizione
realista, condivisa da autori come Weber, gli elitisti ita-
liani, Le Bon e Freud (cui Cavalli esplicitamente si richia-
ma) che è quella della irrazionalità, della mancanza di
autonomia delle masse popolari che risultano incapaci
di esercitare una funzione politica coerente e autonoma
e che pertanto non possono far altro che affidarsi, più o
meno consapevolmente, a leader ed élites politiche. Cavalli in Il leader e il dittatore. Uomini e istituzioni
di governo nel “realismo radicale” del 2003 arriva ad una
puntuale precisazione del suo «realismo politico in chia-
ve radicale» (Cavalli 2003: 41) dichiarando come questo
si fondi «su alcuni presupposti ricavati dall’esperienza
del passato e del presente, analizzati con metodi scienti-
fici» e quindi «in una relazione di libero scambio con le
scienze sociali libere da asservimenti politico-ideologici»
(Ivi: 42). Una concezione «pessimistica della natura umana»,
unita alla consapevolezza della scarsità dei beni che il
contesto ambientale mette a disposizione, porta Cavalli
a concepire l’esistenza come «lotta per la vita» in senso
esteso, sia per quanto riguarda le relazioni personali e le
dinamiche tra gruppi, sia per quanto attiene ai rapporti
internazionali. Un contesto questo, all’interno del qua-
le, trovano una ridefinizione anche i concetti di ordine
sociale e di bene pubblico. Data la fondamentale inegua-
glianza tra gli esseri umani, indotta da caratteristiche sia
«native che acquisite», secondo Cavalli, l’ordine sociale
si «struttura gerarchicamente con posizioni privilegia-
te rispetto ai beni ed in particolare rispetto al bene del
comando». Una occasio-
ne per avvicinarci ad una sociologia “democratica” che, con opportuni riferi-
menti teorici e mediante l’analisi empirica, potesse svolgere quella funzione
di “democratizzazione” della società che mi pare fosse condivisa in quegli SOCIETÀMUTAMENTOPOLITICA 14(27): 139-148, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14345 SOCIETÀMUTAMENTOPOLITICA 14(27): 139-148, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14345 140 Franca Bonichi anni da altri prestigiosi fondatori della sociologia italia-
na come Alberoni, Ferrarotti, Gallino, Pizzorno e che
esercitava su molti di noi, non ancora ventenni, un indi-
scutibile fascino. l’arbitrarietà che lo caratterizza, prima o poi è destina-
to a rivelare la sua precarietà e quindi ad essere esposto
costantemente alla minaccia della instabilità politica e
del conflitto. Il conflitto, anche se latente, finisce per-
tanto per configurarsi come una modalità ricorrente,
e forse ineludibile dei rapporti sociali. Un rischio sem-
pre incombente di disgregazione e di anarchia che però
Cavalli suggerisce di considerare anche come una risorsa
politica, come un indispensabile strumento di mutamen-
to e di innovazione (ivi: 43). Cavalli, come accennavo, giunge negli anni a preci-
sare cosa intenda per un approccio realistico alla politica
arrivando anche a formalizzarne una definizione (“reali-
smo radicale”) e ad individuare i riferimenti teorici cui
questa impostazione si potesse coerentemente richiama-
re. In questo testo però non mi propongo una ricostru-
zione esaustiva e coerente di questo paradigma, avvallata
da una selezione e da un’analisi puntuale di quelle opere
che marchino in modo più significativo questo percorso. Intendo piuttosto accennare ad alcune modalità attraver-
so cui il realismo di Cavalli potrebbe essere attualmente
declinato, in riferimento a tematiche rilevanti per l’ana-
lisi della società contemporanea e comunque in sintonia
con un progetto di ricerca che mi impegna da qualche
tempo. Una opzione forse un po’ arbitraria ma in qual-
che modo legittimata dall’intenzione di esplicitare l’at-
tualità dell’insegnamento di Cavalli che, anche a fron-
te di percorsi intellettuali diversi, continua a proporre
strumenti concettuali preziosi per l’analisi dei fenomeni
politici contemporanei. All’interno di questa condizione, al tempo stes-
so precaria e potenzialmente conflittuale, lo Stato, dal
momento che nel suo sviluppo storico garantisce la
sopravvivenza di una determinata comunità (sia dal
punto di vista materiale che culturale), diventa esso stes-
so un valore. Considerazione che lo porta a ritenere che
i detentori del potere possano esercitare un dominio
anche prevalentemente basato sull’affermazione dei loro
interessi privati, in patente contrasto col bene comune
della comunità cui appartengono. Ma un ordine sociale
fondato su relazioni sociali non consensuali, proprio per Tutto questo considerato e volendo condensa-
re in una unica formula il realismo radicale di Cavalli,
potremmo limitarci a caratterizzarlo come una disposi-
zione a guardare alla società e soprattutto alla politica
(ai suoi processi, al funzionamento delle istituzioni, al
sistema nel suo complesso) come “effettivamente è, qui e
ora”, come di fatto si configura, cogliendone le dinami-
che che concretamente vi operano. Penso però sia inte-
ressante notare come questo approccio possa risultare
assai più articolato e complesso se assumiamo il termi-
ne “reale” nella doppia accezione che lo contrappone, sia
ad “apparente” che ad “ideale”. Quindi se consideriamo
il realismo di Cavalli da un doppio punto di vista: come
una sollecitazione a non accontentarsi della dimensione 141 Il “realismo radicale” di Luciano Cavalli. Appunti per una riflessione puramente fenomenica, istituzionale della realtà politi-
ca, ma anche come un invito a non fare eccessivo affida-
mento sull’ottimismo di ideologie, più o meno salvifiche,
e al moralismo del “dover essere”3. «l’autorità quando se ne abusa sistematicamente a fini
di dominazione, torna a essere mero potere, anche se è
“legittimata”, nei termini di un principio comune» (Ibi-
dem). Secondo questa interpretazione possono essere
quindi gli stessi detentori del potere legittimo ad operare
una violazione, sia della lettera che dello spirito del pat-
to sociale. Un aspetto della manipolazione che Cavalli
dichiara “essenziale” ma poco studiato anche da quegli
scienziati sociali che, come ad esempio Mills fanno rife-
rimento a questa idea-concetto (Ibidem). 2. Reale come opposto di apparente. La democrazia
manipolata (1965), benché sia una delle sue prime ope-
re, mi sembra possa costituire una ottima occasione per
riflettere su alcuni importanti aspetti dell’approccio rea-
lista di Cavalli ai temi della politica. Appare già rilevante la data di pubblicazione del
libro, solo un anno dopo il primo governo di centrosini-
stra, momento in cui, in Italia, con i socialisti al gover-
no si poteva pensare di assistere ad un significativo pro-
gresso del processo di democratizzazione. Invece, già nel
titolo, è presente una tensione tra l’elemento normativo
implicito nel termine “democrazia” e quello, empirica-
mente verificabile nell’esperienza concreta, delle pratiche
di “manipolazione”. Coerentemente col titolo, Cavalli si
propone infatti di indagare, così come aveva fatto Mills
per gli Usa, sul paradosso, per lo meno apparente, dell’e-
sistenza anche all’interno di regimi democratici, di una
«minoranza organizzata che detiene l’autorità [...] per fini
di dominazione» (Cavalli 1965: 9). L’esercizio di un potere
che si affida alla “socializzazione” e al “controllo” socia-
le con lo scopo principale di precludere alla maggioran-
za proprio quella chance di autogoverno su cui si fonda
il principio democratico. Che ricorre quindi alla manipo-
lazione per estorcere un consenso che i meccanismi isti-
tuzionali, previsti dal sistema democratico, renderebbero
assai più difficile, se non impossibile, ottenere (ivi: 19)4. Dal riconoscimento di un possibile impiego abusivo
dell’autorità a fini di dominazione Cavalli deriva il carat-
tere “segreto” della manipolazione, la necessità, che gli è
intrinseca, di agire di nascosto, evitando ogni pubblicità
(Ibidem). Bettin chiarisce il punto sottolineando come
la manipolazione cui fa ricorso la minoranza organiz-
zata non può che essere annoverata tra le pratiche di un
esercizio invisibile del potere dal momento che l’efficacia
di quell’azione fa proprio affidamento sul fatto di poter-
si sottrarre al controllo pubblico, alla trasparenza delle
procedure democratiche che potrebbe limitarne il campo
d’azione e quindi metterne a repentaglio l’autoconserva-
zione (Bettin 2008: 271). Se è certamente vero che la manipolazione ci ricon-
duce ad un esercizio del potere dissimulato e addirittu-
ra segreto, mi pare però interessante notare il fatto che
Cavalli ne proponga una definizione particolare, diffi-
cilmente rubricabile tra le forme “tradizionali” di pote-
re occulto, quelle, per fare un esempio, individuate da
Bobbio in un saggio divenuto ormai un classico, Il futu-
ro della democrazia (1984). Il “criptogoverno” si riferisce
a forme di potere in qualche modo collaterali rispetto al
potere istituzionale; mafia, camorra, ntrangheta, logge
massoniche anomale, servizi segreti deviati sono forze
politiche eversive che agiscono nell’ombra anche se non
di rado in stretto contatto con parti significative dell’ap-
parato dello Stato. Quelle che Bobbio individua come
forme di “sottogoverno” si richiamano invece a prati-
che di lobbing, esercitate soprattutto da grandi centri di
potere economico che sono “invisibili” in quanto si sot-
traggono, se non formalmente, sostanzialmente al con-
trollo democratico e al controllo giurisdizionale (Bobbio
1984). 3 Non mi sfugge la complementarietà di queste due prospettive che ten-
go separate prevalentemente per ragioni espositive, come se volessi zoo-
mare due particolari di una unica foto .
4 Nel libro Il sociologo e la democrazia (1964) Cavalli aveva già chiarito
che per democrazia manipolata «Si intende che i principi della demo-
crazia (liberale) sono formalmente sanciti nelle carte costituzionali e
incorporati nelle istituzioni politiche, ma non vi è democrazia in senso
sostanziale: i principi sono elusi e traditi» (Ivi: 21). La convinzione che certi processi potestativi, e
soprattutto quelli illegali, come la manipolazione, non
possano essere riducibili ad un comando politico visibi-
le, ad un processo decisionale oggettivamente individua-
bile, ma si esercitino anche, e forse soprattutto, attraver-
so strutture e meccanismi diversi. In La democrazia manipolata (1965), forse anche in
controtendenza con quanto avverrà in seguito, Caval-
li invece sembra più interessato a mettere in evidenza
quella che mi verrebbe da definire come “la dimensione
sistemica” del potere invisibile, secondo cui, la mani-
polazione, più che identificarsi con l’espressione di una
“volontà” individuale, si palesa invece come “principio
organizzativo”5, più come una sorta di logos “astrat-
to”, e per certi aspetti impersonale, che come decisione
intenzionalmente assunta. Non solo. Un altro dato rile-
vante è costituito dal fatto che questo sistema di potere
non si configura come una potenza a latere ma nasce e
si sviluppa all’interno delle istituzioni, persino di quelle
democratiche, contribuendo a strutturarle e a regolarne
il funzionamento. Una rappresentazione della dominazione che può
richiamare la celebre dicotomia proposta in quegli stessi
anni da Bachrach e Baratz (1986) per cui il potere può
contare su due facce. Una si esprime nelle forme ritua-
lizzate del diritto e delle regole del gioco democrati-
co, l’altra – in cui sono stabilmente impegnati i fautori
dell’ordine costituito – si manifesta invece come domi-
nio non sottoposto a limiti giuridici, né a regole stipulate
e sottoscritte dalla comunità dei cittadini. La minoranza
può quindi, secondo Bachrach e Baratz, far ricorso ad
un potere invisibile che però non trova la sua espressione
principale in vere e proprie decisioni, in espliciti atti di
volontà, ma opera attraverso il meccanismo della “non
decisione”. Un automatismo che, mediante l’attivazio-
ne di processi di «mobilitazione del pregiudizio», sbarra
di fatto l’ingresso nell’arena della decisione politica alle
issues innovative impedendo che queste si traducano in
temi di lotta politica (Ibidem). In questo senso mi sembra possa essere interpretata
la preminenza che Cavalli accorda all’analisi delle cate-
gorie di socializzazione e di controllo sociale, individuati
come gli strumenti principali attraverso cui la manipo-
lazione si struttura e si manifesta. Un sistema di domi-
nazione dedotto quindi, per così dire a posteriori dall’a-
nalisi di quei processi che rivelano una organica conver-
genza di mezzi e fini per garantire il consenso passivo
dei “dominati”, il contenimento del conflitto e quindi la
conservazione dell’ordine sociale6. Anche in questo caso si tratta quindi di sfere di
potere in qualche modo parallele anche se generalmente
la loro l’efficacia si misura proprio con l’influenza eser-
citata da questi soggetti sull’apparato dello Stato. Ma le
pratiche di manipolazione di cui tratta Cavalli non sono
neppure meccanicamente riconducibili alla categoria
degli arcana imperi: «strumenti di occultamento», quali
il mascheramento e la menzogna utilizzati per sottrarsi
allo sguardo dei dominati «nascondendosi e nasconden-
do». Queste pratiche implicano il riconoscimento di una fi
Il tema principale del libro sembra proporsi allo-
ra come quello classico della teoria delle élites e quello
forse ancor più classico del divario tra democrazia for-
male e democrazia sostanziale che con Bobbio potrebbe
ancora essere declinato come quello degli “insuccessi”
del modello democratico rispetto ad un ideale di “buon
governo”. Luciano Cavalli tratta però la questione da una
prospettiva particolare che offre interessanti spunti di
riflessione su cui vale la pena soffermarsi. lf
Fin dalle primissime pagine dichiara la sua intenzio-
ne di volersi occupare della manipolazione come di un
potere di fatto che può prodursi anche all’interno delle
istituzioni quando l’autorità legittima di una determi-
nata comunità eserciti i propri compiti abusandone: 142 Franca Bonichi sorta di diritto a “simulare” tradizionalmente attribuibi-
le a chi esercita il potere e che presuppone una concezio-
ne del “buon governo” come arte, come personale abilità
del decisore, come capacità di esercitare l’astuzia, non
diversamente dalle “volpi” di Machiavelli. polata (1965), benché ci siano continui riferimenti all’e-
sistenza di una «minoranza organizzata», denominata
d’altronde in vario modo (classe dirigente, classe domi-
nante, classe dei privilegiati, oligarchia istituzionale,
minoranze organizzate, classe superiore, privilegiati...),
manca tuttavia una rigorosa definizione esplicativa di
questa presenza che ne individui i tratti antropologici,
fornisca una indicazione precisa sulla sua collocazione
istituzionale e sulla sua appartenenza sociale. Non solo. Significativo è anche il fatto che Cavalli appaia quasi
ironizzare su una eventuale richiesta che potrebbe esser-
gli rivolta di «provare esaurientemente l’esistenza del-
la minoranza» (questione, come è noto, molto presente,
nel dibattito sociologico di quegli anni) sottolineando
proprio la contraddittorietà della pretesa di «provare l’e-
sistenza di una volontà che ha l’interesse e il potere di
nascondersi» (Cavalli 1965: 17). Risposta che dietro l’i-
ronia, cela probabilmente la consapevolezza dei limiti
di un approccio esclusivamente empirico all’analisi del
potere. 6 «In vari casi io mostrerò come certi aspetti istituzionali siano fun-
zionali rispetto all’ordine esistente, senza poter dimostrare che siamo
coscientemente voluti da una minoranza» (Ivi: 16). 5 Il termine è di Vincenzo Sorrentino (2011). Un invito quindi ancora valido a tener
conto del fatto che gli atteggiamenti, sia individuali che
collettivi, non solo vadano considerati all’interno di
determinati contesti istituzionali, ma anche che il poten-
ziale democratico di una collettività dipenda significa-
tivamente dal funzionamento delle sue istituzioni, dalle
concezioni istituzionalizzate nel sistema politico ed in
particolare nella cultura delle classi dirigenti. Come è evidente, le categorie di socializzazione e
di controllo sociale, oggetto principale di questo libro,
sono in gran parte desunte dalla sociologia funzionalista
parsoniana, anche se è altrettanto evidente come que-
ste categorie vengano rivisitate da Cavalli criticamente. In più occasioni è messa in evidenza la non neutralità
delle istituzioni denunciando il fatto che la minoranza
a queste preposte spesso opera perseguendo finalità che
si è arbitrariamente attribuita piuttosto che adempie-
re ai compiti istituzionalmente previsti. Numerosi nel
testo sono i richiami a queste finalità anche se non mi
pare siano mai sistematicamente precisati. In certi passi
sembrano coincidere con l’autoconservazione del potere
dell’élite, in altri con una generica volontà di preserva-
re l’ordine sociale dominante, in altri ancora, come nel
capitolo sulla Manipolazione del Lavoro, addirittura con
la tutela della «razionalizzazione scientifica» imposta
dal sistema capitalistico di produzione. Nonostante que-
sta indeterminatezza, il carattere arbitrario e costrittivo
della manipolazione non mi pare venga mai messo in
discussione e risulti drammaticamente “provato” anche
(e forse soprattutto) dagli omologanti effetti di sogge-
zione e di sofferenza sociale che produce sui “dominati”
lungo tutto il periodo della loro esistenza, dall’infanzia
all’età matura. Un ordine sociale risultato della coerci-
zione e della manipolazione non può infatti che provoca-
re ingiustizia, privilegi, violenza. Uomini e donne risul-
tano così via espropriati di ogni autonomia di giudizio,
alienati rispetto alle loro possibilità di autorealizzazio-
ne, sfruttati nei rapporti di lavoro, indotti a riconoscersi
prioritariamente nei ruoli di produttori e consumatori. Con spietato realismo Cavalli nega anche le possibilità
di emancipazione che possono offrire la partecipazione
politica e la vita sociale e culturale. Scopo principale,
anche dei partiti “democratici”, è infatti quello di sco-
raggiare ogni pretesa di partecipazione politica. A questo
scopo si favorisce la tendenza dei “sudditi” a credere che
i problemi comuni possano essere affrontati efficacemen-
te solo sulla base di una specifica competenza e quindi
debbano essere affidati unicamente ad “esperti politici”
(Ivi: 192). Non solo. Frequenti sono infatti i riferimenti espliciti alla
dominazione come un “sistema” pervasivo e coerente in
cui non solo sono coinvolte tutte le agenzie più impor-
tanti, ma soprattutto che utilizza per fini propri anche
l’operato di tutta una rete di strutture legalmente legitti-
mate. Un potere (e questo forse è l’aspetto più significati-
vo) che, benché agito da una minoranza organizzata, di
fatto tende a spersonalizzarsi sempre di più, a causa della
burocratizzazione, producendo il risultato di occultarsi e
di rendere sempre più difficile ai dominati identificare «i
soggetti su cui scaricare il proprio risentimento» (Ivi: 17). Una consonanza con la manipolazione di Cavalli
che non è tanto derivata dal comune riferimento ad una
dimensione occulta del potere quanto dalla preminen-
za accordata da entrambe queste analisi, a quelle norme,
quei riti, quelle procedure, quella sorta di inerzia istituzio-
nale che, attraverso processi di preselezione dei temi della
decisione, definiscono preventivamente le regole del siste-
ma politico e i confini all’interno dei quali è consentito il
conflitto, operando di fatto per la conservazione dei criteri
dominanti di allocazione delle risorse e dei valori. Sarebbe davvero interessante poter ricostruire in
dettaglio il funzionamento di alcune di queste agenzie di
socializzazione la cui analisi ci restituisce tutta la com- Una ipotesi interpretativa che mi pare plausibile in
base a diverse considerazioni. In La democrazia mani- 143 Il “realismo radicale” di Luciano Cavalli. Appunti per una riflessione plessità di quei processi senza mai incorrere in sempli-
ficazioni meccanicamente deterministiche. Noto soltanto
come Cavalli, per ogni istituzione che analizza, scenda
nei dettagli, ne individui gli specifici codici di funzio-
namento e metta in guardia contro meccanismi che solo
apparentemente potrebbero presentarsi come “democra-
tici”, come il coinvolgimento degli operai nella gestione
delle imprese, il controllo della devianza, la permissività
in campo sessuale, la diffusione dei media. atteggiamento fondamentalmente passivo» che realizza il
fine principale della manipolazione che proprio quello di
indurre il “suddito” ad astenersi dall’occuparsi dei pro-
blemi della società in cui vive (Ivi: 197). Denunce che possono forse apparire datate ma che
costituiscono comunque un monito salutare a non attri-
buire banalmente alle masse popolari una passività qua-
si naturalmente congenita e a denunciare il ruolo che le
istituzioni possono avere proprio nel creare disimpegno
e apatia politica. Anche i mass media concorrono
significativamente «all’impecorimento» delle masse per-
ché col divertimento di massa «i dominati imparano un g
Tutto questo assunto, è opportuno sottolineare come
questa azione di denuncia per cui Cavalli arriva a defi-
nire la manipolazione addirittura come «un male da
estirpare» (ivi: 19), si accompagni, secondo un approccio
tipicamente elitista, ad una serie di considerazioni vol-
te a rivalutarne realisticamente gli effetti. Innanzitutto,
l’autore ci sollecita a pensare al modello di «democrazia
manipolata», come ad un idea-tipo («un modello puro»)
(Ivi: 15) per cui non solo non è detto che governare
significhi necessariamente dominare, ma che è anche
possibile che questa forma di dominio possa non essere
completamente pervasiva e che quindi ad alcune istitu-
zioni o sub-unità territoriali possano venir lasciati mar-
gini variabili di autonomia (Ibidem). Senza contare poi
che queste pratiche di dominazione non sono presenti
allo stesso modo in tutti i sistemi politici e che una ipo-
tetica rappresentazione lungo un continuum ci potrebbe
restituire una immagine assai differenziata del fenome-
no dove, in prossimità dei due poli opposti, potrebbero
trovar posto i regimi autocratici e democrazie mature,
come quella inglese. Inoltre, e forse questo è l’aspetto più interessante,
la manipolazione può e forse deve essere considerata
anche come una sorta di necessità funzionale rispetto
all’equilibrio del sistema sociale. I meccanismi di socia-
lizzazione e di controllo non solo sono presenti in tutte
le società e ne garantiscono il funzionamento preser-
vandole dai rischi della disgregazione, ma anche quan-
do questi processi vengano agiti in maniera strumentale
(come nel caso della manipolazione) non è detto che non
ne derivi un vantaggio per la comunità. Dato il carat-
tere conflittuale che caratterizza ogni società, il rischio
che un sistema democratico degeneri in anarchia è sem-
pre incombente, consapevolezza che, sostiene Cavalli,
costringerà anche minoranze autenticamente democra- 144 Franca Bonichi sociale, l’ambito decisionale della minoranza di governo,
sia essa istituzionale, democratica, autocratica, si può
supporre allora limitato all’interno da una vasta gamma
di “preselezioni” di cui il sistema delle alleanze geopoli-
tiche, i codici funzionali delle nuove tecnologie, le logi-
che del mercato globale possono essere considerate ai
giorni d’oggi le incarnazioni più salienti. Compatibilità
che agiscono secondo astratte logiche impersonali con lo
scopo di sopprimere quelle issues potenzialmente desta-
bilizzanti prima che queste possano raggiungerei luoghi
della decisione politica, prima cioè che gli antagonismi
sociali possano compiutamente manifestarsi.Una visione
realisticamente inquietante perché ci sollecita a riflettere
sul carattere ambiguo del potere all’interno delle nostre
società contemporanee: sulla sua necessità funzionale,
come requisito per qualunque gruppo sociale organizza-
to ed insieme sulla sua pericolosità rispetto allo statuto
normativo delle democrazie contemporanee. Un potere
che sembra sempre più assumere i tratti di un astratto
algoritmo che la ritualizzazione giuridica ed istituziona-
le non riesce a costringere, se non in modo provvisorio,
parziale e sempre drammatico, alla “visibilità”, e cioè
alla logica della uguaglianza e della non prevaricazione. tiche a ricorrere ad ogni mezzo per contrastare queste
possibili derive degenerative e quindi, paradossalmente,
anche alla manipolazione. «Si noti dunque il paradosso:
per salvare la democrazia, i democratici devono a volte
tradire il codice democratico, e forse fin rallentare tem-
poraneamente lo sviluppo della democrazia» (Ivi: 13-14). Cavalli spiega questo apparente paradosso, in coerenza
con il suo approccio realista, chiarendo che il giudizio
sull’operato delle minoranze al governo deve fondarsi sia
sugli “intenti” che sui “risultati” e che «una classe diri-
gente o meglio gli individui che la compongono debbano
essere valutati in base alle intenzioni, per quanto attiene
ad un giudizio morale, e in base ai risultati per un giu-
dizio politico»(Ibidem). Considerazioni da cui derivano
alcune importanti conseguenze. La necessità per il citta-
dino di accettare che i politici, nei quali riponga la sua
fiducia, si comportino occasionalmente anche in modo
antidemocratico e quella di valutare le singole scelte, non
secondo astratti codici etici, ma politicamente, all’inter-
no di una strategia di potenziamento della democrazia. Un punto di vista che costituisce anche un criterio di
giudizio rispetto all’operato politico dei leader che con-
siste proprio nel domandarsi se quel determinato ricorso
a mezzi antidemocratici sia o meno stato necessario per
raggiungere un fine democratico (Ibidem).i 3. Reale come opposto di ideale8. Nella prima parte
di Il leader e il dittatore. Uomini istituzioni di governo
nel “realismo radicale” (2003) Cavalli, nel precisare una
definizione assai articolata di realismo radicale, sottopo-
ne contestualmente ad una critica severa, «la prospettiva
della modernità» (o costruttivismo) che ritiene «conten-
dere con forza il campo al realismo politico»9. 8 Con questa contrapposizione non intendo ovviamente escludere che
Cavalli abbia un suo ideale di società politica e di modelli istituziona-
li. Cavalli non è stato certo uno scettico disincantato e l’ha dimostra-
to. Non intendo neppure banalizzare idee-concetti così densi di storia
e di significato come utopia, ideologia, realtà. Ritengo piuttosto che
questa contrapposizione possa essere utilizzata per mettere in eviden-
za un monito che può derivare dalla lettura di alcune parti dell’opera
di Cavalli che è quello di sottoporre alla severa critica della fattibilità
e della responsabilità il richiamo in politica ai valori e alla sfera idea-
le. E questo di certo non per legittimare l’esistente come l’unica realtà
possibile ma piuttosto per sottolineare l’esigenza che i valori e gli ide-
ali che condividiamo si traducano in proposta politica piuttosto che
retoricamente declamati per avvallare la nostra impotenza.
9Il tema era già stato introdotto in Il governo del leader e il regime dei
partiti (1992) e nel Primato della politica (2001), ma nella prima parte
di Il leader e il dittatore (2003) è sviluppato in modo più organico ed
esauriente. 7 Questo paradigma è ben rappresentato da David Estlund (2008) e
anche da Nadia Urbinati (2016). Cavalli sostiene queste affermazioni con esem-
pi significativi ricordando come nei due dopoguerra «la
pace ideale dei potenti abbia sempre imposto allo scon-
fitto condizioni di non ritorno» (Ivi: 65) ed elenca una
serie di tragici eventi, dagli gli eccidi di civili ad Ambur-
go e Dresda a Hiroshima e Nagasaki, dalla guerra del
Vietnam alla questione palestinese, in cui i conclamati
valori della pace, dei diritti umani, della giustizia e della
solidarietà tra le genti sono stati clamorosamente violati
e sacrificati sull’altare degli interessi strategici degli Sta-
ti più potenti. Per quanto riguarda la contemporaneità,
l’egemonia del “colosso americano”, realizzatasi dopo il
lungo periodo della guerra fredda, se da un lato limita di
fatto la sovranità degli Stati nazionali non sembra d’al-
tra parte porre le basi per una pacifica convivenza inter-
nazionale, nel rispetto dei diritti umani. Al contrario,
sostiene Cavalli, questa situazione implica di necessità
il ricorso ad una strategia di dominio planetario attivo
ed eventualmente aggressivo, fondato in modo diretto
sull’uso della forza (Ibidem). p
p
p
p
Il costruttivismo, ci ricorda Cavalli, consiste in una
particolare visione del mondo che può contare su una
lunga tradizione di pensiero e le cui componenti princi-
pali possono essere individuate nel cristianesimo e nella
tradizione illuminista. L’autore non ha difficoltà a rico-
noscere che si tratti di impostazioni di pensiero segnate
da indiscutibili differenze, ma tuttavia accomunate dalla
comune convinzione di porre a proprio fondamento due
comuni idee-valori, quella di individuo (o di persona)
e quella di umanità. Particolarmente significativo è il
lascito del pensiero contrattualista da cui Cavalli fa deri-
vare una specifica idea di soggetto e di identità politica
che si sta sempre più affermando nel mondo occidentale. Ad una identità come adesione ad una comunità basa-
ta su legami locali e di tipo ascrittivo si starebbe, a suo
parere, progressivamente sostituendo un senso diver-
so di appartenenza, fondato sulla condivisione di una
morale «genericamente solidaristica» e sulla adesione ad
istituiti obblighi normativi. Una identità quindi definita
nei termini di una appartenenza ad una società costru-
ita, e forse immaginata, piuttosto che ad una comunità
legata da vincoli di tipo etnico e nazionale. In questo
paragrafo è mia intenzione sviluppare alcune considera-
zioni sull’ampia analisi che Cavalli sviluppa nei confronti
della «prospettiva della modernità» ed in particolare sulla
«definizione della realtà» che la sottende. Non mi occu-
però invece dei modelli istituzionali di «Democrazia con
un leader» e di «Democrazia senza leader» che corrispon-
dono rispettivamente alla prospettiva realista e a quella i
Una ammissione particolarmente significativa non
tanto perché vi si potrebbe cogliere una assonanza con
la locuzione “del fine che giustifica i mezzi”, identificata
spesso banalmente come l’assunto più tipico del realismo
politico, quanto piuttosto perché con questa ammissio-
ne Cavalli sembra aderire ad una impostazione elitista
di pensiero che va da Schumpeter fino ai contemporanei
sostenitori delle teorie epistemiche della democrazia7. Una posizione declinata in vari modi, da autori diversi,
in epoche diverse, a volte come un invito a riconside-
rare la teoria democratica, altre il funzionamento del-
le sue istituzioni, ma che si precisa sempre come una
opzione a favore delle esigenze imposte dalla “governa-
bilità” rispetto alle sfide di “disgregazione” che possono
arrivare dalla dinamica sociale. Una impostazione che è
stata efficacemente sintetizzata nella formula secondo la
quale per curare la democrazia si deve poter limitare la
democrazia, che deriva dalla lucida consapevolezza del
carattere arbitrario e assolutamente “partigiano” che è
implicito in ogni criterio di allocazione delle risorse e
dei privilegi sociali e quindi della natura strutturalmente
conflittuale del processo politico, anche all’interno del-
le democrazie contemporanee. Considerata la difficoltà
strutturale di una reale rappresentanza di interessi con-
trapposti e l’esigenza di garantire comunque l’ordine 145 Il “realismo radicale” di Luciano Cavalli. Appunti per una riflessione costruttivista nella convinzione, probabilmente discuti-
bile, che la pars destruens di questa critica possa essere
considerata anche a prescindere dai modelli teorici di lea-
dership e democrazia proposti nella pars construens. siano ben lontani dall’aver realizzato (e come dargli tor-
to!) quella spontanea composizione degli interessi e quel
pacifico equilibrio che la presenza delle istituzioni inter-
nazionali e il diffondersi della ideologia pacifista auspi-
cavano. Sembra invece prevalere ancora la logica della
forza e della sopraffazione per cui l’ordine geopolitico
resta caratterizzato dai conflitti e fondato sull’egemonia
delle nazioni più potenti, sia economicamente che mili-
tarmente. Cavalli cerca di spiegare il successo nel mondo occi-
dentale del diffondersi di questa prospettiva facendo
riferimento ad alcuni eventi che hanno caratterizzato
la nostra storia recente, in particolare al lungo periodo
di pace, susseguito alla tragica esperienza della Secon-
da guerra mondiale e al processo di mondializzazione
del mercato. Questi eventi che sembravano «cancella-
re i confini e affratellare gli uomini» hanno contribuito
al diffondersi della cultura politica delle due tradizio-
ni citate, articolandole e specificandole in precise idee-
valori. Prima tra tutte, quella di uguaglianza che viene
dedotta dalla comune appartenenza all’umanità e da cui
deriva l’obbligo morale di un comportamento ispirato
alla solidarietà e ad una effettiva fratellanza. Si accom-
pagna a questa la fede nella ragione e nel progresso che
contribuiscono a legittimare significativamente quell’
impostazione “cosmopolitica”, di cui l’ Onu e Corti
internazionali di giustizia appaiono i principali vettori e
che mira ad espungere il conflitto e la guerra dalle rela-
zioni internazionali (Cavalli 2003: 48). La critica che Cavalli rivolge al costruttivismo, altri-
menti da lui definito anche come «democraticismo uma-
nitario», non si limita però a rilevarne l’inadeguatezza
analitica ma anche, e forse soprattutto, a denunciare gli
effetti che «questa incursione di sentimenti morali nello
spazio pubblico» può produrre. Innanzitutto, la prospet-
tiva umanitaria e solidaristica, collocando al suo centro
i valori dell’individuo e dell’umanità, produce il risultato
di porre in secondo piano l’importanza dell’appartenenza
alla comunità nazionale e allo Stato. Con l’indebolimento
dei valori di Stato e nazione il rischio, che per Cavalli si
è già sostanzialmente realizzato, è quello del prodursi nei
cittadini una grave crisi di identità collettiva, con conse-
guenti effetti di disgregazione sociale e di sfiducia nelle
istituzioni. Già in Il governo del leader (1992), di qual-
che anno prima, Cavalli aveva messo in guardia contro
il declino del valore di «nazione come comunità di stir-
pe, cultura, storia, destino» (ivi: 241-242) e aveva notato
come, a partire dal secondo dopoguerra, in Italia, si fos-
se assistito ad una profonda trasformazione nella cultu-
ra politica. L’universo valoriale pubblico da quegli anni
sarebbe infatti radicalmente mutato, assumendo come
«pubbliche virtù» qualità «tipicamente femminili» come
la pietà, l’indulgenza, il perdono l’assistenza paziente che Cavalli critica questa prospettiva innanzitutto per
il suo carattere più normativo che analitico, utopistico,
smentito e contraddetto da norme e prassi. fi
A questa politica che si è fondata sulla vacua affer-
mazione (spesso fatta con accenti estremistici) di princi-
pi democratici, Cavalli replica con il monito a confidare
in una visione realistica che assicuri «un ancoramento
alle realtà terrene, lo sforzo di capirle e piegarsi ad esse»,
raccomanda «la prudenza che non consente di crede-
re che ciò che è sempre stato, possa sparire o cambiare
facilmente» e suggerisce di diffidare nella «ideologia e
nella sua figliola preferita, l’utopia» (Ivi: 243). 4. Conclusioni. Da questa sintesi, certamente trop-
po stringata, desidero far emergere un tema che mi pare
rivesta un particolare interesse. Una sorta di sottotesto
che può essere colto nelle opere di Cavalli e letto come
la constatazione di un cambiamento che in anni recenti
sembra essersi prodotto nei modi di concepire e vivere
la cultura e la partecipazione politica nelle democrazie
occidentali e segnatamente da parte delle forze democra-
tiche e progressiste. Un mutamento significativo, colto
anche da alcune analisi contemporanee10, secondo cui ad
una politica, sostenuta da un forte senso di riscatto col-
lettivo e da una visione della storia intesa come continuo
progresso materiale e sociale, sembra essersi progressiva-
mente sostituita una politica “morale”, che si manifesta
più come testimonianza che come disponibilità ad agire
e che ha convertito le grandi passioni collettive in buone
azioni personali. Una convinzione secondo la quale l’a-
zione politica appare caratterizzarsi ai giorni d’oggi più
come una presa di posizione privata, anche se agita in
pubblico, che come comportamento collettivo, su un pro-
getto condiviso. Completa il quadro un ricorrente richia-
mo ai sentimenti morali per legittimare e motivare le
politiche contemporanee che si manifesta prevalentemen-
te attraverso un uso “politico” di valori come la sacralità
della vita umana, la compassione e l’empatia verso la sof-
ferenza, la benevolenza, la solidarietà. Potremmo aggiun-
gere che questa trasformazione trova riscontro anche nel
lessico utilizzato in campo progressista per cui si parla di Una innovazione significativa rispetto ai paradigmi
precedenti di cui è chiara la derivazione dalla tradizione
di pensiero illuminista ma che ha radici più recenti nella
nostra modernità. L’esempio più
eclatante a sostegno di questa sua convinzione l’autore lo
deduce da quanto avviene in ambito internazionale dove
è drammaticamente evidente come i rapporti tra gli Stati 146 Franca Bonichi si sarebbero poi strutturate come veri e propri cardini
dell’organizzazione sociale (Ivi: 245). Convinzioni di cui
è possibile trovare un riscontro nella dura critica che
Cavalli rivolge alle modalità con cui sono state affrontate
questioni come quelle dell’immigrazione e dell’ambien-
talismo. Sfide cui si è risposto, a suo parere, in maniera
velleitaria e moralistica, facendo prevalentemente affida-
mento ad una politica delle buone intenzioni che di fatto
ha sottratto questi problemi ad un vero e proprio dibat-
tito pubblico e pertanto ha impedito che questi temi fos-
sero affrontati responsabilmente, con decisioni politiche
adeguate ed efficaci. fragilità piuttosto che di disuguaglianza e sfruttamento,
di trauma piuttosto che di violenza, di resilienza più che
di resistenza. Un esempio particolarmente significativo
di questo cambiamento può essere colto nelle variazioni
avvenute all’interno del campo semantico della parola
“solidarietà” che dà valore forte del movimento operaio
(solidarietà di classe) è trasmigrata in ambito assistenzia-
le assumendo significati per lo più riconducibile al termi-
ne religioso di “misericordia”. f Sembra quindi essersi affermata una nuova sintas-
si del politico che negli ultimi decenni non si limita a
strutturare le politiche istituzionali (naturalmente quel-
le dei governi democratici-progressisti) ma condizio-
na gran parte dell’agire politico contemporaneo (per lo
meno quello che cerca di opporsi in occidente alle poli-
tiche della destra conservatrice). Come aveva già intuito
Cavalli, idea fondante di questo tipo di visione è quella
di “umanità” nella doppia accezione che si può ricavare
dalla traduzione dei termini inglesi di mankind e huma-
neness. Quindi umanità come insieme di uomini e don-
ne, carattere che li distingue dagli altri esseri viventi
(appartenenza all’umanità), ma anche come sentimen-
to morale nei confronti degli esseri umani (dar prova di
umanità). Significati in parte diversi ma che presuppon-
gono entrambi che l’eguaglianza dei diritti sia naturale,
perché associata in maniera innata alla nostra condizione
di umani, e che convergono nella convinzione che tutte
le vite siano ugualmente sacre e tutte le sofferenze meri-
tino di essere alleviate Dall’esplicito riferimento all’uma-
nità derivano le varie denominazioni assunte da questo
tipo approccio: ragione umanitaria, governo umanita-
rio, umanitarismo, in evidente consonanza col termine
«democraticismo umanitario» utilizzato da Cavalli. 10 Cfr Danilo Zolo (2000) e Didier Fassin (2018). Senza ovviamente volerne ricostruire
la storia, un precedente importante può essere però indi-
viduato nei movimenti per l’abolizione della schiavitù in
Gran Bretagna alla fine del XVIII secolo, ma anche negli
ultimi decenni del secolo scorso, con il moltiplicarsi in
molti paesi occidentali di organizzazioni umanitarie
e di misure e dispositivi creati per i cosiddetti “fragili”
(poveri, disoccupati, senza tetto, stranieri, rifugiati...). Non è questa la sede per sviluppare un discorso
organico sull’umanitarismo contemporaneo, tema su cui
peraltro ritengo varrebbe la pena impegnarsi anche per
cercare di individuarne gli effetti politici: cosa è in gioco
quando si parla di umanitarismo, cosa cambia nella defi-
nizione dei criteri di rilevanza dei bisogni, nella gerar-
chia da assegnare ai valori. Questioni complesse che qui
mi è impossibile anche solo accennare per cui mi limi- 147 Il “realismo radicale” di Luciano Cavalli. Appunti per una riflessione terò a brevi osservazioni su quelli che a mio parere pos-
sano essere i principali aspetti della realtà politica che
attraverso la ragione umanitaria si rendono leggibili, e
quali invece restano in secondo piano o possano addirit-
tura rivelarsi invisibili. frontiere. Il “prima” – le cause che producono questi eso-
di – ma anche il “dopo” – le disumane condizioni in cui
immigranti si trovano troppo spesso a vivere e a lavorare
– risultano scivolare in secondo piano o comunque non
entrano in modo significativo nell’agenda politica neppu-
re dei partiti progressisti. Non mi pare dubbio che l’umanitarismo si caratteriz-
zi per una significativa attenzione rivolta alla sofferenza
sociale e per un’azione diretta rivolta ad alleviare questa
sofferenza. Un atteggiamento giudicato moralmente inat-
taccabile anche dai più severi critici per cui l’ascolto degli
esclusi e dei marginali, l’assistenza verso i poveri, la com-
passione per i malati e per gli sfollati, l’accoglienza verso
i migranti, la testimonianza a favore delle vittime delle
guerre sono sia azioni, che posizioni da giudicare buone
a priori, cause giuste in sé. E questo soprattutto se con-
trapposto al cinismo e all’indifferenza che hanno ispirato
le politiche securitarie che a partire dai primi anni 2000
hanno caratterizzato lo spazio pubblico. Quelle che han-
no attribuito una rilevanza quasi esclusiva ai temi di insi-
curezza, criminalità, terrorismo e a cui si è data risposta
prevalentemente in termini di estensione di poteri di
polizia, di leggi di eccezione e di disposizioni di urgenza. p
p
g
Un uso politico di sentimenti morali che si manife-
sta anche forme che assume la rappresentazione dell’av-
versario politico che in modo sempre più ricorrente
risulta definito attraverso la contrapposizione tra bene e
male. In questo nuovo quadro il nemico politico, soprat-
tutto quello più estremo (ed in questi anni soprattut-
to sulla scena politica interna e internazionale ne sono
apparse varie rappresentazioni) non viene più rappresen-
tato come colui la cui azione politica è orientata da una
visione del mondo e della società inconciliabile e alter-
nativa rispetto a quella “legittima”, ma dal fatto di resta-
re prigioniero di pulsioni talmente intolleranti e basse
da metterne persino in dubbio la umanità. Si potrebbe
quindi sostenere che nelle raffigurazioni odierne della
inimicizia radicale, che pure continuano a far parte della
comunicazione politica, non esista più il nemico politi-
co, ma esista solo il reprobo morale, il fuorilegge. Risulta
infatti oscurata, o posta in secondo piano, quella situa-
zione di potenziale/latente conflitto, nell’ambito di una
particolare contrapposizione di interessi, di definizione
di identità, di opposizione sui criteri di allocazione delle
risorse, che individua la specificità della politica. gg
g
Si possono però cogliere delle importanti implicazio-
ni inerenti all’affermarsi del diffondersi di questo “gover-
no umanitario”. Fassin (2018) facendo riferimento alle
varie «emergenze» degli ultimi anni, nota come il discor-
so umanitario, che opera mobilitando un immaginario
caritatevole rispetto a soggetti sofferenti, si trovi di fatto
ad attivare un particolare sguardo. L’attenzione risulta
spostarsi dalle strutture sociali responsabili di quella sof-
ferenza e rivolgersi ad un soggetto-costruito in termini
morali nel quale è possibile riconoscersi sulla base di una
presunta unità del genere umano, una comunità appunto
morale la cui esistenza si manifesta proprio attraverso la
compassione per le vittime (Ibidem). Questa logica uma-
nitaria, ed è questo forse l’aspetto più interessante, agi-
sce allora, anche se in modo inintenzionale, per privare
gli eventi (le migrazioni, la miseria, la fame, la guerra…)
di ogni specificità storica e politica e quindi finisce per
considerare l’ordine politico all’interno del quale questi
si compiono come una “semplice cornice”. Si può pertan-
to produrre il risultato di una sorta di oscuramento del-
le cause strutturali di disuguaglianza e di sfruttamento
dei fenomeni oggetto di intervento che risultano ridotte
al ruolo di variabili indipendenti su cui non vale la pena
o (nel migliore dei casi) non è possibile intervenire. Un
esempio può essere costituito dal modo in cui in campo
progressista viene affrontato il tema dell’accoglienza dei
migranti troppo spesso limitato alla giusta indignazione
per i tanti morti, per le immense sofferenze e alla denun-
cia della colpevole indifferenza che accompagna i tenta-
tivi di sbarcare sulle nostre coste e di varcare le nostre Corollario interessante di questo approccio ai temi
della politica potrebbe essere individuato in una ricor-
rente enfasi sull’importanza di comportamenti sog-
gettivi eticamente orientati per cui, ex parte populi, il
moralismo politico diventa richiesta (che si cristallizza
poi in senso comune) di impegno e di senso di respon-
sabilità da attivare soprattutto rispetto alle situazioni di
crisi. Ecco che allora assistiamo alle raccomandazioni
di seguire buone pratiche individuali per risolvere situa-
zioni di “emergenza”, come quella ambientale, sanitaria,
energetica; ma anche ad accettare con senso di responsa-
bilità e civismo decisioni impopolari come quelle relative
alle grandi opere. Appelli certamente efficaci e proba-
bilmente anche utili per rinsaldare il legame sociale ma
che parlano implicitamente di una cittadinanza dimez-
zata. Innanzitutto, oscurano il fatto che, all’interno del-
le nostre società, poteri e responsabilità sono ripartiti
in modo disuguale e che, dato il carattere sistemico di
queste “emergenze”, la soluzione non potrà mai risul-
tare dalla sommatoria di tanti comportamenti indivi-
duali, per quanto virtuosi possano essere. Anzi, attivare
comportamenti individuali virtuosi, senza rimuovere
le cause strutturali che sono alla base delle varie emer-
genze, rischia di produrre l’effetto paradossale di creare 148 Franca Bonichi frustrazione ed indifferenza. Inoltre, è forse interessante
notare come la richiesta di adeguati comportamenti civi-
ci raramente si accompagni all’ampliamento di spazi di
dibattito e quindi non solo non produca consapevolezza
sui processi che quelle emergenze hanno determinato,
ma neppure opportunità di decisione politica e quindi di
partecipazione democratica. Tutto questo considerato sarebbe anche interessante
interrogarsi sul perché del successo riscosso dall’umani-
tarismo, sia a livello istituzionale che di senso comune. Una domanda cui non è certo facile trovare una rispo-
sta ma che con molta probabilità è plausibile mettere in
relazione con una condizione umana che nelle nostre
società contemporanee ha raggiunto un livello insoste-
nibile di disuguaglianza. Una condizione che sollecita
un bisogno a non rimanere passivi e che, non trovando
però possibilità di espressione politica, si manifesta come
una necessità etica, per quanto effimera sia questa con-
sapevolezza e per quanto limitato sia l’impatto di que-
sta necessità. Una risposta, come nota Zolo, che ha «la
forza dei deboli» ed in quanto tale non può che risultare
«moralmente motivata, politicamente ambigua e profon-
damente paradossale» (Zolo 2000: 8). 1 Ho conosciuto il professor Luciano Cavalli nell’esperienza della pubblicazione della rivista
“Città & Regione” , di cui fui capo-redattore per nove anni. Una rivista voluta da Lelio Lagorio,
Presidente della Giunta regionale toscana nella prima legislatura delle Regioni a statuto ordina-
rio. L’obiettivo fu quello di analizzare i più importanti problemi istituzionali del Paese, essere
cioè una pubblicazione che creasse un campo aperto di dibattito libero sui temi e sui problemi
che la parte più moderna della società e della cultura poneva al mondo della politica e delle
istituzioni e, in quest’ambito, essere un punto di osservazione – non legato in modo preconcet-
to ai canoni correnti di interpretazione – di tutte le questioni che interessassero l’autonomia e
la pluralità dei centri di governo della società. Insomma, una specie di “tavola rotonda” con-
tinua in cui si affrontavano problemi della società contemporanea sia sul piano nazionale che
internazionale. La parte fondamentale della rivista fu monografica il che la rese – e la rende
ancora – durevole nel tempo. Una rivista, naturalmente, di riflessione approfondita, non di cro-
naca. Per cui il “consiglio direttivo” fu formato da illustri docenti di varie discipline (Gaetano
Arfè, Luciano Cavalli, Enzo Cheli, Renato Curatolo, Mario Cusmano, Furio Diaz, Fabio Merusi
e Alberto Predieri); e dello stesso livello ne furono i collaboratori. Ho voluto ricordare questa
vicenda perché Luciano Cavalli fu una delle “menti” più attive per l’intera durata della pubbli-
cazione e collaborò con tutta la passione civile e culturale che nutriva per il sostrato politico
che circonda e motiva i comportamenti umani, sia privati che sociali. Con particolare riguardo
all’analisi del “capo”, spesso protagonista dei progressi o delle tragedie della storia. RIFERIMENTI BIBLIOGRAFICI Bachrach P. Baratz M. (1986), Le due facce del potere, Liv-
iana, Padova. Bettin Lattes G. (2008), «La democrazia manipolata», in
G. Bettin Lattes e P. Turi (a cura di), La sociologia di
Luciano Cavalli, Firenze University Press, Firenze, pp. 1-16. Bobbio N. (1984), Il Futuro della democrazia, Einaudi,
Torino. Cavalli L. (1965), La democrazia manipolata, Edizioni di
Comunità, Milano. – (1992), Governo del leader e regime dei partiti, il Muli-
no, Bologna. – (2003), Il leader e il dittatore. Uomini istituzioni di gov-
erno nel “realismo radicale”, Ideazione, Roma. Estlund D. (2008), Democratic Authority: A Philosophical
Framework, Princeton University Press, Princeton. Fassin D. (2018), Ragione umanitaria. Una storia morale
del tempo presente, Derive-Approdi, Roma. Sorrentino V. (2011), Il potere invisibile. Il segreto e la
menzogna nella politica contemporanea, Dedalo, Bari.i Urbinati N. (2016), Demcrazia sfigurata: Il popolo tra
opinione e vertà, Università Bocconi, Milano. Zolo D. (2000), Chi dice Umanità, Einaudi, Torino. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a Competing Interests: The Author(s)
declare(s) no conflict of interest. Nella visione di Shakespeare l’uomo non domina la
storia. Né praticamente, né conoscitivamente. Cioè non
sa prevedere, non sa gli sviluppi che verranno. Le sue
azioni contribuiscono certamente a fare la storia ma
attraverso gli effetti non voluti, né intenzionali. Inoltre
c’è il caso, che fa parte di tutto ciò che l’uomo non può
prevedere; e forse il destino. C’è anche l’influenza di Plu-
tarco, del pensiero greco... Gli dèi che regolano le cose
umane... L’analisi dello scienziato sociale parte da un corpo
teorico - tutto formulato in termini concettuali - che
si è costituito attraverso le ricerche di una pluralità di
ricercatori e questi concetti sottopone ulteriormente
alla verifica dei dati empirici, di quelli che normalmen-
te chiamiamo «i fatti»; e d’altra parte l’esperienza che
si acquisisce nella ricerca empirica serve a sviluppare il
corpo teorico. Invece il letterato - poniamo Shakespeare,
per avere un riferimento preciso - si forma certamente
anch’egli delle idee sulla società in cui vive e queste idee
sono alimentate anche da letture, da esperienze altrui,
ma sono sostanzialmente le sue idee e le fa vivere, le sue
rappresentazioni del reale, e mentre le fa vivere le svi-
luppa e le chiarisce. È un procedimento che ha, natural-
mente, punto d’incontro nei risultati. Veniamo espressamente a queste due tragedie: secon-
do lei, in cosa consiste la loro permanente attualità? Sono attuali per due versi. Innanzitutto citerei dei
fatti che a mio avviso mettono già in evidenza la loro
attualità perenne. Intanto, che sia il Giulio Cesare che il
Coriolano siano state rappresentate, nei secoli, con un
continuo ricollegamento alla realtà storica e politica del
momento, è una prova che c’è in esse qualcosa di straor-
dinario che le rende sempre attuali. Però, al di là di que-
sta che mi sembra una prova molto valida, c’è il fatto che
effettivamente in ogni momento di queste due tragedie
c’è un invito a riferirsi a esperienze concrete della sto-
ria e della politica. Personalmente non riesco a leggere
una sola pagina di Shakespeare senza che mi vengano in
mente situazioni storiche e politiche di cui ho letto e che
ho sperimentato nella mia vita insieme alla sollecitazione
a pensare queste vicende vedendone meglio ogni angolo
visuale, aspetti che prima non avevo considerato. Insom-
ma a considerare cose, su cui avevo già a lungo riflettuto,
guardandole da un nuovo angolo visuale. E qui c’è, direi,
la forza del genio. Ci troviamo cioè davanti a una mente
straordinaria. Veniamo, appunto, a Shakespeare. Qual è la sua teo-
ria della storia e della politica? In Shakespeare c’è una visione scettica della società:
non c’è verità, non ci sono certezze per l’uomo. La real-
tà è qualcosa di labile, che ciascuno ridefinisce per farla
diventare la sua realtà. Ma qui s’inserisce la presenza di
uomini che definiscono la realtà per gli altri e questo è il
ruolo tipico del leader politico nei confronti delle masse. Quindi, se il mondo è teatro, la politica è - nella visione
di Shakespeare - teatro. Un teatro in cui il leader gioca
i ruoli dell’autore, del regista e dell’attore protagonista. Questo è un aspetto fondamentale del pensiero politi-
co shakespeariano: in un mondo senza certezze, senza
verità, il grande leader dà le pseudo-certezze, le pseudo-
verità, che sono però quelle che guidano la massa degli
uomini. Il leader recita la parte di attore in tutti i rap-
porti con gli altri uomini del potere, ma soprattutto con
le masse, i pubblici di vario genere che deve - in circo-
stanze diverse - incontrare. Il leader è sempre un uomo
che recita, la sua è una recitazione continua. Non dimen-
tichiamo che siamo nell’epoca elisabettiana, sulla scia
della critica a Machiavelli. Secondo Shakespeare l’uomo
politico deve sempre recitare e se non sa recitare o non
accetta di recitare, non è un uomo politico: è un tenta-
tivo fallito di leader. La sua etica deve consistere nel fare
politica, quindi tale etica diventa quella machiavellica
della dissimulazione, dell’inganno continuo. Posizione
molto ben illustrata in entrambe le tragedie romane e
specialmente nel Coriolano. Nelle tragedie romane Shakespeare si rifaceva certa-
mente ad autori latini che aveva letto tra cui, in primo
luogo, Plutarco...i Shakespeare ha frequentato, almeno fino ai quin-
dici anni, una scuola in cui era richiesta, a quei tempi,
una buona conoscenza del latino, addirittura di parlarlo. Sicuramente vi si leggevano autori come Livio, Svetonio
e Plutarco. Questo significa che erano disponibili fonti
essenziali per la conoscenza del mondo latino. Inoltre,
in Inghilterra c’era una situazione politica molto delica-
ta. Tra l’altro, veniva esercitata un’attenta e stretta cen-
sura sulle rappresentazioni nei teatri, sia pubblici che
«al chiuso». Uno dei timori dell’epoca era: cosa sarebbe
successo alla morte di Elisabetta? Non c’era un erede
diretto. p
Luciano Cavalli1 Citation: Renzo Ricchi (2023) Una lettura
politica di Shakespeare. Intervista a
Luciano Cavalli. SocietàMutamentoPo-
litica 14(27): 149-152. doi: 10.36253/smp-
14346 Renzo Ricchi Copyright: © 2023 Renzo Ricchi. This is
an open access, peer-reviewed article
published by Firenze University Press
(http://www.fupress.com/smp) and dis-
tributed under the terms of the Crea-
tive Commons Attribution License,
which permits unrestricted use, distri-
bution, and reproduction in any medi-
um, provided the original author and
source are credited. Giulio Cesare, Coriolano e il teatro della Repubblica è il titolo dell’ultimo
libro di Luciano Cavalli pubblicato dalla casa editrice Rubbettino. Una lettura
in chiave «politica» delle due grandi tragedie shakespeariane secondo cui i due
testi rappresentano un incisivo intervento, con nuovi punti di vista e suggestive
intuizioni, nella millenaria riflessione dell’Occidente sulla società umana. l
Luciano Cavalli è stato a lungo professore di Sociologia presso la Facol-
tà di Scienze Politiche «Cesare Alfieri» di Firenze, ove ha fondato e diretto
il Dottorato di Sociologia Politica e il Centro Interuniversitario di Sociologia
Politica, che hanno avuto un ruolo da protagonisti nel settore. Ha svolto studi
e ricerche in àmbito internazionale; ha scritto libri e saggi di storia del pen-
siero e di analisi sociologica e politologica. Ha, tra l’altro, riportato al centro
del dibattito scientifico aspetti essenziali del pensiero di Max Weber, con due
libri: Max Weber: religione e società del 1968 e Il capo carismatico del 1981. Ha
dedicato numerosi studi, pubblicati in varie lingue, alla leadership politica, sia
nei regimi totalitari (Carisma e tirannide nel secolo XX, del 1982; e Il leader
e il dittatore, del 2003), sia nelle moderne democrazie (Il presidente america-
no, del 1987). Per primo ha studiato la «personalizzazione della leadership»,
che considera come una tendenza tipica del nostro tempo; e a questo tema ha
dedicato molte pagine in questi ultimi anni. Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information files. Competing Interests: The Author(s)
declare(s) no conflict of interest. SOCIETÀMUTAMENTOPOLITICA 14(27): 149-152, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14346 150 Renzo Ricchi * * * * * * Shakespeare tendeva a personalizzare storia e poli-
tica. Ma la storia dell’uomo è veramente tutta e soltanto
legata alla politica? Prof. Cavalli, in generale qual è la differenza tra l’a-
nalisi della storia delle vicende umane delle moderne
scienze politiche e sociali e l’analisi che si ricava, invece,
da talune opere letterarie? E ancora: e se la regina fosse stata assassinata nel
corso di una delle tante congiure del tempo? Non poten- 151 Una lettura politica di Shakespeare. Intervista a Luciano Cavalli Una lettura politica di Shakespeare. Intervista a Luciano Cavalli Una lettura politica di Shakespeare. Intervista a Luciano Cavalli cinque o sei autori da me prediletti (Marx, gli elitisti -
Mosca, Pareto, Michels -, nonché gli elitisti americani,
- C. Wright Mills, Weber e Durkheim... ). Questi autori
mi hanno aiutato a leggere in profondità certe pagine di
queste due tragedie. Per esempio Durkheim e Freud mi
hanno aiutato a riflettere sull’episodio di Cinna. L’idea
freudiana della folla regredita a stati primitivi e d’altra
parte la constatazione che in queste situazioni di regres-
sione crollano non soltanto i princìpi e le strutture etiche
ma persino quelle logiche e, quindi, non si distinguono
più, in termini logici, gli oggetti, fino a confondere per
ragioni del tutto superficiali le persone e le situazio-
ni. Esempio, appunto, questa folla regredita che uccide
Cinna, il poeta, solo perché ha lo stesso nome del pre-
tore che il giorno precedente aveva fatto un violento
discorso contro Cesare. Si potrebbero citare vari punti
del mio libro influenzati, diciamo così, da Marx oppure
da Weber. L’idea, ad esempio, che sia Bruto che Corio-
lano possano essere visti come uomini ideologici - una
categoria che vale anche oggi - è tipicamente weberiana
così come weberiana è l’idea del conflitto tra un’etica dei
princìpi e un’etica della responsabilità vissuta, in queste
tragedie, sia da Bruto che da Coriolano. do trattare direttamente questo tipo di problemi politici,
Shakespeare adotta situazioni similari della storia roma-
na. Nel 1607-1608 avvengono delle insurrezioni conta-
dine. Sono anni difficili, dominàti da una grave carestia
aggravata dall’usura. Le forze del re - siamo ormai con
Giacomo I - intervengono pesantemente. Non v’è dub-
bio che ci siano analogie tra questi avvenimenti e la sol-
levazione dei contadini e, più in generale, della plebe,
descritta nel Coriolano ed è anche interessante l’imma-
gine che ci viene data delle due classi, anche attraverso i
rispettivi linguaggi che portano l’eco delle posizioni che
caratterizzavano, in Inghilterra, i ceti dirigenti e quelli
in rivolta. Lei comunque prende le distanze dall’identificazio-
ne che qualche regista contemporaneo ha fatto tra Giulio
Cesare e Mussolini, Coriolano e Hitler... Sì perché queste rappresentazioni, che volevano
mostrarci Giulio Cesare e Coriolano come modelli dei
futuri leader fascisti, forzano i tratti di somiglianza che
si possono eventualmente trovare e giungono addirittura
a rappresentarci l’uno e l’altro eroe romano come ante-
signani del tipo di regime che Mussolini e Hitler hanno
creato, cioè dei regimi totalitari moderni, impensabili al
tempo di Shakespeare. Si tratta di suggestioni, esercita-
te sull’ascoltatore odierno, a mio avviso fuorvianti. Anzi
ritengo che le figure di Cesare e di Coriolano, liberate
da queste forzature registiche, siano molto più interes-
santi. Pensi, tanto per fare un esempio, alla differenza
delle personalità. Giulio Cesare era aristocratico, coltis-
simo, scriveva sulla grammatica, sulla lingua, sul teatro,
si occupava di storia, era aggiornatissimo sulle maggiori
correnti di pensiero, sosteneva conversazioni al più alto
livello romano... Come si compie – e come si differenzia nei tempi –
l’identificazione tra capo e massa? i
Nel caso di cui stiano parlando bisogna tornare
allo sfondo scettico di cui parlavamo prima, che vede
il mondo privo di verità e di certezze. In questo mondo
insicuro vivono masse d’uomini che, sì, non hanno cer-
tezze ma le stanno cercando, anelano ad averle. E qui,
appunto, s’inserisce la figura del capo che nella tradi-
zione di Le Bon, Hellpach e di Weber si pone come un
uomo che impone alle masse la sua suggestione, dà loro
una realtà «definita» e ottiene quindi comportamenti di
massa conformi a questa definizione, conformi al suo
volere. La identificazione viene trattata in modi diversi. Per esempio, la massa che Antonio si trova di fronte, al
momento della sua celebre orazione, è una massa infor-
me, che non ha ancora delle opinioni. Tanto vero che il
poeta ce la mostra ambivalente. In un primo momento è
favorevole a Bruto, poi preferisce Antonio cedendo alla
suggestione che questi ottiene anche grazie alla colla-
borazione di uomini particolarmente suggestionabili e
quindi pronti, in mezzo alla massa, a farsi tramiti della
suggestione esercitata dall’oratore. È il «contagio», paro-
la assunta anche da Freud. Nel caso dei tribuni, che par-
lano alla plebe, la situazione è diversa. È una plebe che
è già abituata al loro comando. Si tratta di una massa
organizzata, con un capo. Siamo al Freud dell’analisi
della psicologia delle masse. Si vede anche la differenza
di tecnica. Appendice bio-bibliografica su autori e autrici le e sulla teoria dell’azione nella teoria sociologica classi-
ca e contemporanea, ha pubblicato saggi di teoria socio-
logica su Durkheim, Weber, Eisenstadt, Goffman, Galli-
no, Latour. Nel 2018 ha pubblicato la nuova traduzione
di Stigma di Erving Goffman con introduzione e note al
testo (Ombre Corte, 2018). Arnaldo Bagnasco è professore emerito di Socio-
logia dell’Università di Torino, già professore ordinario
di Sociologia e Sociologia urbana, ha insegnato Sociolo-
gia economica presso l’Università di Firenze (Facoltà di
Scienze Politiche “Cesare Alfieri”) e l’Università Federi-
co II di Napoli (Facoltà di Lettere e Filosofia), sociologia
urbana e sociologia presso l’Università di Torino. Acca-
demico dei Lincei, è socio onorario dell’Associazione
Italiana di Sociologia, della Società geografica italiana e
del Consiglio italiano per le scienze sociali. Ha fondato
e diretto, insieme ad H. Mendras e V. Wright, l’Obser-
vatoire du Changement Social en Europe Occidentale. È noto in ambito internazionale per studi e ricerche in
tema di sviluppo locale e regionale, di sociologia econo-
mica e dell’impresa, di sociologia urbana, di stratifica-
zione sociale. Mario Caciagli è professore emerito di Scienza poli-
tica dell’Università di Firenze. Ha insegnato in prece-
denza materie politologiche nelle università di Catania e
di Padova. È stato visiting professor in varie università
europee: Bonn, Heidelberg, Jena (Germania), Barcellona,
Granada, Santiago di Compostela (Spagna) e Innsbruck
(Austria), nonché nell’Institut de Sciences Politiques di
Parigi. L’Università di Granada gli ha conferito il titolo
di dottore honoris causa. Fra i fondatori della “Società
italiana di studi elettorali”, ne è attualmente presidente
onorario. Dal 1980 al 2019 è stato direttore del seme-
strale Quaderni dell’Osservatorio elettorale. Si è occupato
prevalentemente di elezioni e sistemi elettorali, partiti e
sistemi di partito, politica locale e regionale, nonché di
cultura politica. Gianfranco Bettin Lattes ha insegnato Sociolo-
gia nella Facoltà di Scienze Politiche “Cesare Alfieri” di
Firenze ed ha curato insieme a Paolo Turi il volume La
sociologia di Luciano Cavalli, Firenze University Press,
Firenze, 2008. Antonio Costabile è professore ordinario di Socio-
logia dei fenomeni politici dell’Università della Calabria
dal 2006, dove insegna Etica e politica e Potere politico e
poteri sociali nel mondo globale. Ha diretto il Dottorato
Politica, società e cultura. È Coordinatore nazionale del-
la Sezione di Sociologia Politica dell’AIS dal 2018 (dopo
essere stato per due mandati membro del Direttivo) ed
è Presidente della Consulta scientifica dell’AIS dal 2020. Mentre Antonio acquista, piano piano, que- Lei definisce lo Stato, come s’intravede in queste tra-
gedie, dedito all’organizzazione del dominio e dell’ordine. Un ordine e un dominio, però, sempre discontinui, preca-
ri. Questa non è forse la caratteristica del potere in tutti
i tempi? Certo, ma è interessante che Shakespeare ne abbia
una coscienza così viva. Torniamo alla figura del leader, di cui lei si è occupa-
to molto anche in altre opere. Ebbene, vince sempre quello
più freddo, più cinico, che sa recitare meglio il suo ruolo e
che meglio conosce la psicologia degli uomini. Ma perché
le masse hanno sempre bisogno di un capo? Anche nelle
moderne società democratiche occidentali, in cui le classi
dirigenti non godono né di stima, né di fiducia... i
lo ho dedicato un grosso libro al mutamento stori-
co e sociale prendendo come riferimenti fondamentali 152 Renzo Ricchi sta forza di suggestione, impadronendosi lentamente
dell’animo della folla e usando tra l’altro mezzi retorici
formidabili - il testamento, la veste di Cesare ucciso, il
suo corpo -, i tribuni comandano. E qui entra in cam-
po il tema dell’identifi cazione. Cioè come riesce a fare
Antonio al culmine della sua orazione, così fi n dall’ini-
zio i tribuni, godendo della situazione cui s’è accenna-
to - la massa organizzata -, parlano come coloro i quali esprimono la volontà della massa, come se essi desse-
ro forma, espressione alla massa. Il grande demagogo
populista parla come se fosse la bocca della massa. E la
massa, soggiogata, è pronta a farsi strumento del dema-
gogo. E questo ci riconduce all’attualità permanente di
Shakespeare. Le sue sono rappresentazioni veramente
straordinarie, in profondità, dei processi di avvicina-
mento popolare attraverso la demagogia. Fig. 1. L’immagine ritrae una pagina di appunti di Luciano Cavalli. Fig. 1. L’immagine ritrae una pagina di appunti di Luciano Cavalli. Fig. 1. L’immagine ritrae una pagina di appunti di Luciano Cavalli. societàmutamentopolitica
r i v i s t a i t a l i a n a d i s o c i o l o g i a SOCIETÀMUTAMENTOPOLITICA 14(27): 153-155, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14347 Appendice bio-bibliografica su autori e autrici È stato redattore del quoti-
diano Avanti!, della rete televisiva Rai e addetto stampa
del Presidente della Giunta Regionale Toscana. Carlo A. Marletti è professore emerito dell’Univer-
sità di Torino dove ha insegnato Sociologia generale e ha
diretto il Dipartimento di Studi Politici. È stato Coor-
dinatore nazionale della Sezione di Sociologia Politica
dell’AIS dal 1989 al 1996. Tra le sue numerose pubblica-
zioni a livello nazionale e internazionale si segnalano: La
Repubblica dei media. L’Italia dal politichese alla politica
iperreale, il Mulino, Bologna, 2010; Leadership e demo-
crazia. L’interpretazione neo-weberina di Luciano Cavalli
in G. Bettin Lattes, P. Turi (a cura di), La sociologia di
Luciano Cavalli, Firenze University Press, Firenze, 2008;
Sarkozy e i reali inglesi. Qualche caveat sulla leadership
postmoderna, in «Comunicazione politica», n.1, 2008;
(a cura di) Il leader postmoderno. Studi e ricerche sulla
mediatizzazione delle campagne elettorali locali Franco
Angeli, Milano, 2007; Razionalità e valori. Introduzione
alle teorie dell’azione sociale, Laterza, Roma-Bari, 2006. Sandro Rogari già professore ordinario di Storia
contemporanea presso la Facoltà di Scienze politiche
“Cesare Alfieri” dell’Università di Firenze. È stato vice
direttore del Dipartimento di Studi sullo Stato, Preside
della Facoltà di Scienze Politiche “Cesare Alfieri” e Pro-
rettore alla Didattica e ai servizi agli studenti dell’Uni-
versità di Firenze. È presidente della Società toscana per
la storia del Risorgimento, dell’Accademia “La Colomba-
ria” di Scienze e Lettere e vice presidente della Fondazio-
ne Spadolini-Nuova Antologia. È direttore della Rasse-
gna Storica Toscana. È autore di oltre duecento pubblica-
zioni, è giornalista pubblicista ed editorialista del Quoti-
diano Nazionale. Ambrogio Santambrogio è professore ordinario
di Sociologia presso il Dipartimento di Scienze Politi-
che dell’Università di Perugia di cui è stato direttore. È direttore del Centro di studi internazionale RILES
(RIcerche sul LEgame Sociale). È co-direttore della
rivista Quaderni di teoria sociale e direttore della rivi-
sta Indiscipline – rivista di scienze sociali. Fa parte del
comitato scientifico di diverse riviste e collane di scienze
sociali. Tra le sue più recenti pubblicazioni si segnalano
Utopia senza ideologia, Meltemi, Milano, 2022: Idee per
una sinistra europea, Mondadori, Milano, 2022. Giorgio Marsiglia già professore ordinario di Socio-
logia generale della Facoltà di Scienze politiche “Cesa-
re Alfieri” dell’Università di Firenze, dove ha insegnato
Teoria Sociologica e Sociologia della Modernità presso il
corso di Laurea Magistrale in Sociologia. Appendice bio-bibliografica su autori e autrici Appendice bio-bibliografica su autori e autrici versità di Catania e, come visiting professor, a Warwick,
Barcellona e in altre sedi europee. Ha diretto il Dipar-
timento di Sociologia dell’Università di Catania ed il
DISPO dell’Università di Firenze. È stato membro per
due mandati, in rappresentanza della comunità sociolo-
gica, del Comitato 14 per le Scienze Politiche e Sociali
del CUN. È stato Preside della Facoltà di Scienze Politi-
che “Cesare Alfieri” dell’Università di Firenze dal 1995
al 1998 e Delegato del Rettore prima per le Relazioni sin-
dacali e poi per il Sistema Bibliotecario di Ateneo. Nel
1998 ha fondato presso il DISPO il Laboratorio di Ricer-
ca sulle Trasformazioni Sociali (CAMBIO), che ha diret-
to fino al 2006, e di cui dirige la Collana presso l’editore
Carocci. Nel 2011 ha fondato ed è stato direttore della
rivista Cambio. Rivista sulle trasformazioni sociali. versità di Catania e, come visiting professor, a Warwick,
Barcellona e in altre sedi europee. Ha diretto il Dipar-
timento di Sociologia dell’Università di Catania ed il
DISPO dell’Università di Firenze. È stato membro per
due mandati, in rappresentanza della comunità sociolo-
gica, del Comitato 14 per le Scienze Politiche e Sociali
del CUN. È stato Preside della Facoltà di Scienze Politi-
che “Cesare Alfieri” dell’Università di Firenze dal 1995
al 1998 e Delegato del Rettore prima per le Relazioni sin-
dacali e poi per il Sistema Bibliotecario di Ateneo. Nel
1998 ha fondato presso il DISPO il Laboratorio di Ricer-
ca sulle Trasformazioni Sociali (CAMBIO), che ha diret-
to fino al 2006, e di cui dirige la Collana presso l’editore
Carocci. Nel 2011 ha fondato ed è stato direttore della
rivista Cambio. Rivista sulle trasformazioni sociali. Andrea Millefiorini è professore associato di Socio-
logia politica dell’Università degli studi della Campania
“Luigi Vanvitelli”, dove insegna anche Sociologia gene-
rale. È stato caporedattore e membro della redazione di
“MondOperaio” sotto la direzione di Luciano Pellicani,
dal 2000 al 2008, nonché direttore politico della rivista
“Ragion socialiste”, dal 1995 al 2001. Dal 2018 è membro
del Consiglio scientifico della Sezione di Sociologia poli-
tica dell’AIS. Renzo Ricchi è scrittore e giornalista, attualmente si
occupa di critica letteraria per le riviste Nuova Antologia
e Rivista di Studi Italiani e collabora all’edizione fioren-
tina del quotidiano Corriere della Sera. Ha collaborato
con le riviste Il Ponte, Mondo Operaio, Il Mondo, Città &
Regione e Quaderni di teatro. Appendice bio-bibliografica su autori e autrici I suoi interessi di ricerca riguardano principalmente le
trasformazioni della politica e dello Stato, i rapporti tra
politica e comunità, i legami tra etica e politica e tra
legalità e responsabilità, il sistema politico meridionale. Franca Bonichi già docente di Sociologia e di Socio-
logia dei movimenti presso l’Università di Firenze e
contemporaneamente impegnata nella formazione pro-
fessionale, ha partecipato a numerose ricerche tra cui
quelle sulle istituzioni educative e sui percorsi formati-
vi della popolazione studentesca, sulla militanza sinda-
cale, sull’uso del tempo nella vita quotidiana. Ha scrit-
to numerosi saggi di sociologia politica. In particolare,
sulla teoria politica di Pierre Bourdieu, sui movimenti
sociali, sul concetto di cittadinanza democratica, sulla
teoria delle élites, sul ruolo politico degli attori collettivi. Marco Bontempi è professore ordinario di Socio-
logia e direttore del Dipartimento di Scienze Politiche e
Sociali dell’Università di Firenze. Ha svolto ricerche sul-
la condizione giovanile, il mutamento dei valori politici,
le relazioni tra religioni e sfera pubblica, la dispersione
scolastica. Da anni lavora sui temi del mutamento socia- Paolo Giovannini già professore ordinario di Socio-
logia generale presso Facoltà di Scienze politiche “Cesare
Alfieri” dell’Università di Firenze, insegna attualmente
nel Corso di laurea in Sociologia e politiche sociali. Ha
insegnato, oltre che all’Università di Firenze, all’Uni- SOCIETÀMUTAMENTOPOLITICA 14(27): 153-155, 2023
ISSN 2038-3150 (online) | DOI: 10.36253/smp-14347 154 Appendice bio-bibliografica su autori e autrici Presso la stessa
Facoltà è stato direttore del Corso di Laurea in Servizio
Sociale, dove ha insegnato Sociologia, e successivamente
direttore del Corso di Laurea Magistrale in Organizza-
zione e Gestione degli Interventi Sociali, dove ha inse-
gnato Problemi sociali. È stato membro del Collegio dei
Docenti del Dottorato in Sociologia politica (successiva-
mente in Sociologia) fin dalla sua fondazione. Ha colla-
borato a numerose ricerche del Centro Interuniversitario
di Sociologia Politica (CIUSPO), su finanziamenti nazio-
nali ed europei. Le sue ricerche e pubblicazioni hanno
riguardato temi rilevanti della sociologia nazionale e
internazionali, con l’analisi approfondita, fra gli altri, del
pensiero di Charles Wright Mills e Pierre Bourdieu. Roberto Segatori già professore ordinario di Socio-
logia dei fenomeni politici dell’Università di Perugia,
dove ha insegnato Sociologia dei fenomeni politici e
Governance e politiche pubbliche ed ha diretto il Dipar-
timento Istituzioni e Società. Ha inoltre insegnato Socio-
logia all’Istituto Teologico di Assisi affiliato alla Pontifi-
cia Università Lateranense di Roma. È stato Coordinato- 155 Appendice bio-bibliografica su autori e autrici re nazionale della Sezione di Sociologia politica dell’AIS
dal 2006 al 2013, membro del Centro Interuniversitario
di Sociologia politica (CIUSPO), membro del collegio di
dottorato in Sociologia politica dell’Università di Firen-
ze, Perugia, Genova e Trento. È autore di circa cento-
trenta pubblicazioni scientifiche, e attualmente collabora
come editorialista sul Corriere dell’Umbria e altre testate
locali e nazionali. stessa università ha diretto il Centro Interuniversitario
di Sociologia Politica (CIUSPO), è stato per due mandati
direttore del Dipartimento di Scienze Politiche e Socia-
li e ha fatto parte del collegio dei docenti del Dottorato
in Sociologia politica (poi Sociologia). Le sue attività di
ricerca principali riguardano le élite politiche, la leader-
ship, le biografie politiche, le trasformazioni della classe
politica nei sistemi locali e regionali. È stato segretario
della Sezione di Sociologia politica dell’AIS. Rossana Trifiletti già professoressa associata di
Sociologia dei processi culturali e comunicativi presso
la Facoltà di Scienze politiche “Cesare Alfieri” dell’U-
niversità di Firenze, ha insegnato Sociologia, Sociologia
applicata, Storia del pensiero sociologico, Sociologia del-
la famiglia e Politiche sociali in diversi corsi di laurea. È
stata presidente del Corso di laurea magistrale in Dise-
gno e Gestione degli Interventi sociali dell’Università di
Firenze. I suoi principali interessi di ricerca si articolano
fra la sociologia della famiglia e le politiche sociali, pur
mantenendo un interesse per la storia della sociologia
statunitense contemporanea. Appendice bio-bibliografica su autori e autrici Lorenzo Viviani è professore ordinario di Sociolo-
gia politica dell’Università di Pisa, ha insegnato all’U-
niversità di Firenze dove ha svolto attività di ricerca
nell’ambito Centro Interuniversitario di Sociologia Poli-
tica (CIUSPO). Dal 2018 è segretario della Sezione di
Sociologia politica dell’AIS e partecipa alle attività del-
le principali associazioni internazionali di sociologia
(Esa, Isa, Ipsa, Ecpr). Attualmente è direttore della rivi-
sta SocietàMutamentoPolitica SMP e nel Board di rivi-
ste nazionali e internazionali. Ha svolto e svolge studi e
ricerche sui temi della sociologia politica, con particolare
focalizzazione sui partiti, sulla leadership politica, sulla
democrazia e sul populismo. Le sue pubblicazioni più
recenti si occupano del rapporto tra leadership, populi-
smo e trasformazioni della democrazia. Carlo Trigilia professore emerito di Sociologia eco-
nomica della Facoltà di Scienze Politiche “Cesare Alfieri”
dell’Università di Firenze, ha dedicato molta parte del-
la sua attività di studio e ricerca ai temi dello sviluppo
territoriale e dell’innovazione in Italia e in Europa. Ha
insegnato nelle Università di Palermo e di Trento ed è
stato “Lauro De Bosis professor” presso la Harvard Uni-
versity. È membro della Fondazione Italiani-europei,
presidente della Fondazione RES - Istituto di ricerca su
economia e società in Sicilia e Socio del Consiglio Ita-
liano per le Scienze Sociali (CISS). Ha collaborato con
Il Sole 24 ore e con Il Mattino. Dal 28 aprile 2013 al 22
febbraio 2014 è stato Ministro per la Coesione territo-
riale del Governo Letta. È stato direttore ed è membro
del comitato editoriale della rivista Stato e Mercato, e
del comitato editoriale di Sviluppo locale. Ha studiato lo
sviluppo territoriale in Italia con ricerche sul Mezzogior-
no e sulle regioni di piccola impresa della Terza Italia.,
lo sviluppo locale e dei distretti high-tech in Europa e in
Italia Ha lavorato inoltre sulle origini e gli sviluppi più
recenti della sociologia economica. E’ autore, fra i nume-
rosi testi, dell’Introduzione all’edizione italiana della
Storia Economica di Max Weber (Donzelli, Roma,1993)
e del manuale Sociologia economica, il Mulino, Bologna,
Volumi I e II, 2002-2009. Paolo Turi già professore di Sociologia politica del-
la Facoltà di Scienze politiche “Cesare Alfieri” dell’U-
niversità di Firenze dove ha insegnato, fra i vari corsi,
Sociologia politica e Sociologia della leadership. Nella Finito di stampare da
Logo s.r.l. – Borgoricco (PD) – Italia 21 Il magistero di Luciano Cavalli come sociologo
critico, Giorgio Marsiglia 29 Le tre dimensioni di Luciano Cavalli. Maestro,
studioso, intellettuale pubblico, Roberto Segatori 41 Carisma e leadership nella sociologia della
democrazia, Lorenzo Viviani 61 I leader e le élite. Il contributo di Luciano
Cavalli alla sociologia politica, Carlo A. Marletti 61 I leader e le élite. Il contributo di Luciano
Cavalli alla sociologia politica, Carlo A. Marletti 73 I lati oscuri della democrazia, Gianfranco Bettin
Lattes 85 La democrazia manipolata nella società
digitale, Paolo Giovannini 89 Carisma dei valori e carisma dei fini, Ambrogio
Santambrogio
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https://openalex.org/W2169368996
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https://www.arkat-usa.org/get-file/35752/
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English
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Synthesis of 2-thioxoimidazolin-4-one and thiazolo[3,2-a]-benzimidazole derivatives from substituted maleimides
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ARKIVOC
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cc-by
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Abstract We describe here a hetero Michael-type reaction, involving maleimides and 2-
cyanoacetichydrazide or 2-mercaptobenzimidazole, followed by an opening cyclization reaction
with isothiocyanates. This reaction allows the preparation of new 2-thioxoimidazolidin-4-one
and thiazolo[3,2-a]benzimidazole derivatives depending on the nucleophile used. Keywords:
Thioxoimidazolin-4-one,
hetero
Michael
reaction,
thiazolobenzimidazoles,
maleimide Synthesis of 2-thioxoimidazolin-4-one and thiazolo[3,2-a]-
benzimidazole derivatives from substituted maleimides aLaboratoire de Chimie Organique Appliquée, Université des Sciences et Techniques Houari
Boumediene, BP 3, El-Alia, Bab-Ezzouar, 16111, Alger, Algérie
bCentre de Recherche Scientifique Technique en Analyse Physico-chimique (CRAPC) BP248
Alger RP, 16004, Algérie
cClermont Université, ENSCCF, EA 987, LCHG, BP 10448, F-63000 Clermont-Ferrand, France DOI: http://dx.doi.org/10.3998/ark.5550190.0011.a27 ARKIVOC 2010 (x) 328-337 ARKIVOC 2010 (x) 328-337 General Papers Introduction 2-thioxoimidazolin-4-one (thiohydantoins) framework is often encountered in compounds
dealing with biological activity. As examples, thiohydantoin 1 is an efficient inhibitor of muscle
and liver glycogen phosphorylases1 and has also showed herbicidal activity by inhibition of
purine biosynthesis.2 Substituted 4-methylene-2-thiohydantoins 2 have displayed Cycline
Dependant Kinases (CDK) inhibition in a micromolar range3a or antileishmanial activity3b
whereas compounds 3 are Fatty Acid Amide Hydrolase (FAAH) inhibitors4 and compounds 4
are claimed to be active for treatment of hormone refractory prostate cancer.5 So, it is not surprising if a lot of patents have described the use of this attractive skeleton for
a range of activity which cover from compounds for biological use6 till fire retardants materials.7
Besides conventional methods used for their synthesis, solid phase,8 use of ionic liquids9 and
microwaves-assisted approaches have been published.10 On the other hand, the benzimidazole
structure is also a scaffold often endowed with different types of biological activities including ARKAT USA, Inc. ISSN 1551-7012 ISSN 1551-7012 Page 328 ARKIVOC 2010 (x) 328-337 General Papers antitumor,11 antimicrobial12 or antiviral13 properties. Thiazole nucleus fused with a
benzimidazole skeleton, namely thiazolobenzimidazoles, have been reported to exhibit biological
properties too, such as anti-HIV14 or anti-microbial15 agents. We wish to report here the synthesis
of new 2-thioxoimidazolin-4-one and thiazolobenzimidazoles derivatives by hetero Michael
reactions on N-substituted maleimides. Figure 1. Thiohydantoins with described biological activities. Figure 1. Thiohydantoins with described biological activities. 2-Thioxoimidazolin-4-one derivatives Previous investigations from our laboratory16 have shown that 2-thioxoimidazolin-4-one
derivatives 7 could be easily obtained by Michael addition of 3-ethylhydrazinoacetate on N-
substituted maleimide 5, leading to 3,4-dihydro maleimides 6 which reacted smoothly with
substituted isothiocyanates to give compounds 7 (Scheme1). Scheme 1. Synthesis of compounds 7a-h. Scheme 1. Synthesis of compounds 7a-h. Scheme 1. Synthesis of compounds 7a-h. ISSN 1551-7012
Page 329 ARKAT USA, Inc. ARKIVOC 2010 (x) 328-337 General Papers We wish to report here an extension of this methodology using 2-cyanoacetic hydrazide as
nucleophile. This compound, by its carbonyl and cyano functions, is enabling to react with
bidendate reagents. Moreover, the active hydrogen of this molecule can also take part in a wide
range of condensation and substitutions reactions.17 Thus, condensation of maleimide 5a-d with 2-cyanoacetic hydrazide in refluxing ethanol led
to compounds 8a-d in fair to good yields. Spectral data (1H and 13C NMR) were in complete
accordance with the proposed structures and showed that these compounds existed under a
tautomeric equilibrium between the keto (I) and enol (II) form in an average ratio of 3/1. For
example concerning succinimide 8d, protons of CH2-CH fragment showed characteristic patterns
of an ABX system (Scheme 2). Scheme 2. Preparation of 2-thioxoimidazolidines 9a-d. Scheme 2. Preparation of 2-thioxoimidazolidines 9a-d. The chemical shift observed for the doublet of doublet corresponding to proton Ha was δ=
2.69 ppm (J = 4 and 18 Hz) in the form (I) while it was δ= 2.85 ppm (J = 5 and 18 Hz) in the
form (II). For proton Hb, the observed chemical shift was δ= 3.05 ppm (J = 8 and 18 Hz) in form
(I) and 2.95 ppm (J = 8 and 18 Hz) in form (II). Same observations could also be made on
protons He and Hf. In form (I) He and Hf give two doublets respectively at 3.62 (He) and 3.67 (Hf)
ppm (J = 18 Hz), while in form (II) the signals corresponding to these protons was shifted to
3.90 (He) and 4.08 (Hf) ppm (J = 19 Hz). As described previously, condensation of succinimides
8a-d with phenylisothiocyanate furnished the corresponding 2-thioxoimidazolidines 9a-d
resulting of the opening-recyclization reaction. For these compounds 9, it should be noted that no
tautomeric form was observed, contrary to compounds 8. 2-Thioxoimidazolin-4-one derivatives Here too, the structures of these new
compounds were confirmed by careful examination of spectral data, showing mainly in the IR
spectra a band around ν = 1500 cm-1 and in the 13C NMR spectra a signal at 184 ppm attributed
to the thiocarbonyl group. Next, we envisaged to test the reactivity of the 2-cyanoacetic appendage. So, Knoevenagel
condensation of compound 8d with benzaldehyde in refluxing ethanol containing a catalytic
amount of piperidine18 resulted in the formation of benzylidene 10 which reacted smoothly with
phenylisothiocyanate to furnish the 2-thioxoimidazolidine derivative 11 in good yield (Scheme
3), the structure of which was in accordance with all spectral data. ISSN 1551-7012 ARKAT USA, Inc. Page 330 ARKIVOC 2010 (x) 328-337 General Papers Scheme 3. Synthesis of compound 11. Scheme 3. Synthesis of compound 11. Experimental Section General. Unless otherwise specified, reagents were obtained from commercial suppliers. Solvents were dried and freshly distilled following the usual procedures. Product organic
solutions were dried over sodium sulfate prior to evaporation of the solvents under reduced
pressure on a rotatory evaporator. Thin layer chromatography was performed on TLC precoated
aluminium backed silica plates and spots were visualized using UV light (254 nm) before using
ethanolic phosphomolybdic acid solution (heating). Column chromatography was carried out on
silica gel (70-230 mesh).Melting points were measured in open capillary tubes on a BÜCHI B-
540 apparatus. 1H and 13C NMR spectra were measured at 400.13 and 100.61 MHz respectively
using TMS as internal reference. Chemicals shifts are reported in ppm relative to TMS. Signals
are quoted s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) and coupling
constant (J) values are given in Hz. Infrared spectra were recorded on a Shimadzu FTIR 8400s
spectrophotometer. High Resolution Electro Spray Impact Mass Spectra (HR-ESI-MS) were
obtained from the Centre Régional de Mesures Physiques de l'Université Blaise Pascal
(Clermont II), France. Elemental analyses (C, H, O, N) were performed at the Centre de
Microanalyses du CNRS, Lyon, France. Thiazolo[3,2-a]benzimidazole derivatives Finally we envisaged to test the reactivity of other nucleophiles on hetero Michael addition to
maleimide and for this purpose we tried the addition of 2-mercaptobenzimidazole 12. This latter
was chosen as this framework is endowed with a lot of biological activities.11,13 Several methods
have been developed for the synthesis of thiazolo[3,2-a]framework using the nucleophilicity of
the thiol function of 2-mercaptobenzimidazole.19 A recent work has also described the synthesis
of target molecule by opening thiiranes with 2-chlorobenzimidazole.20 Thus maleimides 1a,b,d
reacted with 12 in refluxing acetic acid in presence of concentrated sulphuric acid to afford in
each case the corresponding thiazolobenzimidazole 13a,b,d in average yields (Scheme 4). Scheme 4. Synthesis of thiazolobenzimidazole 13a,b,d. Scheme 4. Synthesis of thiazolobenzimidazole 13a,b,d. Formation of compounds 13 resulted from initial attack of the mercapto group on the double
bond of maleimide followed, as described previously, by ring opening of the maleimide by the
amino group of the benzimidazole ring. Spectral data obtained from compounds 13 were in
complete accordance with the expected thiazolobenzimidazole structures: in 1H NMR spectrum
of each derivative, together with the expected signals, one can noticed the presence of three new
signals at around 2.95, 3.30-4.95 and 11.30 ppm, due respectively to CH, CH2 and NH2 protons. Moreover in 13C NMR spectrum, signals at 38.6, 62.4 and 177.9 ppm were assigned to CH, CH2
and C=O carbons beside all others carbons at the expected regions. In conclusion, we have described new 2-thioxoimidazolidine and new thiazolo[3,2-a]-
benzimidazole derivatives. The use of 2-cyanoacetic hydrazide as nucleophile in the hetero- ISSN 1551-7012 Page 331 ARKAT USA, Inc. ARKAT USA, Inc. ARKIVOC 2010 (x) 328-337 General Papers Michael reaction on maleimide, followed by the reaction with isothiocyanates, has permitted an
entry to polyfunctionalized heterocyclic structures due to its intrinsic reactivity. The extension of
this methodology, together with the use of the variety of reactions which could be realized21
starting from this brick, is actually under progress. General procedure for preparation of compounds (8) Yield 44%; white solid;
mp 99-100 °C, IR (KBr, ν cm-1) 3348 (w), 3306 (m) , 3267(m), 2258 (m), 1774 (s), 1701 (s),
1624 (m); 1H NMR (400 MHz, DMSO-d6) 9.78 (1H, br s, Hd(I)), 9.22 (1H, br s, Hd(II)), 5.79
(2H, m, Hc(I, II)), 4.20 (1H, dd, 3J = 8.0 and 4.0 Hz, Hx(II)), 4.15 (1H, dd, 3J = 8.0 and 4.0 Hz,
Hx(I)), 3.90 (1H, d, 2J = 18.0 Hz, Hf(II)), 3.85 (1H, d, 2J = 18.0 Hz, He(II)), 3.65 (1H, d, 2J = 18.0
Hz, Hf(I)), 3.58 (1H, d, 2J = 18.0 Hz, He(I)), 3.40 (4H, q, 3J = 7.0 Hz, CH2(I, II)), 2.82 (1H, dd, 2J
= 18.0, 3J = 8.0 Hz, Hb(I)), 2.80 (1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(II)), 2.64 (1H, dd, 2J = 18.0,
3J = 4.0 Hz, Ha(II)), 2.40 (1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(I)), 1.02 (6H, t, 3J = 7.0 Hz, CH3(I,
II)); 13C NMR (100 MHz, DMSO-d6) 176.2 (C=O imide, II), 176.0 (C=O imide, I), 175.2
(C=O imide, I), 175.1 (C=O imide, II), 167.3 (C=O amide, I), 162.0 (C(OH)=N, II), 116.4 (CN,
II), 115.8 (CN, I), 57.7 (CHx, II), 57.1 (CHx, I), 33.8 (CH2, I, II), 33.0 (CHaHb, II), 32.8 (CHaHb,
I), 23.7 (CHeHf, I), 23.5 (CHeHf, II), 12.6 (CH3, I, II); HRMS m/z calcd. for C9H12N4O3Na
(M+Na)+: 247.0808, found : 247.0807; Anal. Calcd. for C9H12N4O3: C, 48.21; H, 5.39; N, 24.99;
O, 21.41. Found: C, 48.37; H, 5.43; N, 25.07; O, 21.13. 2-Cyano-N’-(2,5-dioxo-1-phenylpyrrolidin-3-yl)acetohydrazide (8d). Yield 55%; white solid;
mp 179-180 °C, IR (KBr, ν cm-1) 3483 (m), 3288 (m), 3211 (m), 2258 (m), 1784 (s), 1710 (s),
1680 (s); 1H NMR (400 MHz, DMSO-d6) 9.77 (1H, d, 3J = 5.0 Hz, Hd(I)), 9.17 (1H, br s,
Hd(II)), 7.53-7.25 (10H, m, Harom. General procedure for preparation of compounds (8) Yield 44%; white solid;
mp 99-100 °C, IR (KBr, ν cm-1) 3348 (w), 3306 (m) , 3267(m), 2258 (m), 1774 (s), 1701 (s),
1624 (m); 1H NMR (400 MHz, DMSO-d6) 9.78 (1H, br s, Hd(I)), 9.22 (1H, br s, Hd(II)), 5.79
(2H, m, Hc(I, II)), 4.20 (1H, dd, 3J = 8.0 and 4.0 Hz, Hx(II)), 4.15 (1H, dd, 3J = 8.0 and 4.0 Hz,
Hx(I)), 3.90 (1H, d, 2J = 18.0 Hz, Hf(II)), 3.85 (1H, d, 2J = 18.0 Hz, He(II)), 3.65 (1H, d, 2J = 18.0
Hz, Hf(I)), 3.58 (1H, d, 2J = 18.0 Hz, He(I)), 3.40 (4H, q, 3J = 7.0 Hz, CH2(I, II)), 2.82 (1H, dd, 2J
= 18.0, 3J = 8.0 Hz, Hb(I)), 2.80 (1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(II)), 2.64 (1H, dd, 2J = 18.0,
3J = 4.0 Hz, Ha(II)), 2.40 (1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(I)), 1.02 (6H, t, 3J = 7.0 Hz, CH3(I,
II)); 13C NMR (100 MHz, DMSO-d6) 176.2 (C=O imide, II), 176.0 (C=O imide, I), 175.2
(C=O imide, I), 175.1 (C=O imide, II), 167.3 (C=O amide, I), 162.0 (C(OH)=N, II), 116.4 (CN,
II), 115.8 (CN, I), 57.7 (CHx, II), 57.1 (CHx, I), 33.8 (CH2, I, II), 33.0 (CHaHb, II), 32.8 (CHaHb,
I), 23.7 (CHeHf, I), 23.5 (CHeHf, II), 12.6 (CH3, I, II); HRMS m/z calcd. for C9H12N4O3Na
(M+Na)+: 247.0808, found : 247.0807; Anal. Calcd. for C9H12N4O3: C, 48.21; H, 5.39; N, 24.99;
O, 21.41. Found: C, 48.37; H, 5.43; N, 25.07; O, 21.13. 2-Cyano-N’-(2,5-dioxo-1-phenylpyrrolidin-3-yl)acetohydrazide (8d). Yield 55%; white solid;
mp 179-180 °C, IR (KBr, ν cm-1) 3483 (m), 3288 (m), 3211 (m), 2258 (m), 1784 (s), 1710 (s),
1680 (s); 1H NMR (400 MHz, DMSO-d6) 9.77 (1H, d, 3J = 5.0 Hz, Hd(I)), 9.17 (1H, br s,
Hd(II)), 7.53-7.25 (10H, m, Harom. (I, II)), 5.88 (2H, m, Hc(I, II)), 4.20 (1H, dd, 3J = 8.0 and 4.0
Hz Hx(II)) 4 17 (1H dd
3J = 8 0 and 4 0 Hz Hx(I)) 3 90 (1H d
2J = 19 0 Hz Hf(II)) 3 87 219.0491, found : 219.0494; Anal. Calcd. for C7H8N4O3: C, 42.86; H, 4.11; N, 28.56; O, 24.47. Found: C, 42.75; H, 4.25; N, 28.65; O, 24.59. 219.0491, found : 219.0494; Anal. Calcd. for C7H8N4O3: C, 42.86; H, 4.11; N, 28.56; O, 24.47. Found: C, 42.75; H, 4.25; N, 28.65; O, 24.59. 2-Cyano-N'-(1-methyl-2,5-dioxopyrrolidin-3-yl)acetohydrazide (8b). General procedure for preparation of compounds (8) Yield 56%; white solid;
mp 121-122 °C, IR (KBr, ν cm-1) 3348 (w), 3308 (m), 3198 (w), 2258 (m), 1786 (w), 1705 (s),
1654 (s); 1H NMR (400 MHz, DMSO-d6) 9.69 (1H, d, 3J = 5.5 Hz, Hd(I)), 9.29 (1H, br s,
Hd(II)), 5.78 (2H, m, Hc(I, II)), 4.08-3.99 (2H, m, Hx(I, II)), 3.98 (1H, d, 2J = 18.0 Hz, Hf(II)),
3.87 (1H, d, 2J = 18.0 Hz, He(II)), 3.63 (1H, d, 2J = 18.0 Hz, He(I)), 3.58 (1H, d, 2J = 18.0 Hz,
Hf(I)), 2.87-2.75 (8H, m, Hb(I), Hb(II), CH3 (I, II)), 2.67 (1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(II)),
2.54 (1H, dd, 2J = 18.0 3J = 4.0 Hz, Ha(I)); 13C NMR (100 MHz, DMSO-d6) δ 176.5 (C=O imide,
II), 176.3 (C=O imide, I), 175.5 (C=O imide, I), 175.3 (C=O imide, II), 162.0 (C=O amide, I),
161.9 (C(OH)=N, II), 116.0 (CN, I), 115.8 (CN, II), 57.7 (CHx, I), 57.2 (CHx, II), 33.7 (CHaHb,
I), 33.0 (CHaHb, II), 24.3 (CHeHf, II), 24.2 (CHeHf, I), 23.7 (CH3, II), 23.5 (CH3, I); HRMS m/z
calcd. for C8H10N4O3Na (M+Na)+: 233.0651, found : 233.0651; Anal. Calcd. for C8H10N4O3: C,
45.71; H, 4.80; N, 26.66; O, 22.84. Found: C, 45.49; H, 4.88; N, 26.41; O, 22.78. 2-Cyano-N'-(1-ethyl-2,5-dioxopyrrolidin-3-yl)acetohydrazide (8c). General procedure for preparation of compounds (8) A mixture of cyanoacetic acid hydrazide (1 equivalent) and the corresponding maleimide 5a-d
(1 equivalent) in ethanol was refluxed for 12 hours. After cooling the precipitate was filtered and
the resulting solid was crystallized from methanol. 2-Cyano-N'-(2,5-dioxopyrrolidin-3-yl)acetohydrazide (8a). Yield 52%; white solid; mp 102-
103 °C, IR (KBr, ν cm-1) ) 3348 (m), 3282 (w), 3194 (m), 3136 (w), 2260 (m), 1703 (s), 1685 (s),
1622 (m); 1H NMR (400 MHz, DMSO-d6) 11.25 (1H, br s, NH (I, II)), 9.68 (1H, d, 3J = 5.5
Hz, Hd(I)), 9.07 (1H, s, Hd(II)), 5.67 (1H, d, 3J = 3.5 Hz, Hc(II)), 5,64 (1H, t, 3J = 4.5 Hz, Hc(I)),
4.05-3.95 (3H, m, Hx(I, II) and Hf(II)), 3.86 (1H, d, 2J = 19.0 Hz, He(II)), 3.63 (1H, d, 2J = 18.0
Hz, Hf(I)), 3.58 (1H, d, 2J = 18.0 Hz, He(I)), 2.80 (1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(I)), 2.77
(1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(II)), 2.65 (1H, dd, 2J = 18.0, 3J = 5.0 Hz, Ha(II)), 2.43 (1H,
dd, 2J = 18.0, 3J = 4.0 Hz, Ha(I)); 13C NMR (100 MHz, DMSO-d6) δ 178.0 (C=O imide, I), 177.8
(C=O imide, II), 176.8 (C=O imide, II), 176.6 (C=O imide, I), 167.4 (C=O amide, I), 162.0
(C(OH)=N, II), 116.2 (CN, II), 115.8 (CN, I), 58.9 (CHx, II), 58.3 (CHx, I), 34.9 (CHaHb, I), 34.2
(CHaHb,II), 23.7 (CHeHf, I), 23.6 (CHeHf, II); HRMS m/z calcd. for C7H8N4O3Na (M+Na)+: ARKAT USA, Inc. ISSN 1551-7012 Page 332 Page 332 ARKIVOC 2010 (x) 328-337 General Papers 219.0491, found : 219.0494; Anal. Calcd. for C7H8N4O3: C, 42.86; H, 4.11; N, 28.56; O, 24.47. Found: C, 42.75; H, 4.25; N, 28.65; O, 24.59. 2-Cyano-N'-(1-methyl-2,5-dioxopyrrolidin-3-yl)acetohydrazide (8b). General procedure for preparation of compounds (8) Yield 56%; white solid;
mp 121-122 °C, IR (KBr, ν cm-1) 3348 (w), 3308 (m), 3198 (w), 2258 (m), 1786 (w), 1705 (s),
1654 (s); 1H NMR (400 MHz, DMSO-d6) 9.69 (1H, d, 3J = 5.5 Hz, Hd(I)), 9.29 (1H, br s,
Hd(II)), 5.78 (2H, m, Hc(I, II)), 4.08-3.99 (2H, m, Hx(I, II)), 3.98 (1H, d, 2J = 18.0 Hz, Hf(II)),
3.87 (1H, d, 2J = 18.0 Hz, He(II)), 3.63 (1H, d, 2J = 18.0 Hz, He(I)), 3.58 (1H, d, 2J = 18.0 Hz,
Hf(I)), 2.87-2.75 (8H, m, Hb(I), Hb(II), CH3 (I, II)), 2.67 (1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(II)),
2.54 (1H, dd, 2J = 18.0 3J = 4.0 Hz, Ha(I)); 13C NMR (100 MHz, DMSO-d6) δ 176.5 (C=O imide,
II), 176.3 (C=O imide, I), 175.5 (C=O imide, I), 175.3 (C=O imide, II), 162.0 (C=O amide, I),
161.9 (C(OH)=N, II), 116.0 (CN, I), 115.8 (CN, II), 57.7 (CHx, I), 57.2 (CHx, II), 33.7 (CHaHb,
I), 33.0 (CHaHb, II), 24.3 (CHeHf, II), 24.2 (CHeHf, I), 23.7 (CH3, II), 23.5 (CH3, I); HRMS m/z
calcd. for C8H10N4O3Na (M+Na)+: 233.0651, found : 233.0651; Anal. Calcd. for C8H10N4O3: C,
45.71; H, 4.80; N, 26.66; O, 22.84. Found: C, 45.49; H, 4.88; N, 26.41; O, 22.78. 2-Cyano-N'-(1-ethyl-2,5-dioxopyrrolidin-3-yl)acetohydrazide (8c). General procedure for preparation of compounds (8) (I, II)), 5.88 (2H, m, Hc(I, II)), 4.20 (1H, dd, 3J = 8.0 and 4.0
Hz, Hx(II)), 4.17 (1H, dd, 3J = 8.0 and 4.0 Hz, Hx(I)), 3.90 (1H, d, 2J = 19.0 Hz, Hf(II)), 3.87
(1H, d, 2J = 19.0 Hz, He(II)), 3.67 (1H, d, 2J = 18.0 Hz, Hf(I)), 3.62 (1H, d, 2J = 18.0 Hz, He(I)),
3.05 (1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(I)), 3.00 (1H, dd, 2J = 18.0, 3J = 8.0 Hz, Hb(II)), 2.85
(1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(II)), 2.69 (1H, dd, 2J = 18.0, 3J = 4.0 Hz, Ha(I)); 13C NMR
(100 MHz, DMSO-d6) 175.7 (C=O imide, II), 175.4 (C=O imide, I), 174.6 (C=O imide, I),
174.4 (C=O imide, II), 167.4 (C(OH)=N, II), 162.1 (C=O amide, I), 132.2 (Cipso, I), 132.1
(Cipso, II), 128.9 (CHarom.., II), 128.8 (CHarom., I), 128.3 (CHarom., II), 128.2 (CHarom., I), 127.0
(CHarom., I), 126.9 (CHarom., II), 116.4 (CN, I), 115.8 (CN, II), 57.9 (CHx, II), 57.4 (CHx, I), 34.0 Page 333 ISSN 1551-7012 ARKAT USA, Inc. ARKIVOC 2010 (x) 328-337 General Papers (CHaHb, I), 33.4 (CHaHb, II), 23.8 (CHeHf, I), 23.5 (CHeHf, II); HRMS m/z calcd. for
C13H12N4O3Na (M+Na)+: 295.0811, found : 295.0807; Anal. Calcd. for C13H12N4O3: C, 57.35;
H, 4.44; N, 20.58; O, 17.63. Found: C, 57.25; H, 4.46; N, 20.29; O, 17.87. (CHaHb, I), 33.4 (CHaHb, II), 23.8 (CHeHf, I), 23.5 (CHeHf, II); HRMS m/z calcd. for
C13H12N4O3Na (M+Na)+: 295.0811, found : 295.0807; Anal. Calcd. for C13H12N4O3: C, 57.35;
H, 4.44; N, 20.58; O, 17.63. Found: C, 57.25; H, 4.46; N, 20.29; O, 17.87. General procedure for preparation of compounds 9 To a solution of compounds 8a-d (10 mmol) in ethanol (20 mL) was added phenyl
isothiocyanate (10 mmol). The resulting mixture was brought to reflux for 12 hours. The solid
obtained after cooling was filtered and crystallized from ethanol to give compounds 9a-d. isothiocyanate (10 mmol). The resulting mixture was brought to reflux for 12 hours. The solid
obtained after cooling was filtered and crystallized from ethanol to give compounds 9a-d. N-[5-(2-Amino-2-oxoethyl)-4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-2-cyanoacetamide
(9a). Yield 57%; white solid; mp 158-160 °C; IR (KBr, ν cm-1) 3340 (m), 3284 (m), 2262 (m),
1710 (m), 1697 (s), 1670 (s), 1512 (m); 1H NMR (400 MHz, DMSO-d6) 11.25 (1H, s, Hd),
7.56 (2H, s, NH2), 7.60-7.35 (m, 5H, Harom.), 4.67 (1H, t, 3J = 4.0 Hz, Hx), 3.89 (1H, d, 2J = 18.0
Hz, He), 3.85 (1H, d, 2J = 18.0 Hz, Hf), 2.87 (2H, d, 3J = 4.0 Hz, Ha, Hb); 13C NMR (100 MHz,
DMSO-d6) δ 183.2 (C=S), 171.5 (NH2C=O), 169.7 (C=O thioimidazole), 162.2 (NNHC=O),
133.8 (Cipso), 128.9(CHarom.), 128.8 (CHarom.), 128.5 (CHarom.), 115.1 (CN), 59.5 (CHx), 34.9
(CHaHb), 23.9 (CHeHf); HRMS m/z calcd. for C14H13N5O3S (M+H)+: 332.0824, found :
332.0817; Anal. Calcd. for C14H13N5O3S: C, 50.75; H, 3.95; N, 21.14; O, 14.49; S, 9.68. Found:
C, 50.84; H, 4.09; N, 21.35; O, 14.98; S, 9.74. g
y
g
p
N-[5-(2-Amino-2-oxoethyl)-4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-2-cyanoacetamide
(9a). Yield 57%; white solid; mp 158-160 °C; IR (KBr, ν cm-1) 3340 (m), 3284 (m), 2262 (m),
1710 (m), 1697 (s), 1670 (s), 1512 (m); 1H NMR (400 MHz, DMSO-d6) 11.25 (1H, s, Hd),
7.56 (2H, s, NH2), 7.60-7.35 (m, 5H, Harom.), 4.67 (1H, t, 3J = 4.0 Hz, Hx), 3.89 (1H, d, 2J = 18.0
Hz, He), 3.85 (1H, d, 2J = 18.0 Hz, Hf), 2.87 (2H, d, 3J = 4.0 Hz, Ha, Hb); 13C NMR (100 MHz,
DMSO-d6) δ 183.2 (C=S), 171.5 (NH2C=O), 169.7 (C=O thioimidazole), 162.2 (NNHC=O),
133.8 (Cipso), 128.9(CHarom.), 128.8 (CHarom.), 128.5 (CHarom.), 115.1 (CN), 59.5 (CHx), 34.9
(CHaHb), 23.9 (CHeHf); HRMS m/z calcd. for C14H13N5O3S (M+H)+: 332.0824, found :
332.0817; Anal. Calcd. for C14H13N5O3S: C, 50.75; H, 3.95; N, 21.14; O, 14.49; S, 9.68. Found:
C, 50.84; H, 4.09; N, 21.35; O, 14.98; S, 9.74. 2-Cyano-N-{5-[2-(methylamino)-2-oxoethyl]-4-oxo-3-phenyl-2-thioxoimidazolidin-1-
yl}acetamide (9b). General procedure for preparation of compounds 9 Yield 45%; yellow solid; mp 216-218 °C; IR (KBr, ν cm-1) 3258 (w), 3129 (w), 2220
(m), 1753 (s), 1686 (s), 1647 (m), 1595 (m), 1271 (w); 1H NMR (400 MHz, DMSO-d6) 11.62
(1H, s, Hd), 10.19 (1H, s, NHPh), 8.32 (1H, s, Hg), 7.95 (2H, d, 3J = 6.5 Hz, Harom.), 7.65-7.55
(7H, m, Harom.), 7.50 (1H, t, 3J = 7.0 Hz, Harom.), 7.45 (2H, d, 3J = 7.5 Hz, Harom.), 7.35 (2H, t, 3J
= 7.5 Hz, Harom.), 7.05 (1H, t, 3J = 7.5 Hz, Harom.), 4.87 (1H, t, 3J = 4.0 Hz, Hx), 3.20 (2H, dd, 2J =
17.9 Hz, 3J = 4.0 Hz, Ha); 3.17 (1H, dd, 2J = 17.9, 3J = 4.0 Hz, Hb); 13C NMR (100 MHz,
DMSO-d6) δ 184.7 (C=S), 170.0 (C=O thioimidazole), 166.0 (C=O amide), 160.7 (NNHC=O),
153.1 (CH=C), 138.8 (Cipso), 133.7 (Cipso), 131.3 (Cipso), 130.2 (CHarom.), 129.3 (CHarom.),
128.9 (CHarom.), 128.8 (CHarom.), 128.7 (CHarom.), 128.6 (CHarom.), 128.4 (CHarom.), 123.2
(CHarom.), 119.0(CHarom.), 115.3 (CN), 103.3 (C=CH), 59.9 (CHx), 35.0 (CHaHb); HRMS m/z
calcd. for C27H22N5O3S (M+H)+: 496.1443, found : 496.1448. 2-Cyano-N'(2,5-dioxo-1-phenylpyrrolidin-3-yl)-3-phenylacrylo hydrazide (10). To a solution
of 8d (1 mmol) in ethanol (20 mL) containing a catalytic amount of piperidine, benzaldehyde (10
mmol) was added. The reaction mixture was heated at reflux for 12 hours. The resulting solid
formed was collected by filtration. Yield 58%; yellow solid; mp 163-165 °C; IR (KBr, ν cm-1)
3298 (w), 3059 (w), 1780 (s), 1708 (s), 1651 (m), 1604 (m); 1H NMR (400 MHz, DMSO-d6)
10.25 (1H, br s, Hd), 8.16 (1H, s, Hg), 7.95 (2H, m, Harom.), 7.65-7.55 (3H, m, Harom.), 7.50
(2H, t, 3J = 7.5 Hz, Harom.), 7.30 (2H, d, 3J = 7.5 Hz, Harom.), 6.09 (1H, br s, Hc); 4.28 (1H, dd, 3J
= 8.5 and 4.0 Hz, Hx), 3.10 (1H, dd, 2J = 18.0, 3J = 8.5 Hz, Ha), 2.80 (1H, dd, 2J = 18.0, 3J = 4.0
Hz, Hb); 13C NMR (100 MHz, DMSO-d6) δ 175.3 (C=O imide), 174.7 (C=O imide), 161.0
(NNHC=O), 150.8 (CH=C), 132.4 (Cipso), 132.2 (Cipso), 131.8 (CHarom.), 130.0 (CHarom.),
129.2 (CHarom.), 128.9 (CHarom.), 128.5 (CHarom.), 126.9 (CHarom.), 115.8 (CN), 104.8 (C=CH),
57.3 (CHx), 34.4 (CHaHb); HRMS m/z calcd. for C20H16N4O3: 383.1106, found : 383.1120. N-(4-(2-Anilino-2-oxoethyl)-5-oxo-1-phenyl-2-thioxopyrrolidin-3-yl)-2-cyano-3-phenyl-
acrylamide (11). A solution of compound 10 (1 mmol.) in ethanol (20 mL) was refluxed with a
large excess of phenyl isothiocyanate during 5 hours. General procedure for preparation of compounds 9 Yield 66%; white solid; mp 222-224 °C, IR (KBr, ν cm-1) 3379 (m), 3286
(w), 2260(m), 1766 (s), 1654 (s), 1500 (m); 1H NMR (400 MHz, DMSO-d6) 11.20 (1H, s,
Hd), 8.05 (1H, q, 3J = 4.5 Hz, NH), 7.65-7.35 (5H, m, Harom.), 4.75 (1H, t, 3J = 4.0 Hz, Hx), 3.95
(1H, d, 2J = 18.0 Hz, He), 3.88 (1H, d, 2J = 18.0 Hz, Hf), 2.93 (2H, d, 3J = 4.0 Hz, Ha, Hb), 2.65
(3H, d, J = 4.5 Hz, NCH3); 13C NMR (100 MHz, DMSO-d6) δ 183.1 (C=S), 171.3
(NH(CH3)C=O), 167.8 (C=O thioimidazole), 162.1 (NNHC=O), 133.7 (Cipso), 128.8 (CHarom.),
128.7 (CHarom.), 128.4 (CHarom.), 115.0 (CN), 59.5 (CHx), 33.3 (CHaHb), 25.5 (NCH3), 23.8
(CHeHf); HRMS m/z calcd. for C15H16N5O3S (M+H)+: 346.0971, found : 346.0974; Anal. Calcd. for C15H15N5O3S: C, 52.16; H, 4.38; N, 20.28; O, 13.90; S, 9.28. Found: C, 51.95; H, 4.29; N,
20.37; O, 14.24; S, 9.15. 2-Cyano-N-{5-[2-(ethylamino)-2-oxoethyl]-4-oxo-3-phenyl-2-thioxoimidazolidin-1-
yl}acetamide (9c). Yield 53%; white solid; mp 145-146 °C, IR (KBr, ν cm-1) 3369 (m), 3169
(w), 2258 (m), 1766 (s), 1708 (s), 1647 (s), 1500 (m); 1H NMR(400MHz, DMSO-d6) 11.20
(1H, s, Hd), 8.05 (1H, t, 3J = 4.5 Hz, NHC2H5), 7.60-7.30 (5H, m, Harom.), 4.75 (1H, t, 3J = 4.0
Hz, Hx), 3.89 (1H,d, 2J = 18.0 Hz, He), 3.85 (1H, d, 2J = 18.0 Hz, Hf), 3.10 (2H, m, NCH2), 2.86
(2H, d, 3J = 4.0 Hz, Ha, Hb), 1.00 (3H, t, 3J = 7.0 Hz, CH3); 13C NMR (100 MHz, DMSO-d6) δ
183.1 (C=S), 171.4 (NH(C2H5)C=O), 167.1 (C=O thioimidazole), 162.2 (NNHC=O), 133.8
(Cipso), 128.9 (CHarom.), 128.8 (CHarom.), 128.5 (CHarom.), 115.1 (CN), 59.6 (CHx), 33.5 (CHaHb),
33.1 (NCH2), 23.9 (CHeHf), 14.5 (CH3); HRMS m/z calcd. for C16H18N5O3S (M+H)+: 360.3541,
found : 360.3543; Anal. Calcd. for C20H17N5O3S: C, 53.47; H, 4.77; N, 19.49; O, 13.35; S, 8.92. Found: C, 52.70; H, 4.73; N, 19.53; O, 13.82; S, 8.73. Page 334 ARKAT USA, Inc. ISSN 1551-7012 ISSN 1551-7012 ARKIVOC 2010 (x) 328-337 General Papers N-[5-[2-Anilino-2-oxoethyl)-4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-2-cyanoacetamide)
(9d). General procedure for preparation of compounds 9 Yield 57%; white solid; mp 258-259 °C; IR (KBr, ν cm-1) 3335 (m), 3190 (m), 2260 (m),
1763 (s), 1710 (s), 1668 (s), 1502 (m); 1H NMR (400 MHz, DMSO-d6)
11.29 (1H, s, Hd),
10.20 (1H, s, NHPh)), 7.60–7.45 (4H, m, Harom.), 7.50 (1H, tt, 3J = 7.0 Hz, 4J = 1.5 Hz, Harom.),
7.40-7.30 (4H, m, Harom.), 7.05 (1H, tt, 3J = 7.5 Hz, 4J = 1.0 Hz, Harom.), 4.81 (1H, dd, 3J = 4.0
Hz and 3.5 Hz, Hx), 3.90 (1H, d, 2J = 18.0 Hz, He), 3.85 (1H, d, 2J = 18.0 Hz, Hf), 3.17 (1H, dd,
2J = 18.0 Hz, 3J = 3.5 Hz, Ha), 3.13 (1H, dd, 2J = 18.0 Hz, 3J = 4.0 Hz, Hb); 13C NMR (100
MHz, DMSO-d6) δ 183.1 (C=S), 171.3 (NH(Ph)C=O), 166.6 (C=O thioimidazole), 162.3
(NNHC=O), 138.7 (Cipso), 133.7 (Cipso), 129.0 (CHarom.), 128.8 (CHarom.), 128.7 (CHarom.),
128.5 (CHarom.), 123.4 (CHarom.), 119.1 (CHarom.), 115.1 (CN), 59.5 (CHx), 34.5 (CHaHb), 23.9
(CHeHf); HRMS m/z calcd. for C20H18N5O3S (M+H)+: 408.1131, found : 408.1130; Anal. Calcd. for C20H17N5O3S: C, 58.96; H, 4.21; N, 17.19; O, 11.78; S, 7.87. Found: C, 58.83; H, 4.26; N,
17.14; O, 12.03; S, 7.36 N-[5-[2-Anilino-2-oxoethyl)-4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-2-cyanoacetamide)
(9d). Yield 57%; white solid; mp 258-259 °C; IR (KBr, ν cm-1) 3335 (m), 3190 (m), 2260 (m),
1763 (s), 1710 (s), 1668 (s), 1502 (m); 1H NMR (400 MHz, DMSO-d6)
11.29 (1H, s, Hd),
10.20 (1H, s, NHPh)), 7.60–7.45 (4H, m, Harom.), 7.50 (1H, tt, 3J = 7.0 Hz, 4J = 1.5 Hz, Harom.),
7.40-7.30 (4H, m, Harom.), 7.05 (1H, tt, 3J = 7.5 Hz, 4J = 1.0 Hz, Harom.), 4.81 (1H, dd, 3J = 4.0
Hz and 3.5 Hz, Hx), 3.90 (1H, d, 2J = 18.0 Hz, He), 3.85 (1H, d, 2J = 18.0 Hz, Hf), 3.17 (1H, dd,
2J = 18.0 Hz, 3J = 3.5 Hz, Ha), 3.13 (1H, dd, 2J = 18.0 Hz, 3J = 4.0 Hz, Hb); 13C NMR (100
MHz, DMSO-d6) δ 183.1 (C=S), 171.3 (NH(Ph)C=O), 166.6 (C=O thioimidazole), 162.3
(NNHC=O), 138.7 (Cipso), 133.7 (Cipso), 129.0 (CHarom.), 128.8 (CHarom.), 128.7 (CHarom.),
128.5 (CHarom.), 123.4 (CHarom.), 119.1 (CHarom.), 115.1 (CN), 59.5 (CHx), 34.5 (CHaHb), 23.9
(CHeHf); HRMS m/z calcd. for C20H18N5O3S (M+H)+: 408.1131, found : 408.1130; Anal. Calcd. for C20H17N5O3S: C, 58.96; H, 4.21; N, 17.19; O, 11.78; S, 7.87. General procedure for preparation of compounds 9 Found: C, 58.83; H, 4.26; N,
17.14; O, 12.03; S, 7.36 17.14; O, 12.03; S, 7.36
2-Cyano-N'(2,5-dioxo-1-phenylpyrrolidin-3-yl)-3-phenylacrylo hydrazide (10). To a solution
of 8d (1 mmol) in ethanol (20 mL) containing a catalytic amount of piperidine, benzaldehyde (10
mmol) was added. The reaction mixture was heated at reflux for 12 hours. The resulting solid
formed was collected by filtration. Yield 58%; yellow solid; mp 163-165 °C; IR (KBr, ν cm-1)
3298 (w), 3059 (w), 1780 (s), 1708 (s), 1651 (m), 1604 (m); 1H NMR (400 MHz, DMSO-d6)
10.25 (1H, br s, Hd), 8.16 (1H, s, Hg), 7.95 (2H, m, Harom.), 7.65-7.55 (3H, m, Harom.), 7.50
(2H, t, 3J = 7.5 Hz, Harom.), 7.30 (2H, d, 3J = 7.5 Hz, Harom.), 6.09 (1H, br s, Hc); 4.28 (1H, dd, 3J
= 8.5 and 4.0 Hz, Hx), 3.10 (1H, dd, 2J = 18.0, 3J = 8.5 Hz, Ha), 2.80 (1H, dd, 2J = 18.0, 3J = 4.0
Hz, Hb); 13C NMR (100 MHz, DMSO-d6) δ 175.3 (C=O imide), 174.7 (C=O imide), 161.0
(NNHC=O), 150.8 (CH=C), 132.4 (Cipso), 132.2 (Cipso), 131.8 (CHarom.), 130.0 (CHarom.),
129.2 (CHarom.), 128.9 (CHarom.), 128.5 (CHarom.), 126.9 (CHarom.), 115.8 (CN), 104.8 (C=CH),
57.3 (CHx), 34.4 (CHaHb); HRMS m/z calcd. for C20H16N4O3: 383.1106, found : 383.1120. N-(4-(2-Anilino-2-oxoethyl)-5-oxo-1-phenyl-2-thioxopyrrolidin-3-yl)-2-cyano-3-phenyl-
acrylamide (11). A solution of compound 10 (1 mmol.) in ethanol (20 mL) was refluxed with a
large excess of phenyl isothiocyanate during 5 hours. The yellow solid formed is collected by
filtration. General procedure for preparation of compounds 9 The yellow solid formed is collected by
filtration. Yield 45%; yellow solid; mp 216-218 °C; IR (KBr, ν cm-1) 3258 (w), 3129 (w), 2220
(m), 1753 (s), 1686 (s), 1647 (m), 1595 (m), 1271 (w); 1H NMR (400 MHz, DMSO-d6) 11.62
(1H, s, Hd), 10.19 (1H, s, NHPh), 8.32 (1H, s, Hg), 7.95 (2H, d, 3J = 6.5 Hz, Harom.), 7.65-7.55
(7H, m, Harom.), 7.50 (1H, t, 3J = 7.0 Hz, Harom.), 7.45 (2H, d, 3J = 7.5 Hz, Harom.), 7.35 (2H, t, 3J
= 7.5 Hz, Harom.), 7.05 (1H, t, 3J = 7.5 Hz, Harom.), 4.87 (1H, t, 3J = 4.0 Hz, Hx), 3.20 (2H, dd, 2J =
17.9 Hz, 3J = 4.0 Hz, Ha); 3.17 (1H, dd, 2J = 17.9, 3J = 4.0 Hz, Hb); 13C NMR (100 MHz,
DMSO-d6) δ 184.7 (C=S), 170.0 (C=O thioimidazole), 166.0 (C=O amide), 160.7 (NNHC=O),
153.1 (CH=C), 138.8 (Cipso), 133.7 (Cipso), 131.3 (Cipso), 130.2 (CHarom.), 129.3 (CHarom.),
128.9 (CHarom.), 128.8 (CHarom.), 128.7 (CHarom.), 128.6 (CHarom.), 128.4 (CHarom.), 123.2
(CHarom.), 119.0(CHarom.), 115.3 (CN), 103.3 (C=CH), 59.9 (CHx), 35.0 (CHaHb); HRMS m/z
calcd. for C27H22N5O3S (M+H)+: 496.1443, found : 496.1448. ARKAT USA, Inc. Page 335 Page 335 ISSN 1551-7012 ARKIVOC 2010 (x) 328-337 General Papers General procedure for preparation of compounds 13 Found: C, 48.05; H, 3.61; N, 9.94; O, 22.82; S, 15.53. 1. He, S.; Kuang, R.; Venkatamaran, R.; Tu, J.; Truong, T. M.; Chan, H. T.; Groutas, W.
Bioorg. Med. Chem. 2000, 8, 1713. General procedure for preparation of compounds 13 To a mixture of 2-mercapto benzimidazoles 12 (1 mmol), appropriate substituted maleimide 5
(1.1 mmol) and sulphuric acid (1.1 mmol) was added glacial acetic acid (5 mL). The resulting
solution was heated under reflux for 10 hours. After cooling, the solution was treated with
NH4OH until neutralization. The resulting solid formed was collected by filtration, washed
several times with water, dried and crystallized from ethanol to give the corresponding
compounds 13a-d. 2-(3-Oxo-2,3-dihydro[1,3]thiazolo[3,2-a]benzimidazol-2-yl) acetamide (13a). Yield 43%;
white solid; mp 190-192 °C, IR (KBr, ν cm-1) 3227 (w), 1780 (s), 1708 (s); 1H NMR (400MHz,
DMSO-d6) δ 12.70 (1H, s, NH), 11.50 (1H, s, NH), 7.42 (2H, m, Harom.), 7.25 (2H, m, Harom.),
4.73 (1H, dd, 3J = 9.5 and 5.5 Hz, Hx), 3.23 (1H, dd, 2J = 18.0 Hz, 3J = 9.5 Hz, Ha), 2.95 (1H, dd,
2J = 18, 3J = 5.5 Hz, Hb); 13C NMR (100 MHz, DMSO-d6) δ 176.4 (C=O amide), 176.3 (C=O
thioimidazole), 147.5 (C=N), 143.4 (Cipso), 135.1 (Cipso), 121.9 (CHarom.), 121.2 (CHarom.), 117.5
(CHarom.), 110.5 (CHarom.), 42.6 (CHx), 37.5 (CHaHb); HRMS m/z calcd. for C11H10N3O2S
(M+H)+: 248.0504, found : 248.0494; Anal. Calcd. for C11H9N3O2S: C, 53.43; H, 3.67; N, 16.99;
O, 12.94; S, 12.97. Found: C, 53.04; H, 3.75; N, 16.80; O, 13.08; S, 12.88. 2-(3-Oxo-2,3-dihydro[1,3]thiazolo[3,2-a]benzimidazol-2-yl) acetamide (13a). Yield 43%;
white solid; mp 190-192 °C, IR (KBr, ν cm-1) 3227 (w), 1780 (s), 1708 (s); 1H NMR (400MHz,
DMSO-d6) δ 12.70 (1H, s, NH), 11.50 (1H, s, NH), 7.42 (2H, m, Harom.), 7.25 (2H, m, Harom.),
4.73 (1H, dd, 3J = 9.5 and 5.5 Hz, Hx), 3.23 (1H, dd, 2J = 18.0 Hz, 3J = 9.5 Hz, Ha), 2.95 (1H, dd,
2J = 18, 3J = 5.5 Hz, Hb); 13C NMR (100 MHz, DMSO-d6) δ 176.4 (C=O amide), 176.3 (C=O
thioimidazole), 147.5 (C=N), 143.4 (Cipso), 135.1 (Cipso), 121.9 (CHarom.), 121.2 (CHarom.), 117.5
(CHarom.), 110.5 (CHarom.), 42.6 (CHx), 37.5 (CHaHb); HRMS m/z calcd. for C11H10N3O2S
(M+H)+: 248.0504, found : 248.0494; Anal. Calcd. for C11H9N3O2S: C, 53.43; H, 3.67; N, 16.99;
O, 12.94; S, 12.97. Found: C, 53.04; H, 3.75; N, 16.80; O, 13.08; S, 12.88. N-Methyl-2-(3-oxo-2,3-dihydro[1,3]thiazolo[3,2-a]benzimidazol-2-yl)
acetamide
(13b). General procedure for preparation of compounds 13 Yield 46%; white solid; mp 156-157 °C, IR (KBr, ν cm-1) 3195 (m), 1766 (s), 1697 (s); 1H NMR
(400MHz, DMSO-d6) δ 12.61 (1H, br s, NH), 7.40 (2H, m, Harom.), 7.30 (2H, m, Harom.), 4.76
(1H, dd, 3J = 9.5 and 5.0 Hz, Hx), 3.31 (1H, dd, 2J = 18.0 Hz, 3J = 9.5 Hz, Ha), 2.90 (1H, dd, 2J =
18.0 Hz, 3J = 5.0 Hz, Hb), 2.85 (3H, s, CH3); 13C NMR (100 MHz, DMSO-d6) δ 175.4 (C=O
amide), 175.2 (C=O thioimidazole), 147.6 (C=N), 134.5 (Cipso), 132.1 (Cipso), 122.2 (CHarom.),
109.4 (CHarom.), 41.2 (CHx), 36.5 (CHaHb), 24.9 (CH3); HRMS m/z calcd. for C12H12N3O2S
(M+H)+: 262.2805, found : 262.2803; Anal. Calcd. for C12H11N3O2S: C, 55.16; H, 4.24; N,
16.08; O, 12.25; S, 12.27. Found: C, 54.59; H, 4.23; N, 15.92; O, 12.64; S, 12.60. 2-(3-Oxo-2,3-dihydro[1,3]thiazolo[3,2-a]benzimidazol-2-yl)-N-phenylacetamide (13d). Yield
42%; white solid; mp 258-260 °C, IR (KBr, ν cm-1) 3169 (m), 1751 (s), 1668 (s); 1H NMR
(400MHz, DMSO-d6) δ 12.63 (1H,
s, NH), 7.56-7.13 (9H, m, Harom.), 4.86 (1H, dd, 3J = 9.5
and 5.0 Hz, Hx), 3.46 (1H, dd, 2J = 18.0 Hz, 3J = 9.5 Hz, Ha), 3.09 (1H, dd, 2J = 18.0 Hz, 3J =
5.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ 174.5 (C=O amide), 174.2 (C=O thioimidazole),
148.9 (C=N), 138.9 (Cipso), 138.5 (Cipso), 130.8 (Cipso), 128.7 (CHarom.), 124.4 (CHarom.), 123.2
(CHarom.), 121.6 (CHarom.), 115.4 (CHarom.), 41.4 (CHx), 36.6 (CHaHb); HRMS m/z calcd. for
C17H14N3O2S (M+H)+: 324.0813, found : 324.0807; Anal. Calcd. for C17H13N3O2S: C, 48.45; H,
3.59; N, 9.97; O, 22.78; S, 15.22. Found: C, 48.05; H, 3.61; N, 9.94; O, 22.82; S, 15.53. 2-(3-Oxo-2,3-dihydro[1,3]thiazolo[3,2-a]benzimidazol-2-yl)-N-phenylacetamide (13d). Yield
42%; white solid; mp 258-260 °C, IR (KBr, ν cm-1) 3169 (m), 1751 (s), 1668 (s); 1H NMR
(400MHz, DMSO-d6) δ 12.63 (1H,
s, NH), 7.56-7.13 (9H, m, Harom.), 4.86 (1H, dd, 3J = 9.5
and 5.0 Hz, Hx), 3.46 (1H, dd, 2J = 18.0 Hz, 3J = 9.5 Hz, Ha), 3.09 (1H, dd, 2J = 18.0 Hz, 3J =
5.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ 174.5 (C=O amide), 174.2 (C=O thioimidazole),
148.9 (C=N), 138.9 (Cipso), 138.5 (Cipso), 130.8 (Cipso), 128.7 (CHarom.), 124.4 (CHarom.), 123.2
(CHarom.), 121.6 (CHarom.), 115.4 (CHarom.), 41.4 (CHx), 36.6 (CHaHb); HRMS m/z calcd. for
C17H14N3O2S (M+H)+: 324.0813, found : 324.0807; Anal. Calcd. for C17H13N3O2S: C, 48.45; H,
3.59; N, 9.97; O, 22.78; S, 15.22. References 1. He, S.; Kuang, R.; Venkatamaran, R.; Tu, J.; Truong, T. M.; Chan, H. T.; Groutas, W. Bioorg. Med. Chem. 2000, 8, 1713. ARKAT USA, Inc. ARKAT USA, Inc. ISSN 1551-7012 Page 336 Page 336 ARKIVOC 2010 (x) 328-337 General Papers 2. Fuentes, J.; Salameh, B. A. B.; Pradera, A.; Fernandez de Cordoba, F. J.; Gash, G. Tetrahedron 2006, 62, 97 and references cited therein. 3. (a) Roue, N.; Bergman, J Tetrahedron 1999, 55, 14729. (b) Cherouvrier, J. R.; Carreaux, F.; 3. (a) Roue, N.; Bergman, J Tetrahedron 1999, 55, 14729. (b) Cherouvrier, J. R.; Carreaux, F.;
Bazureau, J. P. Tetrahedron Lett. 2002, 43, 8745. Renault, S.; Bertrand, S.; Carreaux, F.;
Bazureau, J. P. J. Comb. Chem. 2007, 9, 935. (c) Porwal, S.; Chauhan, S. S.; Chauhan, P. M. S.; Shakya, N.; Verma, A.; Gupta, S. J. Med. Chem. 2009, 52, 5793. J. P. Tetrahedron Lett. 2002, 43, 8745. Renault, S.; Bertrand, S.; Carreaux, F. Bazureau, J. P. J. Comb. Chem. 2007, 9, 935. (c) Porwal, S.; Chauhan, S. S.; Chauhan, P. M. S.; Shakya, N.; Verma, A.; Gupta, S. J. Med. Chem. 2009, 52, 5793. 4. Muccioli, G. G.; Fazio, N.; Sciba, G. K. E.; Poppitz, W.; Cannata, F.; Poupaert, J. H.;
Wouters, J.; Lambert, D. M. J. Med. Chem. 2006, 49, 417. 5. Sawyers, C.; Jung, M. E.; Chen, C. D.; Ouk, S.; Welsbie, D.; Tran, C.; Wongvipat, J.; Yoo,
D. WO 2006 124118. 6. Claussner, A.; Goubet, F.; Teutsch, J. G. WO 1997 19064. Thurieau, C.; Poitout, L. WO
2001 09090. Binet, J.; Boubia, B.; Chaput, E.; Edgar, A.; Ou, K.; Ratel, P.; Samreth, S.;
Thomas, D. WO 2004 031160. Wan, Y.; Pan, S.; Zhang, G.; Wang, X.; Xie, Y. F.; Jiang, J.;
Phillips, D.; Yang, Y. WO 2009 123948. 7. Lewalter, J.; Rottmaier, L.; Merten, R.; Dunwald, W.; Schulte, B. EP 1980 0013727. 8. Bélai, I. Tetrahedron Lett. 2003, 44, 7475. Severinsen, R.; Lau, J. F.; Bondensgaard, K.;
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https://www.nature.com/articles/ncomms6758.pdf
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English
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Arteries are formed by vein-derived endothelial tip cells
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Nature communications
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cc-by
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ARTICLE Received 10 Apr 2014 | Accepted 4 Nov 2014 | Published 15 Dec 2014 Received 10 Apr 2014 | Accepted 4 Nov 2014 | Published 15 Dec 2014 Received 10 Apr 2014 | Accepted 4 Nov 2014 | Published 15 Dec 2014 Arteries are formed by vein-derived endothelial
tip cells Cong Xu1, Sana S. Hasan1, Inga Schmidt1, Susana F. Rocha1, Mara E. Pitulescu1, Jeroen Bussmann1,
Dana Meyen2, Erez Raz2, Ralf H. Adams1,3 & Arndt F. Siekmann1 Cong Xu1, Sana S. Hasan1, Inga Schmidt1, Susana F. Rocha1, Mara E. Pitulescu1, Jeroen Bussmann1,
Dana Meyen2, Erez Raz2, Ralf H. Adams1,3 & Arndt F. Siekmann1 Tissue vascularization entails the formation of a blood vessel plexus, which remodels into
arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration
and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endo-
thelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip
cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the
relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even
when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine
gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to devel-
oping arteries when in association with wild-type cells, suggesting collective migration of
endothelial cells. Together, our findings reveal specific cell migratory behaviours in the
developing blood vessel plexus and uncover a conserved mode of artery formation. 1 Max Planck Institute for Molecular Biomedicine, Roentgenstr. 20, 48149 Muenster, Germany. 2 Institute of Cell Biology, ZMBE, Von-Esmarch-Str. 56, 48149
Muenster, Germany. 3 University of Mu¨nster, Faculty of Medicine, D-48149 Mu¨nster, Germany. Correspondence and requests for materials should be
addressed to A.F.S. (email: arndt.siekmann@mpi-muenster.mpg.de). 1 NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. & 2014 Macmillan Publishers Limited. All rights reserved. These maintain the connection to the pre-
existing vasculature, are less motile and subsequently establish a
hierarchical network of arteries and veins to allow for efficient
tissue perfusion12. Further studies have investigated the dynamics
of endothelial cell migration during sprout outgrowth. Jakobsson
et al.13 showed in an embryoid body sprouting assay that
endothelial cells compete for the tip cell position. Another study
using a mouse aortic ring assay suggested that this competition
and the ensuing ‘cell-mixing’ are important for the proper
outgrowth of angiogenic sprouts14. Time-lapse imaging reveals vein cells contribute to arteries. To
detect endothelial cell movements, and arterial differentiation, we
combined Tg(fli1a:nEGFP)y7, labelling endothelial cell nuclei20
with Tg( 0.8flt1:RFP)hu5333 fish and performed time-lapse
imaging for 24 h, starting at 9 d.p.a. (Supplementary Movie 1). Representative images taken every 6 h can be seen in Fig. 2. At
this stage, the fin ray started to bifurcate and consisted of two
arteries flanked by a shared vein in the middle and two lateral
veins. We then colour coded endothelial cells located in arterial
and venous positions and followed their migratory paths
(Fig. 2b,c). This analysis revealed prominent differences in
migratory behaviours between venous- and arterial-derived
endothelial cells during tissue vascularization. Cells located in
venous
positions
migrated
more
extensively
towards
the
sprouting front than arterial-derived endothelial cells (compare
blue/white dots and tracks with pink ones in Fig. 2b,c). Furthermore, some of these vein-derived endothelial tip cells
subsequently changed their orientation, turned medially and
finally migrated against the direction of the moving vascular front
(arrows in Fig. 2c, time stamp in d, schematic drawings in e, f). Strikingly, we observed that the cells that had turned medially
formed the remodelling artery in the centre of each fin ray
(Fig. 2b, 23:45 h time point, inset). By contrast, we did not
observe endothelial cells coming from the artery in venous blood
vessels. We furthermore quantified proliferation of vein- and
artery-derived
endothelial
cells
(Supplementary
Fig. 3a,b;
Supplementary Movie 2). Whereas about 50% of venous
endothelial cells divided within 24 h, less than 20% of artery-
derived endothelial cells proliferated (Supplementary Fig. 3e). We
detected both dividing tip- and stalk cells (Supplementary Fig. 4
and Supplementary Movie 3). Thus, our time-lapse analysis of
blood vessel growth reveals a venous origin of arterial endothelial
cells and shows that distinct migratory properties of endothelial
tip cells contribute to proper blood vessel morphogenesis during
tissue regeneration. & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 T
he formation of new blood vessels is a crucial process
during
embryogenesis
and
growth1–4,
but
also
in
regenerative processes, such as wound healing and tissue
repair5. It entails the tight coordination of different cellular
processes, such as proliferation, migration and lumen formation. In the early embryo, vascular development is often stereotypical,
resulting in the formation of identical looking vascular networks6. By contrast, at later stages many vascular beds form via a plexus
intermediate, which remodels into a hierarchical network of
arteries and veins. Examples are the emerging skin blood vessels7
or the postnatally forming blood vessels of the mouse retina8. In
addition, the vasculature in regenerating tissues, for instance in
the regenerating zebrafish fin9 or during wound healing10, forms
via a plexus. T imaging of fins of double transgenic animals revealed that, in the
distal part, each fin ray contained a medially located artery, which
was flanked by two veins (Fig. 1a–d). These vessels furthermore
showed distinct topologies in respect to the bones of the fin rays. While the artery ran within the bone, the veins were located
outside of the bony rays (Supplementary Fig. 1a–g). Thus, the
vasculature of the zebrafish fin consists of a regular pattern of
arteries and veins, which can be visualized using different
transgenic zebrafish lines. g
We next amputated the fins of double transgenic animals and
observed blood vessel growth within the regenerating tissue
(Fig. 1e). In agreement with previous observations9, we detected
the formation of a dense vascular plexus at 3 days post
amputation (Fig. 1f, 3 d.p.a.). This plexus, after bifurcating,
subsequently remodelled into a single medial artery and two
lateral veins (Fig. 1f, 5–14 d.p.a., see also Supplementary Fig. 2). We could distinguish different areas within the vasculature
distally to the established artery and vein during this outgrowth
phase. Most distally, we observed a sprouting front invading the
avascular tissue, followed by a remodelling vascular plexus
(Fig. 1f’). Within this plexus, we could distinguish arterial fated
endothelial
cells
by
virtue
of
RFP
expression
(see
also
Supplementary
Fig. 2c). Therefore,
our
double
transgenic
zebrafish allowed for the detailed observation of the forming
vasculature via a plexus intermediate during tissue regeneration. In the current concepts of blood vessel formation, an initial
pro-angiogenic cue specifies endothelial tip cells, which become
motile and navigate through the avascular tissue11. Tip cells are
followed by stalk cells. & 2014 Macmillan Publishers Limited. All rights reserved. It also shows that pronounced differences
exist in endothelial cell proliferation between arteries and veins. Despite
the
insights
these
studies
provided,
they
were
performed in in vitro settings, which lack proper arterial-venous
differentiation of the forming vascular plexus and tissue perfusion. So far, in vivo imaging of the forming vasculature has been mainly
performed in transparent zebrafish embryos15–17. However, these
studies have not included vascular beds that form via a plexus
intermediate. Therefore, despite the significance of vascular plexus
formation and subsequent remodelling for tissue perfusion, we
still have a poor understanding of endothelial cell dynamics
during these processes. In particular, we do not understand how
endothelial cells coordinate the sprouting of new vessels with the
establishment of larger arteries and veins. In this study, we take advantage of the optical clarity of the
adult zebrafish fin to perform in vivo time-lapse imaging of the
complex cell migratory behaviours during blood vessel formation
in regenerating tissues. Our results show that endothelial tip cells
not only invade avascular tissues, but that they can subsequently
change their direction of migration and ultimately migrate against
the advancing vascular front. Furthermore, we show that this
behaviour is necessary for the proper formation of arteries. In
addition, we observe proliferating endothelial cells in tip and stalk
cells of venous origin, while arterial endothelial cells proliferate
less. Through genetic lineage tracing in the mouse retina, we
provide evidence that this mode of artery formation is conserved
in other vascular beds that form via a plexus intermediate. We
implicate the chemokine receptor Cxcr4a in regulating these
migratory behaviours and suggest that a tissue-intrinsic mode is
responsible for the generation of a Cxcl12a chemokine gradient. We finally demonstrate through cell transplantation experiments
that endothelial cells show a collective cell migration behaviour. Results
l
d Blood vessels regenerate via an intermediate vascular plexus. To visualize regenerating blood vessels, we analysed vascular
dynamics during fin regeneration in adult transgenic zebrafish. In
Tg(fli1a:EGFP)y1 zebrafish, all endothelial cells are labelled by
EGFP expression18, while, in Tg( 0.8flt1:RFP)hu5333 fish, arterial
endothelial cells are labelled by high RFP expression19. Confocal NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
014 Macmillan Publishers Limited All rights reserved Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other Inset (f0) shows remodelling vascular plexus, which is located proximal
to the sprouting front and distally to the established arteries and veins. Scale bar in a is 80 mm, in a0 is 20 mm and 200 mm in f. Representative images from
a total of three zebrafish imaged per stage. 0 d.p.a. 3 d.p.a. 7 d.p.a. 14 d.p.a. 5 d.p.a. A
V
V
Dorsal
Ventral
Distal
Proximal
Regenerated Fin
Area imaged
Overlay
Distal
Proximal
Lobes
Cleft
Tg(–0.8flt1:RFP)hu5333
Tg(fli1a:EGFP)y1
Ray1
Ray2
Remodelling
plexus
Sprouting
front
Established
artery/vein
Bifurcation
Bony ray
′ Dorsal
Ventral
Distal
Proximal
Regenerated Fin
Area imaged
Distal
Proximal
Lobes
Cleft
Bony ray al
Regenerated Fin Dorsal Figure 1 | Visualizing blood vessels in the regenerating zebrafish fin. (a,a0) Tg(fli1a:EGFP)y1 labels all fin blood vessels. (b,b0) Tg( 0.8flt1:RFP)hu5333
preferentially labels arterial endothelial cells. (c,d) Schematic drawings of the zebrafish fin vasculature, indicating the ray bifurcation, arteries (A) and veins
(V). (e) Schematic of zebrafish fin, indicating plane of amputation and imaged area. (f) Overlay of Tg(fli1a:EGFP)y1 and Tg( 0.8flt1:RFP)hu5333 at different
stages of fin regeneration highlighting the medially located arterial cells in yellow. Inset (f0) shows remodelling vascular plexus, which is located proximal
to the sprouting front and distally to the established arteries and veins. Scale bar in a is 80 mm, in a0 is 20 mm and 200 mm in f. Representative images from
a total of three zebrafish imaged per stage. cells within the vasculature after 24 or 48 h of tamoxifen
injection (Fig. 3e–g, 48 h time point shown). We observed a
striking bias of EGFP-positive cells towards the developing
arteries, while we did not find a significant contribution of
EGFP-positive cells to developing veins (Fig. 3g, arteries are
marked by arrows, veins by arrowheads). After 48 h, about 15% of
the arterial vessel area was occupied by EGFP-positive cells,
while only about 1.5% of the venous vessel area was occupied by
EGFP-positive cells (Fig. 3h). This suggests that similar to the
vasculature in the regenerating zebrafish fin, endothelial tip cells
in the mouse retina can contribute to nascent arteries, while
they
only
marginally
contribute
to
forming
veins. EdU
incorporation
experiments
in
recombined
endothelial
cells
and their descendants indicated proliferation of these cells vascular beds that are established via a plexus intermediate
occurs by a similar mechanism. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. & 2014 Macmillan Publishers Limited. All rights reserved. Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 0 d.p.a. 3 d.p.a. 7 d.p.a. 14 d.p.a. 5 d.p.a. Tg(fli1a:EGFP)y1
Tg(–0.8flt1:RFP)hu5333
Vein
Vein
A
V
V
Dorsal
Ventral
Distal
Proximal
Regenerated Fin
Area imaged
Artery
Overlay
Distal
Proximal
Lobes
Cleft
Tg(–0.8flt1:RFP)hu5333
Tg(fli1a:EGFP)y1
Ray1
Ray2
Remodelling
plexus
Sprouting
front
Established
artery/vein
Bifurcation
Artery
Bony ray
′
′
′
Figure 1 | Visualizing blood vessels in the regenerating zebrafish fin. (a,a0) Tg(fli1a:EGFP)y1 labels all fin blood vessels. (b,b0) Tg( 0.8flt1:RFP)hu5333
preferentially labels arterial endothelial cells. (c,d) Schematic drawings of the zebrafish fin vasculature, indicating the ray bifurcation, arteries (A) and veins
(V). (e) Schematic of zebrafish fin, indicating plane of amputation and imaged area. (f) Overlay of Tg(fli1a:EGFP)y1 and Tg( 0.8flt1:RFP)hu5333 at different
stages of fin regeneration highlighting the medially located arterial cells in yellow. Inset (f0) shows remodelling vascular plexus, which is located proximal
to the sprouting front and distally to the established arteries and veins. Scale bar in a is 80 mm, in a0 is 20 mm and 200 mm in f. Representative images from
a total of three zebrafish imaged per stage ′ ′ 0 d.p.a. 3 d.p.a. 7 d.p.a. 14 d.p.a. 5 d.p.a. Tg(fli1a:EGFP)y1
Tg(–0.8flt1:RFP)hu5333
Vein
Vein
A
V
V
Dorsal
Ventral
Distal
Proximal
Regenerated Fin
Area imaged
Artery
Overlay
Distal
Proximal
Lobes
Cleft
Tg(–0.8flt1:RFP)hu5333
Tg(fli1a:EGFP)y1
Ray1
Ray2
Remodelling
plexus
Sprouting
front
Established
artery/vein
Bifurcation
Artery
Bony ray
′ 0 d.p.a. 3 d.p.a. 7 d.p.a. 14 d.p.a. 5 d.p.a. Tg(fli1a:EGFP)y1
Tg(–0.8flt1:RFP)hu5333
A
V
V
Dorsal
Ventral
Distal
Proximal
Regenerated Fin
Area imaged
y
Overlay
Distal
Proximal
Lobes
Cleft
Tg(–0.8flt1:RFP)hu5333
Tg(fli1a:EGFP)y1
Ray1
Ray2
Remodelling
plexus
Sprouting
front
Established
artery/vein
Bifurcation
Bony ray
′
Figure 1 | Visualizing blood vessels in the regenerating zebrafish fin. (a,a0) Tg(fli1a:EGFP)y1 labels all fin blood vessels. (b,b0) Tg( 0.8flt1:RFP)hu5333
preferentially labels arterial endothelial cells. (c,d) Schematic drawings of the zebrafish fin vasculature, indicating the ray bifurcation, arteries (A) and veins
(V). (e) Schematic of zebrafish fin, indicating plane of amputation and imaged area. (f) Overlay of Tg(fli1a:EGFP)y1 and Tg( 0.8flt1:RFP)hu5333 at different
stages of fin regeneration highlighting the medially located arterial cells in yellow. Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other regenerating wild-type fin vessels
Vessel front
Bifurcated fin rays
Regenerated
vessels
Vessel regeneration
Vein
Artery
Shared vein
Vein
Artery
Shared vein
Regenerated artery
Lateral vein derived
Artery derived
Medial vein derived
Artery
Time stamp
Vein
Vein
Vein
Artery
Artery
0 h
24 h
00:00:00.000
06:00.00.000
12:00.00.000
18:00.00.000
23:45:00.000 Vessel front
Bifurcated fin rays
Vein
Artery
Shared vein Regenerated
vessels
Vessel regeneration
Vein
Artery
Shared vein
Regenerated artery Time stamp
0 h
24 h Figure 2 | Time-lapse imaging of regenerating blood vessels reveals contribution of vein-derived tip cells to the forming artery. (a) Still images taken
every 6 h from a 24-h time-lapse movie on 9 d.p.a. fin regenerate of wild-type fish. (b) Tracking of individual cells deriving form the lateral vein (white dots),
the artery (pink dots) or the medial vein (blue dots). While arterial cells hardly contribute to the advancing front, vein-derived cells contribute to the newly
forming artery (inset at 23:45 h time point). (c) Tracks of labelled cells in (b). White arrows indicate migration paths of lateral vein-derived endothelial cells,
while blue arrows indicate migration paths of medial vein-derived endothelial cells. Note change in migration direction. (d) Time stamp of tracked
endothelial cells. (e) Schematic drawing of vessel front in the regenerated fin of wild-type fish at 10 d.p.a. Arteries and veins are indicated. (f) Schematic drawing of wild-type fin vasculature regenerated within 24 h from 9 d.p.a. Scale bar, 100 mm; d.p.a. ¼ days post amputation. Representative
movie of a total of three movies is shown. (Supplementary Fig. 5), accounting for an increase in labelled
endothelial cells at later time points. (Supplementary Fig. 5), accounting for an increase in labelled
endothelial cells at later time points. Defects of cxcr4a and cxcl12a mutants in arterial patterning. To address the relevance of cxcr4a in endothelial cell migration,
we
analysed
the
regenerating
vasculature
in
homozygous
cxcr4aum20 mutant zebrafish27. Although the total endothelial cell
numbers were not different between wild type and cxcr4aum20
mutant fish at any of the analysed time points, we detected a
strong reduction in endothelial cell numbers, vascular coverage
and vessel length in the centre of the fin rays in cxcr4aum20
mutants (Supplementary Fig. 7), suggesting that artery formation
was severely impaired (Fig. 4d–f, 14 d.p.a. time point shown,
Supplementary Tables 1 and 2). We also observed ectopically
located Tg( 0.8flt1:RFP)hu5333-positive arterial cells in mutant
fish, often running in parallel with veins (Fig. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other We chose to investigate the
mouse retinal vasculature due to the availability of genetic tools
that allow for lineage tracing of different cell populations. To
genetically label endothelial tip cells located at the vascular front,
we generated double transgenic mice containing a R26-tomato-
EGFP reporter21 in conjunction with a tamoxifen-inducible
iCre-ERT2 cassette under the control of the endothelial-specific
molecule-1 (Esm-1) promoter22 (Fig. 3a). Previous studies
showed Esm-1 mRNA expression mainly in endothelial tip
cells23,24. Accordingly,
we
observed
specific
expression
of
EGFP in endothelial cells at the vascular front 12 h after
tamoxifen injection (Fig. 3b–d, bracket). To track labelled
cells over time, we analysed the location of EGFP expressing 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Dot tracking
Dot and line
cell tracking
No tracking
Tg(fli1a:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
24-h live imaging of 9-d.p.a. regenerating wild-type fin vessels
Vessel front
Bifurcated fin rays
Regenerated
vessels
Vessel regeneration
Vein
Artery
Shared vein
Vein
Artery
Shared vein
Regenerated artery
Lateral vein derived
Artery derived
Medial vein derived
Artery
Time stamp
Vein
Vein
Vein
Artery
Artery
0 h
24 h
00:00:00.000
06:00.00.000
12:00.00.000
18:00.00.000
23:45:00.000
Figure 2 | Time-lapse imaging of regenerating blood vessels reveals contribution of vein-derived tip cells to the forming artery. (a) Still images taken
every 6 h from a 24-h time-lapse movie on 9 d.p.a. fin regenerate of wild-type fish. (b) Tracking of individual cells deriving form the lateral vein (white dots),
the artery (pink dots) or the medial vein (blue dots). While arterial cells hardly contribute to the advancing front, vein-derived cells contribute to the newly
forming artery (inset at 23:45 h time point). (c) Tracks of labelled cells in (b). White arrows indicate migration paths of lateral vein-derived endothelial cells,
while blue arrows indicate migration paths of medial vein-derived endothelial cells. Note change in migration direction. (d) Time stamp of tracked
endothelial cells. (e) Schematic drawing of vessel front in the regenerated fin of wild-type fish at 10 d.p.a. Arteries and veins are indicated. (f) Schematic drawing of wild-type fin vasculature regenerated within 24 h from 9 d.p.a. Scale bar, 100 mm; d.p.a. ¼ days post amputation. Representative
movie of a total of three movies is shown. 24-h live imaging of 9-d.p.a. regenerating wild-type fin vessels Dot tracking
Dot and line
cell tracking
No tracking
Tg(fli1a:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
24-h live imaging of 9-d.p.a. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 RFP
EGFP
iCre-ERT2
EGFP
+Tamoxifen
at P4
Ubiquitous promoter
Esm1 promoter
Ubiquitous promoter
EGFP
V A
A
A
V A
IsolectinB4
IsolectinB4
Overlay
EGFP
Overlay
48 h after tamox. 12 h after tamox. V
V
P6
P6
Vein
Artery
% of vessel area occupied
by EGFP-positive cells
12 h
24 h
48 h
3
9
15
21
*
*
NS
100 μm
Figure 3 | Genetic lineage tracing of tip cells in the mouse retinal vascular plexus. (a) Schematic of genetic lineage tracing and transgenic lines used. (b) Isolectin B4 staining of the retinal vasculature at P6. Bracket indicates vascular front. (c) Location of EGFP expressing cells at the vascular front 12 h after
tamoxifen administration. (d) Overlay of isolectin B4 and EGFP channels. Remodelling arteries (A) and veins (V) are marked. (e) Isolectin B4 staining
of the retinal vasculature at P6. (f) Location of EGFP expressing cells at the vascular front 48 h after tamoxifen administration. (g) Overlay of isolectin
B4 and EGFP channels. Remodelling arteries (A) and veins (V) are marked. Arrows indicate EGFP-positive endothelial cells in arteries, while arrowheads
indicate EGFP-negative veins. (h) Percentage of EGFP-expressing endothelial cells in arteries and veins at different time points after tamoxifen
administration. Scale bar, 100 mm. For each time point five mice were analysed. *Po0.05; t-test, error bars indicate s.e.m. RFP
EGFP
iCre-ERT2
EGFP
+Tamoxifen
at P4
Ubiquitous promoter
Esm1 promoter
Ubiquitous promoter Vein
Artery
% of vessel area occupied
by EGFP-positive cells
12 h
24 h
48 h
3
9
15
21
*
*
NS IsolectinB4
IsolectinB4
P6
P6 A
V A
Overlay
V EGFP
12 h after tamox. Ov Ov EGFP
48 h after tamox. P6 48 h after tamox. Figure 3 | Genetic lineage tracing of tip cells in the mouse retinal vascular plexus. (a) Schematic of genetic lineage tracing and transgenic lines used. (b) Isolectin B4 staining of the retinal vasculature at P6. Bracket indicates vascular front. (c) Location of EGFP expressing cells at the vascular front 12 h after
tamoxifen administration. (d) Overlay of isolectin B4 and EGFP channels. Remodelling arteries (A) and veins (V) are marked. (e) Isolectin B4 staining
of the retinal vasculature at P6. (f) Location of EGFP expressing cells at the vascular front 48 h after tamoxifen administration. (g) Overlay of isolectin
B4 and EGFP channels. Remodelling arteries (A) and veins (V) are marked. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Arrows indicate EGFP-positive endothelial cells in arteries, while arrowheads
indicate EGFP-negative veins. (h) Percentage of EGFP-expressing endothelial cells in arteries and veins at different time points after tamoxifen
administration. Scale bar, 100 mm. For each time point five mice were analysed. *Po0.05; t-test, error bars indicate s.e.m. mutants specifically affected forming arteries, the proliferative
defects were apparent both in arteries and veins. migration to the gonad and in collective cell migration during the
formation of the lateral line30. To address whether Cxcr4a
signalling within endothelial cells was necessary for single cell or
collective
cell
migration,
we
generated
chimeric
embryos
consisting of cxcr4aum20 mutant and wild-type cells and let
them grow to adulthood. To visualize donor-derived endothelial
cells, we transplanted double Tg(fli1a:EGFP)y1; Tg( 0.8flt1:
RFP)hu5333 cells into single Tg( 0.8flt1:RFP)hu5333 hosts. We
subsequently analysed fish that had donor- and host-derived
blood vessels in their fins after amputation (Fig. 6a). cxcr4aum20 mutant endothelial cells show defective migration. To understand how Cxcr4a signalling influences artery formation,
we performed time-lapse imaging of cxcr4aum20 mutant fish for
24 h, starting at 9 d.p.a. These studies revealed that in the absence
of Cxcr4a signalling, endothelial tip cells failed to turn medially,
and instead continued to migrate as a vascular front (Fig. 5a–c,
time stamp in d, schematic drawings in e,f, Supplementary Movie
5, and compare with Fig. 2a–f). Consequently, no artery formed
in the centre of the regenerating fin ray. By contrast, we observed
Tg( 0.8flt1:RFP)hu5333-positive endothelial cells adjacent to the
laterally located veins. Thus, Cxcr4a signalling is indispensible
during the coordination of the complex endothelial cell move-
ments that are necessary for artery formation during tissue
regeneration. Of a total of about 5,200 transplanted embryos, 14 showed
donor-derived cells in different adult fins (Supplementary
Table 3). We observed two distinct patterns of donor cell
contribution: either the vasculature of an entire given fin ray was
donor-derived (Fig. 6b–d00, arrows mark artery) or only a fraction
of venous and/or arterial cells was donor-derived (Fig. 6e–j00). In
situations, where the entire fin ray vasculature was donor-derived,
transplanted endothelial cells behaved according to the donor
genotype. Wild-type cells transplanted to either wild-type hosts
(Fig. 6b–b00) or to cxcr4aum20 mutants (Fig. 6c–c00) formed
normal arteries, while cxcr4aum20 mutant endothelial cells
transplanted into wild-type hosts did not form arteries in the
proper location (Fig. 6d–d00). These observations suggest that Cell transplantations reveal collective cell migration. Tip cells in the mouse retinal plexus contribute to arteries. We
next wanted to know whether blood vessel formation in other 4e, white
arrowheads). We observed similar defects in arterial patterning
in cxcl12at30516 mutant zebrafish28 (Fig. 4g–i, 14 d.p.a. time point
shown, Supplementary Fig. 8, Supplementary Tables 1 and 2). By
contrast,
cxcl12bmu100
mutant
zebrafish15
did
not
show
differences in arterial patterning when compared with wild-type
siblings (Supplementary Fig. 9, Supplementary Tables 1 and 2). Despite these patterning defects, differentiation of arterial cells
appeared unaffected, as we observed Tg( 0.8flt1:RFP)hu5333-
positive endothelial cells in cxcr4aum20 mutants, albeit in
ectopic
locations. Thus,
Cxcr4a-Cxcl12a
signalling
appears
to be necessary for proper arterial morphogenesis, but not
differentiation. Quantifying endothelial cell proliferation in
cxcr4aum20
mutants
(Supplementary
Fig. 3c,d
and
Supplementary Movie 4), we detected a reduction in endothelial
cell proliferation in both veins and arteries in these mutants by
about 50% (Supplementary Fig. 3e). Therefore, while the
endothelial
cell
migratory
defects
observed
in
cxcr4aum20 Expression of cxcr4a and its ligands cxcl12a and cxcl12b. We
reasoned that pro-migratory genes specifically expressed by
endothelial tip cells might control the observed distinct migratory
behaviours of arterial-fated endothelial cells. We and others
had previously shown that the chemokine receptor Cxcr4 was
specifically expressed in endothelial tip cells, both in mouse23,24
and zebrafish (cxcr4a)15. We therefore analysed expression of
cxcr4a during fin regeneration. To achieve cellular resolution,
we generated transgenic animals expressing fluorescent proteins
under the control of the cxcr4a, cxcl12a and cxcl12b promoters25,
respectively (see Methods). To visualize the vasculature in
addition to transgene expression, we crossed these fish into the
Tg( 0.8flt1:RFP)hu5333
background. In
Tg(cxcr4a:YFP)mu104
zebrafish, we observed YFP expression mainly in the centre of
regenerating fin rays, where it overlapped with individual
endothelial cells in a salt and pepper distribution (Fig. 4a–a00,
compare
cells
marked
by
arrows
and
arrowheads). Tg(cxcl12a:CFP)mu146 fish showed expression of CFP in non-
endothelial cells located in the centre of regenerating fin rays, in
the
area
of
the
forming
artery
(Fig. 4b–b00). Finally, in
Tg(cxcl12b:YFP)mu105 fish,
we observed YFP expression in
filamentous structures, most probably nerve fibres (Fig. 4c–c00)26. These staining patterns were mirrored by in situ hybridization
results (Supplementary Fig. 6). Thus, mRNAs for the chemokine
receptor Cxcr4a and for the ligands Cxcl12a and Cxcl12b are
expressed in the regenerating zebrafish fin. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. 4 4 ARTICLE & 2014 Macmillan Publishers Limited. All rights reserved. |
|
|
& 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Twenty individual fin rays from five fish were analysed. Transgenic reporter fish, 2 d.p.a. Tg(cxcr4a:YFP)mu104
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(cxcl12b:YFP)mu105
Tg(cxcl12a:CFP)mu146
centre
centre
14-d.p.a. regenerated vessels
Tg(fli1a:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
cxcl12at30516
cxcr4aum20
Sibling
Sibling
′
′
′′
′′
′
′′ EC number (total)
EC number (centre)
Vessel length (centre)
Vessel coverage (centre)
Sib
Mut
0
50
100
150
200
250
300
NS
Sib
Mut
0
10
20
30
40
50
60
70
****
cxcr4aum20 EC number (total)
Vessel coverage (centre)
Sib
Mut
0
50
100
150
200
250
300
NS
cxc EC number (centre)
Vessel length (centre)
Sib
Mut
0
10
20
30
40
50
60
70
****
cr4aum20 Vessel coverage (centre) Vessel length (centre) Vessel length (centre)
el coverage (centre)
Sib
Mut
0
200
400
600
800
1,000
****
μm
Sib
Mut
** Vessel coverage (centre)
Sib
Mut
0
2,000
4,000
6,000
8,000
10,000
**
μm2 Sib
Mut
0
200
400
600
800
000
**** EC number (centre)
V
l l
th (
t
)
Sib
Mut
0
10
20
30
40
50
60
70
****
cl12at30516 EC number (total)
Sib
Mut
0
50
100
150
200
250
300
NS
cxcl EC number (total)
EC num
V
l
(
t
)
V
l l
Sib
Mut
0
50
100
150
200
250
300
NS
Sib
0
10
20
30
40
50
60
70
cxcl12at30516 EC number (centre) Vessel length (centre) Vessel coverage (centre) Vessel coverage (centre)
Sib
Mut
0
2,000
4,000
6,000
8,000
10,000
****
μm2 Vessel length (centre)
Sib
Mut
0
200
400
600
800
1,000
μm
**** Figure 4 | The chemokine receptor cxcr4a and its ligand cxcl12a are required for proper arterial patterning during fin regeneration. Dashed lines
indicate amputation planes in a–c00. (a) Tg(cxcr4a:YFP)mu104 fish reveal YFP expression in individual cells in the centre of the regenerating fin ray. (a0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (a00) Overlay of red and yellow channels reveals expression of YFP in individual endothelial cells (arrows),
while neighbouring endothelial cells do not express YFP (arrowheads). (b) CFP expression in the centre of the regenerating fin in Tg(cxcl12a:CFP)mu146
transgenic zebrafish. (b0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (b00) Overlay of red and blue channels. (c) YFP expression in filamentous structures
(arrowheads) extending into the regenerating fin in Tg(cxcl12b:YFP)mu105 transgenic zebrafish. (c0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (c00) Overlay of red and yellow channels. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Transgenic reporter fish, 2 d.p.a. Tg(cxcr4a:YFP)mu104
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(–0.8flt1:RFP)hu5333
Overlay
Tg(cxcl12b:YFP)mu105
Tg(cxcl12a:CFP)mu146
centre
centre
14-d.p.a. regenerated vessels
Tg(fli1a:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
cxcl12at30516
cxcr4aum20
Sibling
Sibling
EC number (total)
EC number (centre)
Vessel coverage (centre)
Vessel length (centre)
Sib
Mut
0
50
100
150
200
250
300
NS
Sib
Mut
0
10
20
30
40
50
60
70
****
Sib
Mut
0
2,000
4,000
6,000
8,000
10,000
****
Sib
Mut
0
200
400
600
800
1,000
μm
****
EC number (total)
EC number (centre)
Vessel length (centre)
Vessel coverage (centre)
Sib
Mut
0
50
100
150
200
250
300
NS
Sib
Mut
0
10
20
30
40
50
60
70
****
Sib
Mut
0
200
400
600
800
1,000
****
μm
Sib
Mut
0
2,000
4,000
6,000
8,000
10,000
**
μm2
cxcr4aum20
cxcl12at30516
μm2
′
′
′′
′′
′
′′
Figure 4 | The chemokine receptor cxcr4a and its ligand cxcl12a are required for proper arterial patterning during fin regeneration. Dashed lines
indicate amputation planes in a–c00. (a) Tg(cxcr4a:YFP)mu104 fish reveal YFP expression in individual cells in the centre of the regenerating fin ray. (a0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (a00) Overlay of red and yellow channels reveals expression of YFP in individual endothelial cells (arrows),
while neighbouring endothelial cells do not express YFP (arrowheads). (b) CFP expression in the centre of the regenerating fin in Tg(cxcl12a:CFP)mu146
transgenic zebrafish. (b0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (b00) Overlay of red and blue channels. (c) YFP expression in filamentous structures
(arrowheads) extending into the regenerating fin in Tg(cxcl12b:YFP)mu105 transgenic zebrafish. (c0) Tg( 0.8flt1:RFP)hu5333 labelled blood vessels. (c00) Overlay of red and yellow channels. (d) Fin vasculature in wild-type sibling 14 d.p.a. Arrows indicate Tg( 0.8flt1:RFP)hu5333-positive endothelial cells in
the centre of the fin ray. (e) cxcr4aum20 mutant; arrowheads indicate ectopic Tg( 0.8flt1:RFP)hu5333-positive endothelial cells. (f) Quantification of artery
formation defects in cxcr4aum20 mutants. Endothelial cell numbers, vessel coverage and length are reduced in the centre. (g) Fin vasculature in wild-type
sibling 14 d.p.a. Arrows indicate Tg( 0.8flt1:RFP)hu5333-positive endothelial cells in the centre of the fin ray. (h) cxcl12at30516 mutant; arrowheads
indicate ectopic Tg( 0.8flt1:RFP)hu5333-positive endothelial cells. Scale bar (a–c,e,h), 100mm. (i) Quantification of artery formation defects in cxcl12at30516
mutants. Endothelial cell numbers, vessel coverage and length are reduced in the centre. NS, not significant, **Po0.01, ****Po0.0001; Mann–Whitney
U-test. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 (d) Fin vasculature in wild-type sibling 14 d.p.a. Arrows indicate Tg( 0.8flt1:RFP)hu5333-positive endothelial cells in
the centre of the fin ray. (e) cxcr4aum20 mutant; arrowheads indicate ectopic Tg( 0.8flt1:RFP)hu5333-positive endothelial cells. (f) Quantification of artery
formation defects in cxcr4aum20 mutants. Endothelial cell numbers, vessel coverage and length are reduced in the centre. (g) Fin vasculature in wild-type
sibling 14 d.p.a. Arrows indicate Tg( 0.8flt1:RFP)hu5333-positive endothelial cells in the centre of the fin ray. (h) cxcl12at30516 mutant; arrowheads
indicate ectopic Tg( 0.8flt1:RFP)hu5333-positive endothelial cells. Scale bar (a–c,e,h), 100mm. (i) Quantification of artery formation defects in cxcl12at30516
mutants. Endothelial cell numbers, vessel coverage and length are reduced in the centre. NS, not significant, **Po0.01, ****Po0.0001; Mann–Whitney
U-test. Twenty individual fin rays from five fish were analysed. cxcr4a functions endothelial cell autonomously during artery
formation in regenerating fins. of Cxcl12a-mCherry (Fig. 7b, Supplementary Table 4). Surpris-
ingly, we detected strong accumulation of the fusion protein in
newly forming bone segments in the regenerates of both siblings
and mutants (Fig. 7b, white arrowheads). We did not detect
Cxcl12a-mCherry in the uninjured bone proximal to the ampu-
tation plane (Fig. 7b, proximal to the dotted line). The remainder
of the regenerated fin, especially the most distal tissues, displayed
faint Cxcl12a-mCherry signal, which accumulated in a dot-like
pattern. Thus, although ubiquitously expressed under the control
of a heat-shock promoter, Cxcl12a-mCherry accumulated in a
tissue-restricted manner in regenerating fin tissue, with higher
concentration detected within newly forming bones. In mosaic
cases
(Fig. 6e–j00),
we observed
that
donor
(Fig. 6e,g,i,f–f00,h–h00,j–j00, white arrowheads) and host cells
(Fig. 6e0,g0,i0,f–f00,h–h00,j–j00, blue arrowheads) could contribute
to arteries, irrespective of their genotypes. These findings suggest
that endothelial cells of the forming artery engage in collective
migration, which allows for wild-type cells to rescue the
migratory phenotype of cxcr4aum20 mutant cells. They further-
more suggest that, while showing migration defects, cxcr4aum20-
deficient endothelial cells can properly differentiate into the
arterial lineage. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Migrating
cells can either follow guidance cues individually or migrate as
groups of cells29. Cxcr4 signalling in zebrafish has been described
to play a role in single-cell migration regulating germ cell 5 ARTICLE NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. Discussion
I
hi
d In the mouse skin, only endothelial cells that are in
close proximity to VEGF providing nerves can differentiate into
arteries7,36. One reason for this difference might be that, during
regeneration, tissue VEGF levels are high enough to allow for
arterial differentiation throughout the entire fin. chemokine receptor Cxcr4 (refs 11,23,24,33). Although activation
of Notch signalling via Dll4 in neighbouring cells leads to the
suppression of the tip cell phenotype34, Cxcr4a induction would
change the tip cells’ responsiveness to guidance cues present in the
environment. As we show that the chemokine ligand Cxcl12a is
likely enriched in the central area of the fin ray, Cxcr4a expressing
endothelial cells would respond to this chemokine and change
their direction of migration towards the centre of the ray, allowing
for proper arterial morphogenesis. Accordingly, our cxcr4aum20
mutant analysis revealed that in this setting, endothelial cells failed
to turn medially, but continued to migrate in the direction of the
avascular area. Therefore, Cxcr4a-mediated medial migration of
tip cells would be necessary to balance the directionality imposed
on the outgrowing vasculature by VEGF. g
g
y
A dependence on Cxcr4 signalling for artery formation was
recently observed in the mouse skin vasculature. In this setting,
nerves secrete Cxcl12, while a subset of endothelial cells in the
surrounding vascular plexus expresses Cxcr4 (ref. 35). In the
absence of Cxcr4 signalling vessel nerve alignment is disrupted. This
suggests that in the developing mouse skin vasculature, endothelial
cell migration might occur similarly to what we observe in the
regenerating zebrafish fin. In addition, Strasser et al.24 observed
that, in retinae treated with the Cxcr4 inhibitor AMD3100, long
sprouts formed that failed to connect laterally to neighbouring
sprouts, a phenotype that corresponds to our observations during
the live imaging of cxcr4aum20 mutants. This, together with our
genetic lineage tracing of tip cells in the retina, suggests that the
morphogenetic movements we observed during vascular plexus
formation in the regenerating fin might be a general principle. Our data furthermore show that endothelial cell proliferation
during vascular plexus formation differs between arterial and
venous endothelial cells. We detect higher proliferation in venous
endothelial cells. These findings are in agreement with previous
studies in the mouse retina, which found persistent endothelial cell
proliferation in veins, but not in arteries during vascular
maturation37. Therefore, veins appear to constitute the main
source of endothelial cells for newly forming blood vessels. Discussion
I
hi
d Note persistent migration of tracked endothelial cells in the direction of the growing
regenerate. (d) Time stamp of tracked endothelial cells. (e) Schematic drawing of vessel front in the regenerated fin of cxcr4aum20 fish at 10 d.p.a. Arteries
and veins are indicated. (f) Schematic drawing of cxcr4aum20 fin vasculature regenerated within 24 h from 9 d.p.a. Scale bar, 100 mm; d.p.a., days post
amputation. Representative movie of a total of three movies. 24-h live imaging of 9-d.p.a. regenerating cxcr4a mutant fin vessels Vessel front
Bifurcated fin rays
Vein
Artery
Shared vein Regenerated
vessels
Vessel regeneration
Vein
Artery
Shared vein
Regenerated artery 0 h
24 h
Time stamp Figure 5 | Time-lapse imaging of regenerating blood vessels in cxcr4aum20 mutant fish. (a) Still images taken every 6 h from a 24 h time-lapse movie on
9 d.p.a. fin regenerate of cxcr4aum20 fish. (b) Tracking of individual cells deriving form the lateral vein (white dots), the artery (pink dots) or the
medial vein (blue dots). (c) Tracks of labelled cells in (b). White arrows indicate migration paths of lateral vein-derived endothelial cells, while blue arrows
indicate migration paths of medial vein-derived endothelial cells. Note persistent migration of tracked endothelial cells in the direction of the growing
regenerate. (d) Time stamp of tracked endothelial cells. (e) Schematic drawing of vessel front in the regenerated fin of cxcr4aum20 fish at 10 d.p.a. Arteries
and veins are indicated. (f) Schematic drawing of cxcr4aum20 fin vasculature regenerated within 24 h from 9 d.p.a. Scale bar, 100 mm; d.p.a., days post
amputation. Representative movie of a total of three movies. unaffected in cxcr4aum20 mutant zebrafish. We readily observe
Tg( 0.8flt1:RFP)hu5333-positive blood vessels in cxcr4aum20 mutant
fins, albeit in random locations. Proper arterial differentiation of
endothelial cells lacking Cxcr4a function is furthermore supported
by our cell transplantation experiments, where cxcr4aum20 mutant
cells could contribute to arteries when surrounded by wild-type
cells. This is in line with observations on Cxcr4-expressing
endothelial cells from the skin of mouse embryos. In this setting,
Cxcl12 exposure did not lead to arterial differentiation, while
VEGF-A exposure could induce the expression of the arterial
marker gene ephrinB2 also in Cxcr4 mutant endothelial cells35. One
important difference between the mouse skin and the regenerating
fin vasculature is that, in the fin, arterial differentiation does not
seem to depend on the proper location of the arterial-fated
endothelial cells. Discussion
I
hi
d Ubiquitous overexpression of Cxcl12a rescues cxcl12a mutants. Our analysis of cxcl12a expression during fin regeneration
(Fig. 4b–b00) suggested that Cxcl12a might be distributed in a
graded manner with higher concentrations in the centre of the
fin. To determine whether graded Cxcl12a distribution was
indeed necessary for proper artery formation, we ubiquitously
overexpressed
Cxcl12a-mCherry
for
14
days
during
fin
regeneration
using
Tg(Cry.kop.HSP:mutSDF1a.mCherry. globin30UTR)mu4 fish. Overexpression of Cxcl12a-mCherry did
not affect fin regeneration in wild-type siblings (Fig. 7, compare
values for sibling in Fig. 7a with heat-shock sibling fish in
Fig. 7b). Importantly, the arterial defects in cxcl12at30516 mutant
zebrafish were completely rescued by ubiquitous overexpression In this study, we have analysed endothelial cell migration during
blood vessel plexus formation and remodelling in an in vivo
setting: the regenerating zebrafish fin. Our results show that
endothelial cells within growing blood vessel sprouts initially
invade the avascular area. The signalling process that is most likely
responsible for this behaviour is the VEGF pathway. During fin
regeneration, vegf-a mRNA is being expressed in the fin regenerate
and inhibition of VEGF signalling abolishes fin vessel outgrowth31. In addition, a VEGF gradient was postulated to drive retinal
angiogenesis32. Studies in the mouse retina and in cell culture have
shown that the exposure to VEGF can lead to the induction of tip
cell specific genes, such as the Notch ligand Dll4 and the NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. 6 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 0 h
24 h
Dot tracking
Dot and line
cell tracking
No tracking
Vessel front
Bifurcated fin rays
Vein
Artery
Shared vein
Regenerated
vessels
Vessel regeneration
Vein
Artery
Shared vein
Regenerated artery
Lateral vein derived
Artery derived
Medial vein derived
Time stamp
Vein
Vein
Vein
Artery
Artery
24-h live imaging of 9-d.p.a. regenerating cxcr4a mutant fin vessels
Tg(fli1a:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
00:00:00.000
06:00.00.000
12:00.00.000
18:00.00.000
23:45:00.000
Figure 5 | Time-lapse imaging of regenerating blood vessels in cxcr4aum20 mutant fish. (a) Still images taken every 6 h from a 24 h time-lapse movie on
9 d.p.a. fin regenerate of cxcr4aum20 fish. (b) Tracking of individual cells deriving form the lateral vein (white dots), the artery (pink dots) or the
medial vein (blue dots). (c) Tracks of labelled cells in (b). White arrows indicate migration paths of lateral vein-derived endothelial cells, while blue arrows
indicate migration paths of medial vein-derived endothelial cells. & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. Discussion
I
hi
d Dashed box in (g) indicates magnified area in h–h00 (n ¼ 2 adult zebrafish). (i–j00) In a mosaic situation, both donor-derived cxcr4aum20 mutant cells
(white arrowheads in j–j’’) and host-derived wild-type cells (j–j00, blue arrowheads) contributed to forming arteries. Dashed box in i indicates magnified area
in j–j00 (n ¼ 3 adult zebrafish). Scale bars are 50 mm in i00 and 20 mm in j00. Mosaic ray vasculature Donor: Figure 6 | Analysis of cxcr4a function in chimeric fin vasculature. (a) Schematic drawing of transplantation procedure. Cells from double transgenic
Tg(fli1a:EGFP)y1; Tg( 0.8flt1:RFP)hu5333 donors were transplanted into single Tg( 0.8flt1:RFP)hu5333 hosts. Two different scenarios were observed: Either the
vasculature of the whole ray was donor-derived (b–d00), or a mosaic ray vasculature, consisting of donor and host-derived endothelial cells formed (e–j00). (b–b00) In wild type to wild-type transplants, normal fin blood vessels formed. Arrow in b00 marks artery (n ¼ 8 adult zebrafish). (c–c00) When wild-type cells
were transplanted into cxcr4aum20 mutant hosts, normal arteries could form (arrow in c00, n ¼ 2 adult zebrafish). (d–d00) When cxcr4aum20 mutant cells were
transplanted into wild-type hosts and formed the vasculature of an entire fin ray, arteries showed similar patterning defects as in cxcr4aum20 mutants (arrow
in d00, n ¼ 3 adult zebrafish). (e–f00) In a control mosaic situation, both donor (white arrowhead, f–f00) and host-derived (blue arrowhead, f–f00) wild-type
cells contributed to forming arteries. Dashed box in (e) indicates magnified area in f–f00 (n ¼ 8 adult zebrafish). (g–h00) In a mosaic situation, both donor-
derived wild-type cells (white arrowheads in h–h00) and host-derived cxcr4aum20 mutant cells (h–h00, blue arrowheads) contributed to forming arteries. Dashed box in (g) indicates magnified area in h–h00 (n ¼ 2 adult zebrafish). (i–j00) In a mosaic situation, both donor-derived cxcr4aum20 mutant cells
(white arrowheads in j–j’’) and host-derived wild-type cells (j–j00, blue arrowheads) contributed to forming arteries. Dashed box in i indicates magnified area
in j–j00 (n ¼ 3 adult zebrafish). Scale bars are 50 mm in i00 and 20 mm in j00. stalk cells can proliferate during vascular outgrowth. This is in
agreement with previous studies in developing zebrafish inter-
segmental vessels38–40. We observe a reduction in endothelial cell
proliferation both in arteries and veins in cxcr4aum20 mutants. This is in contrast to the observed cell migration defects in
cxcr4aum20 mutants, which specifically affect newly forming
arteries. Discussion
I
hi
d In the
mouse retina we find that descendants of previously labelled
endothelial tip cells can proliferate during blood vessel formation
and thereby account for an increase in the number of labelled
endothelial cells. Our zebrafish data also show that both tip and Despite the important role of Cxcr4a signalling during arterial
morphogenesis, differentiation of arterial cells appears to be 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 Transplant
Grow to adulthood
Analyse fin vasculature
Vasculature of whole
ray donor derived
Mosaic ray vasculature
WT --> WT
WT --> cxcr4aum20
cxcr4aum20 --> WT
Donor
cells
Donor
+host
Overlay
Donor
cells
Donor
+host
Overlay
Donor
cells
Donor
+host
Overlay
Regenerated fin
Donor: Tg(fli1a:EGFP)y1;Tg(–0.8flt1:RFP)hu533 Host: Tg(–0.8flt1:RFP)hu533
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
| Vasculature of whole
ray donor derived
Mosaic ray vasculature
WT --> WT
WT --> cxcr4aum20
cxcr4aum20 --> WT
Donor
cells
Donor
+host
Overlay
Donor
cells
Donor
+host
Overlay
Donor
cells
Donor
+host
Overlay
egenerated fin
Donor: Tg(fli1a:EGFP)y1;Tg(–0.8flt1:RFP)hu533 Host: Tg(–0.8flt1:RFP)hu533
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
′
′′
Figure 6 | Analysis of cxcr4a function in chimeric fin vasculature. (a) Schematic drawing of transplantation procedure. Cells from double transgenic
Tg(fli1a:EGFP)y1; Tg( 0.8flt1:RFP)hu5333 donors were transplanted into single Tg( 0.8flt1:RFP)hu5333 hosts. Two different scenarios were observed: Either the
vasculature of the whole ray was donor-derived (b–d00), or a mosaic ray vasculature, consisting of donor and host-derived endothelial cells formed (e–j00). (b–b00) In wild type to wild-type transplants, normal fin blood vessels formed. Arrow in b00 marks artery (n ¼ 8 adult zebrafish). (c–c00) When wild-type cells
were transplanted into cxcr4aum20 mutant hosts, normal arteries could form (arrow in c00, n ¼ 2 adult zebrafish). (d–d00) When cxcr4aum20 mutant cells were
transplanted into wild-type hosts and formed the vasculature of an entire fin ray, arteries showed similar patterning defects as in cxcr4aum20 mutants (arrow
in d00, n ¼ 3 adult zebrafish). (e–f00) In a control mosaic situation, both donor (white arrowhead, f–f00) and host-derived (blue arrowhead, f–f00) wild-type
cells contributed to forming arteries. Dashed box in (e) indicates magnified area in f–f00 (n ¼ 8 adult zebrafish). (g–h00) In a mosaic situation, both donor-
derived wild-type cells (white arrowheads in h–h00) and host-derived cxcr4aum20 mutant cells (h–h00, blue arrowheads) contributed to forming arteries. NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE fin regenerated vessel
cxcl12at30516 wild-type sibling and mutant ****
μm
μm2
****
****
0
50
0
10
20
30
40
50
60
70
0
200
400
600
800
1,000
Sib
Mut
Sib
Mut
Sib
Mut
Sib
Mut
0
2,000
4,000
6,000
8,000
10,000
Tg(fli:nEGFP)y7;
Tg(–0.8flt1:RFP)hu5333
Tg(fli:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin3′UTR)mu4
300
250
200
150
100
0
50
0
10
20
30
40
50
60
70
0
200
400
600
800
1,000
0
2,000
4,000
6,000
8,000
10,000
300
250
200
150
100
EC number (centre)
EC number (total)
Vessel coverage (centre)
Vessel length (centre)
hs sib
hs mut
hs sib
hs mut
hs sib
hs mut
hs sib
hs mut
NS
NS
NS
NS
EC number (total)
EC number (centre)
Vessel length (centre)
Vessel coverage (centre)
Sib
Mut
Sib
Mut
Sib
Mut
Sib
Mut
NS
NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/
& 2014 Macmillan Publishe μm2
****
Sib
0
50
0
2,000
4,000
6,000
8,000
10,000
300
250
200
150
100
EC number (total)
Vessel coverage (centre)
Sib
Mut
Sib
Mut
NS Mut 2
Sib
Mut b
Mut Sib Mut p
Vascular malformations and patterning defects, including
stenosis and arteriovenous shunts, can lead to life-threatening
conditions in humans, underscoring the importance of under-
standing the mechanisms that lead to proper blood vessel
sprouting and artery formation. Previous studies have used
in vitro blood vessel sprouting assays to study endothelial cell
dynamics during blood vessel growth. Arima et al.14 used an aortic
ring sprouting assay and elegant cell tracking methods to show
that endothelial cells in elongating branches are highly dynamic. The authors observed cell mixing and overtaking at the tip
position. Interestingly, they also noticed individual endothelial
cells that migrated opposite to the direction of the elongating
sprout. The authors attributed this behaviour to loss of VEGF
gradient formation in their culture setting. Our results now argue
that this change in migratory direction could rather reflect the
initiation of artery formation, not readily assessable in the aortic
ring assay. In an embryoid body sprouting assay, Jakobsson et al.13
also observed endothelial cell mixing and shuffling of tip cells. These behaviours suggested competition between endothelial cells
for the tip cell position, which was dependent on the relative levels
of VEGF receptors and the Notch ligand Dll4. However, also in
this assay, normal artery differentiation does not take place,
precluding the analysis of the complete sprouting and remodelling
programme. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 homologues30. In the embryo, cxcl12bmu100 mutants recapitulate
the vascular phenotypes observed in cxcr4aum20 mutants15,27. Surprisingly, blood vessel formation during fin regeneration was
unaffected in cxcl12bmu100 mutants, while cxcl12at30516 mutants
showed phenotypes similar to those of cxcr4aum20 mutants. It will
be interesting in the future to determine the reason for this
apparent switch in Cxcr4a ligand usage between embryonic stages
and during regeneration. the surrounding tissue by a decoy chemokine receptor, Cxcr7
(ref. 45). More recent reports show that migrating tissues
can self-generate chemokine gradients46,47. We find selective
accumulation of ubiquitously overexpressed Cxcl12a-mCherry
protein in newly forming bones during zebrafish fin regeneration,
which can fully rescue artery formation in cxcl12at30516 mutants. This finding suggests that, while the precise mechanism is
currently unknown, the regenerating fin tissue is either able to
selectively degrade Cxcl12a protein outside of the bone or to
stabilize it within the bone. Further studies will help to distinguish
between these two options. The formation of chemokine gradients has been under intense
investigation in recent years. One breakthrough study described
the generation of chemokine gradients via ligand sequestration in Our cell transplantation experiments showed that cxcr4aum20
mutant cells could contribute to the forming artery when
surrounded by wild-type cells. This demonstrates that endothelial
cells can also cooperate during plexus remodelling and that
collective cell migration occurs during this process. Similar cell
behaviours play a role during Cxcr4b controlled lateral line
migration in zebrafish embryos48. In this setting, cxcr4bt26035
mutant cells can migrate normally in a wild-type primordium49. This indicates that, also in the vasculature, the organization of
collective cell migration might be dependent on competition and
cooperation between endothelial cells29. 14 d.p.a. fin regenerated vessel
****
μm
μm2
****
****
Sib
Mut
Sib
Mut
cxcl12at30516 wild-type sibling and mutant
Tg(fli:nEGFP)y7;
Tg(–0.8flt1:RFP)hu5333
Tg(fli:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin3′UTR)mu4
0
50
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10
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40
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EC number (total)
EC number (centre)
Vessel length (centre)
Vessel coverage (centre)
Sib
Mut
Sib
Mut
Sib
Mut
Sib
Mut
NS 14 d.p.a. Discussion
I
hi
d At present it is not clear whether the observed cell
proliferation defects are due to a direct effect of cxcr4a signalling in both endothelial cell populations or due to perfusion defects,
which might result from arterial malformations. Chemokines play important roles in guiding the migration of
various cell types, such as leukocytes41, endothelial cells42,
endodermal cells43, germ cells30 and neurons44. They mainly
function through the formation of a gradient, which provides
positional information or through the activation of integrin
signalling30. The
zebrafish
genome
contains
two
cxcl12 NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. 8 Methods The removal of extra tricaine solution in the imaging chamber was
achieved by connecting it to a fluid aspiration system for cell culture. Image
acquisition was done for a maximum of 24h every 15 min using a Leica SP5 confocal
microscope equipped with a 20 dipping lens. Confocal stacks and movies were
assembled using Imaris software (Bitplane). The Linear Stack Alignment with SIFT
plugin of ImageJ (NIH) was used for aligning image stacks. The MtrackJ plugin as
implemented in ImageJ was used to label movies. EdU labelling. Esm1-iCreERT2 þ /T, R26mTmG þ /T double heterozygous mice
were injected intragastric with 50 ml of 4-hydroxytamoxifen solution
(0.25 mg ml 1; H7904 Sigma), at P2. Fifty microlitres of freshly prepared EdU
nucleotides (2 mg ml 1 in PBS) was administered intraperitoneally at P6 and pups
were dissected after 2 h. Whole-mount retina immunostaining and EdU detection. Retina immunos-
taining was performed according to Pitulescu et al.53 with some modifications. Whole
eyes were fixed for 10min at RT followed by 1h on ice, with freshly prepared 4%PFA/
PBS. Retinas were dissected and blocked/permeabilized (1% BSA, 0.3% Triton, PBS) for
30min at RT. After rinsing twice with modified Pblec buffer (1mM CaCl2, 1mM
MgCl2, 1mM MnCl2, 0.4% Triton X-100 in PBS), retinas were incubated overnight at
4C with biotinylated Griffonia simplicifolia lectin I (isolectin B4) (1:50, VectorLabs,
B-1205) and the following primary antibodies: rabbit anti-Erg (1:200, Santa-Cruz, sc-
353) and chicken anti-GFP (1:300, Aves Labs, GFP-1010). On the next day, retinas were
washed for 20min in blocking buffer diluted once with PBS and three times (10min
per wash) in PBS. Retinas were then incubated for 2h with CFTM405M-streptavidin
antibody (1:100, Biotrend, 29033) or Alexa Fluor 546 streptavidin-conjugated (1:100,
Invitrogen, S11225) and the following species-specific secondary antibodies: Alexa Fluor
488-donkey anti-chicken (1:500, Jackson ImmunoResearch Lab., 703-545-155), Alexa
Fluor 647-goat anti-rabbit (1:500, Invitrogen, A21244) or Alexa Fluor 594-donkey anti-
rabbit (1:500, Invitrogen, A21207). Retinas were then washed as described above. To
detect EdU incorporation Click-IT EdU detection reactions were performed according
to the manufacturer (Life Technologies, C103339 or C10340), using either Alexa Fluor
594 or 647 azide. Retinas were washed twice in PBS and flat-mounted on glass
microscope slides using Fluoromount-G (Southern Biotech, 0100-01). Confocal microscopy and imaging processing. Fin regenerates were embedded
in 1% low melting point agarose (Invitrogen) and imaged immediately after
amputation. Methods The endothelial cell contribution of transplanted cells was
assessed by visualization of EGFP and RFP expression. Adult fish containing EGFP
and RFP expressing endothelial cells in the fin vasculature were selected for
analysis. Genetic lineage tracing of endothelial tip cells. Esm1(BAC)-iCreERT2 trans-
genics22 were bred into a R26-tomato-EGFP21 reporter background. Cre activity in
newborn mice was induced by a single intraperitoneal injection of 50 mg 4-hydroxy
tamoxifen solution (Sigma, H7904; 1 mg ml 1 in ethanol/peanut oil) at 12, 24 or
48 h before analysis at postnatal stage P6. The Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin30UTR)mu4 fish line was generated
using the Tol2 transposon system. The transgene contains the HSP70/4 promoter
(AF158020.1, GI:7108904) cloned upstream of the Danio rerio Cxcl12a coding
sequence (FJ915063.1, GI:239596165) mutated in a way rendering it resistant for the
morpholino oligonucleotide (ATGgacttgaaggtcatcg) and fused in frame to the
mCherry open-reading frame (AB971706.1 GI:662033815). The Xenopus laevis
globin30UTR (J00978.1 GI:214209) was cloned downstream of the mCherry sequence. Retina immunostaining for genetic lineage labelling. For retina staining, eyes
were dissected and fixed in 4% PFA for 2 h at room temperature. Retinas were
dissected, permeabilized and blocked in 1% BSA (Sigma, A4378-25G) and 0.3%
Triton X-100 2 h at room temperature with agitation. Biotinylated isolectin B4
(Vector, B-1205, Griffonia simlicifolia lectin I; 1:25) in blocking buffer was added
and incubated overnight at 4 C with agitation. Next day, Retinas were washed
three times in PBS and incubated with Alexa-Fluor-coupled streptavidin 647
(Invitrogen, 1:100) in blocking buffer together with GFP Alexa-Fluor-coupled 488
antibody (A21311, Invitrogen, 1:300) for 2 h at RT. Retinas were flat-mounted
using Fluromount-G (SouthernBiotech, 0100-01) and images were taken with a
Leica SP5 confocal microscope. Volocity software (PerkinElmer) was used for
image processing and quantitative analysis. In vivo imaging of adult fish. The in vivo imaging of adult fish was carried out as
described with the following modifications51. Tg(fli1a:nEGFPy7; 0.8flt1:RFPhu5333)
fish were anaesthetized in 0.02% tricaine until they stopped swimming, transferred
to the home-made chamber for live imaging, and immobilized by covering the trunk
and caudal fin with 1% agarose. Afterwards, fish were orally administered with
126 mg l 1 tricaine in fish water at the speed of 5.5ml min 1. We inserted a
Silicone tubing (ID mm, OD 6 mm) into the fish mouth for tricaine delivery. The
speed of tricaine delivery was controlled by a peristaltic pump (ISM-795C,
ISMATEC). Methods Zebrafish Strains and Fin amputations. Previously described zebrafish lines were
Tg(fli1a:EGFP)y1 (ref. 18), (Tg(fli1a:nEGFP)y7 (ref. 20), Tg( 0.8flt1:RFP)hu5333
(ref. 19), Tg(cxcl12bBAC:YFP)mu105 (ref. 25), cxcr4aum20 (ref. 27), cxcl12bmu100
(ref. 15), cxcl12at30516 (ref. 28). Zebrafish of 5–18 months of age were used. Fin
amputations were performed as previously described50. All animal experiments
were performed in compliance with the relevant laws and institutional guidelines
and were approved by local animal ethics committees of the Landesamt fu¨r Natur,
Umwelt und Verbraucherschutz Nordrhein-Westfalen. Zebrafish Strains and Fin amputations. Previously described zebrafish lines were
Tg(fli1a:EGFP)y1 (ref. 18), (Tg(fli1a:nEGFP)y7 (ref. 20), Tg( 0.8flt1:RFP)hu5333
(ref. 19), Tg(cxcl12bBAC:YFP)mu105 (ref. 25), cxcr4aum20 (ref. 27), cxcl12bmu100
(ref. 15), cxcl12at30516 (ref. 28). Zebrafish of 5–18 months of age were used. Fin
amputations were performed as previously described50. All animal experiments
were performed in compliance with the relevant laws and institutional guidelines
and were approved by local animal ethics committees of the Landesamt fu¨r Natur,
Umwelt und Verbraucherschutz Nordrhein-Westfalen. Generation of transgenic lines. We used Bacterial artificial chromosome (BAC)
recombineering as described previously to generate transgenic lines25. To generate
Tg(cxcr4aBAC:YFP)mu104 and Tg(cxcl12aBAC:CFP)mu146 animals, the start codon
of the cxcr4a and cxcl12a gene in the BAC clone CH73-268G8 and CH73-353M13
was replaced with a Citrine or Cerulean cassette using Red/ET recombineering
(GeneBridges). The Citrine cassette was amplified by PCR from pCS2 þ Citrine_kanR
with the primers cxcr4a _HA1_GFP_fw (50-ttattttttatttttacggctggtggggtagactttcgaga-
aaatcggttACCATGGTGAGCAAGGGCGAGGAG-30) and cxcr4a_HA2_kanR_rev
(50-gtgttgggctggggcgccgggggcttcagctcggtttccatcatgttataTCAGAAGAACTCGTCA-AG
AAGGCG-30). The Cerulean cassette was amplified by PCR from pCS2þ Cerulean_
kanR with the primers cxcl12a_HA1_gfp_fw (50-cacagttgctcctggat-tctacacagtgcggatc
tcttcttcacactgcaAC-CATG-GTGAGCAAGGGCGAGGAG-30) and cxcl12a_HA2_
kanR_rev (50-gcatgaatggcgaccgccatcagagcgactactacgatcactttgagatc-TCAGAAGAAC
TCGTCAAGAAGGCG -30); homology to the BAC vector is depicted in lower
case. Tg(cxcl12bBAC:YFP)mu105 was generated as previously described25. BAC DNA
was isolated by Midiprep (Invitrogen) and injected at 100 pg per embryo together
with 50pg per embryo of tol2 transposase mRNA into wild-type embryos. The Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin30UTR)mu4 fish line was generated
using the Tol2 transposon system. The transgene contains the HSP70/4 promoter
(AF158020.1, GI:7108904) cloned upstream of the Danio rerio Cxcl12a coding
sequence (FJ915063.1, GI:239596165) mutated in a way rendering it resistant for the
morpholino oligonucleotide (ATGgacttgaaggtcatcg) and fused in frame to the
mCherry open-reading frame (AB971706.1 GI:662033815). The Xenopus laevis
globin30UTR (J00978.1 GI:214209) was cloned downstream of the mCherry sequence. Blastomere transplantation. Cell transplantation was performed as described40. About 40 cells were transplanted from donor embryos to the margin of host
embryos at sphere stage. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6758 to forming arteries. Thus, our results provide a new framework for
an understanding of the coordinate migratory behaviours of
endothelial cells that allow for the proper sprouting and
subsequent remodelling of arteries and veins in a vascular plexus. Heat-shock experiments. Heat shocks were carried out every 12 h on 14 con-
secutive days. For this purpose, fish were placed in system water, which was heated
gradually for about 10–15 min from 28 C to 37 C in a standard waterbath. Subsequently, fish were exposed to 37 C for 1 h, before gradual return to 28 C. In situ hybridization. Whole-mount in situ hybridization was carried out as
described27. Previously described probes were cxcr4a27, cxcl12b27 and cxcl12a52. Probes were generated from plasmid DNA. The plasmid containing cxcr4a was
digested with NotI. The plasmids containing cxcl12a or cxcl12b were digested with
NcoI. Sp6 was used to generate DIG-labelled antisense RNA for all three probes. DNA template for in vitro synthesis of cxcr4a sense probe was amplified from
pCRII-cxcr4a by PCR. The forward primer 50-GCTTGATTTAGGTGACACTAT
AGAATTATCGGGAACGGACTGG-30 containing SP6 promoter and reverse
primer 50-AGGCGTACAGGATCGGG-30 were used for PCR amplification. Proteinase K (30 mg ml 1) was used to permeabilize 2 d.p.a. fin regenerates for
20 min at room temperature before proceeding to the in situ hybridization with
aforementioned probes. Images were acquired using a Leica M205 C Microscope
and a Zeiss Axio Imager microscope. Methods Images were acquired using a Leica SP5 confocal microscope and a
Zeiss LSM780 confocal microscope. Maximum Projection images were generated
with Imaris software (Bitplane). ImageJ (NIH) and Volocity 6.1 (PerkinElmer)
were used to quantify cell number, vessel area and vessel length. We chose the third
fin ray from the dorsal and/or ventral edge for our measurements. A region of
interest (40 250 mm, width height) in the centre of the growing vessel in the
distal end of regenerating fin rays at 3 d.p.a. and two regions of interest of the same
size in bifurcating fin rays from 5 d.p.a. onwards were measured. In total, the fin
vasculature of at least ten fin rays from at least five fish was analysed for the each
animal group of different regeneration stages. The Mann–Whitney non-parametric
test as implemented in Prism 6.0b (Graphpad) was used to analyse the data. Quantification of endothelial cell proliferation in zebrafish fins by in vivo live
imaging. Dividing endothelial cells were counted over 24 h in three independent
movies each for wild-type and cxcr4aum20 mutants. Differences in proliferative
behaviour between wild types and mutants were analysed in venous (lateral vein
and medial vein combined) and arterial cell populations. The unpaired t-test as
implemented in Prism 6.0b (Graphpad) was used for statistic analysis; significance
(*Po0.05, **Po0.01) in unpaired t-test; NS ¼ not significant. ARTICLE We now show that during zebrafish fin regeneration
and in the developing mouse retinal vasculature, endothelial tip
cells are derived from veins and that these tip cells can contribute ****
0
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Sib
Mut Vessel coverage (centre) Vessel length (centre) μm2
****
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Sib
Mut μm
****
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200
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600
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1,000
g
(
)
Sib
Mut Mut Tg(fli:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin3′UTR)mu4 Tg(fli:nEGFP)y7; Tg(–0.8flt1:RFP)hu5333
Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin3′UTR)mu4 Tg(fli:nEGFP)y ; Tg(–0.8flt1:RFP) u5333
Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin3′UTR)mu 0
50
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EC number (centre)
EC number (total)
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Vessel length (centre)
hs sib
hs mut
hs sib
hs mut
hs sib
hs mut
hs sib
hs mut
NS
NS
NS
NS Sib
Mut Mut Figure 7 | Global overexpression of Cxcl12a-mCherry rescues the
vascular phenotype of cxcl12t30516 mutant fish. White dashed boxes
indicate central areas quantified. Fin vasculature was analysed at 14d.p.a. Dashed lines indicate amputation planes. (a) Heat-shocked control wild type
and cxcl12at30516 mutant fish not carrying the Tg(Cry.kop.HSP:mutSDF1a. mCherry.globin30UTR)mu4 transgene. White arrowheads indicate ectopic
Tg( 0.8flt1:RFP)hu5333-positive endothelial cells in cxcl12at30516 mutants. Endothelial cell numbers, vessel coverage and vessel length are reduced in
cxcl12at30516 mutant fish. (b) Heat-shocked wild type and cxcl12at30516
mutants carrying the Tg(Cry.kop.HSP:mutSDF1a.mCherry.globin30UTR)mu4
transgene. None of the assayed parameters differs between heat-shocked
wild type and cxcl12at30516 mutants. Note accumulation of ubiquitously
overexpressed Cxcl12a-mCherry protein in bony rays (white arrowheads) but
not in the joints (blue arrowheads) between bone segments. ****Po0.0001;
Mann–Whitney U-test, NS, not significant; n ¼ 8 adult zebrafish per stage. Scale bar, 100 um. 0
50
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hs sib
hs mut
NS
NS Vessel length (centre) hs sib NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. 9 & 2014 Macmillan Publishers Limited. All rights reserved. References
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fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007). 22. Rocha, S. F. et al. Esm1 modulates endothelial tip cell behavior and vascular Acknowledgements This work was funded by the Max Planck Society (A.F.S.), the Deutsche For-
schungsgemeinschaft (DFG SI-1374/3-1; A.F.S.), the Sonderforschungsbereich (SFB) 629
an ERC advanced grant (268806 - CellMig; E.R.) and an ERC starting grant (260794-
ZebrafishAngio; A.F.S.). This work was supported by the Deutsche For-
schungsgemeinschaft (DFG) Cells-in-Motion Cluster of Excellence (EXC 1003-CIM),
University of Mu¨nster, Germany. Jeroen Bussmann was supported by a European
Molecular Biology Organization (EMBO) long-term fellowship. This work was funded by the Max Planck Society (A.F.S.), the Deutsche For-
schungsgemeinschaft (DFG SI-1374/3-1; A.F.S.), the Sonderforschungsbereich (SFB) 629
an ERC advanced grant (268806 - CellMig; E.R.) and an ERC starting grant (260794-
ZebrafishAngio; A.F.S.). This work was supported by the Deutsche For- 22. Rocha, S. F. et al. Esm1 modulates endothelial tip cell behavior and vascular
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Molecular Biology Organization (EMBO) long-term fellowship. 24. Strasser, G. A., Kaminker, J. S. & Tessier-Lavigne, M. Microarray analysis of
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25. Bussmann, J. & Schulte-Merker, S. Rapid BAC selection for tol2-mediated Author contributions 26. Montes, G. S., Becerra, J., Toledo, O. M., Gordilho, M. A. & Junqueira, L. C. Fine structure and histochemistry of the tail fin ray in teleosts. Histochemistry
75, 363–376 (1982). C.X., R.H.A. and A.F.S. planned the experiments. C.X., I.S. and M.P. carried out the
experiments. C.X., S.S.H., I.S., M.P., R.H.A. and A.F.S. analysed the data. S.F.R. generated
the esm1(BAC)-iCreERT2 transgenic mouse line. J.B. generated the Tg(cxcr4aBA-
C:YFP)mu104 transgenic zebrafish line. D.M. and E.R. contributed the Tg(Cry.-
kop.HSP:mutSDF1a.mCherry.globin30UTR)mu4 fish line. C.X. and A.F.S. wrote the paper. All authors commented on the manuscript. C.X., R.H.A. and A.F.S. planned the experiments. C.X., I.S. and M.P. carried out the
experiments. C.X., S.S.H., I.S., M.P., R.H.A. and A.F.S. analysed the data. S.F.R. generated
the esm1(BAC)-iCreERT2 transgenic mouse line. J.B. generated the Tg(cxcr4aBA-
C:YFP)mu104 transgenic zebrafish line. D.M. and E.R. contributed the Tg(Cry.-
kop.HSP:mutSDF1a.mCherry.globin30UTR)mu4 fish line. C.X. and A.F.S. wrote the paper. All authors commented on the manuscript. 27. Siekmann, A. F., Standley, C., Fogarty, K. E., Wolfe, S. A. & Lawson, N. D. Chemokine signaling guides regional patterning of the first embryonic artery. Gene Dev. 23, 2272–2277 (2009). 28. Valentin, G., Haas, P. & Gilmour, D. The chemokine SDF1a coordinates tissue
migration through the spatially restricted activation of Cxcr7 and Cxcr4b. Curr. Biol. 17, 1026–1031 (2007). References 1. Eilken, H. M. & Adams, R. H. Dynamics of endothelial cell behavior in
sprouting angiogenesis. Curr. Opin. Cell. Biol. 22, 617–625 (2010). 2. Herbert, S. P. & Stainier, D. Y. Molecular control of endothelial cell behaviour
during blood vessel morphogenesis. Nat. Rev. Mol. Cell. Biol. 12, 551–564 (2011). 1. Eilken, H. M. & Adams, R. H. Dynamics of endothelial cell behavior in
sprouting angiogenesis. Curr. Opin. Cell. Biol. 22, 617–625 (2010). 2. Herbert, S. P. & Stainier, D. Y. Molecular control of endothelial cell behaviour
during blood vessel morphogenesis. Nat. Rev. Mol. Cell. Biol. 12, 551–564 (2011). NATURE COMMUNICATIONS | 5:5758 10 NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. & 2014 Macmillan Publishers Limited. All rights reserved. Additional information Supplementary Information accompanies this paper at http://www.nature.com/
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naturecommunications 29. Rorth, P. Fellow travellers: emergent properties of collective cell migration. EMBO Rep. 13, 984–991 (2012). p
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New players tune in for a common theme. Exp. Cell. Res. 319, 1255–1263 (2013). migration. Curr. Opin. Neurobiol. 18, 237–244 (2008). 45. Boldajipour, B. et al. Control of chemokine-guided cell migration by ligand
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gradient. Nature 503, 285–289 (2013). 13. Jakobsson, L. et al. Endothelial cells dynamically compete for the tip cell
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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/ 34. Benedito, R. & Hellstrom, M. Notch as a hub for signaling in angiogenesis. Exp. Cell Res. 319, 1281–1288 (2013). 35. Li, W. et al. Peripheral nerve-derived CXCL12 and VEGF-A regulate the 35. Li, W. et al. Peripheral nerve-derived CXCL12 and VEGF-A regulate the
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359–371 (2013). 11 NATURE COMMUNICATIONS | 5:5758 | DOI: 10.1038/ncomms6758 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved. & 2014 Macmillan Publishers Limited. All rights reserved.
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Logical Decision-Making Method Relating to Innovation Management
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IFIP advances in information and communication technology
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cc-by
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To cite this version: Nélio Reis, Priscila Tavares, Cristina Oliveira, Jair Minoro Abe. Logical Decision-Making Method
Relating to Innovation Management. IFIP International Conference on Advances in Production Man-
agement Systems (APMS), Sep 2016, Iguassu Falls, Brazil. pp.12-19, 10.1007/978-3-319-51133-7_2. hal-01615717 Distributed under a Creative Commons Attribution 4.0 International License 1
Introduction Many companies claim to be innovative, creative and be ahead of your competitors,
despite the fact that they are not using appropriate metrics and methods to innovative
processes and results. The model for this purpose is built through an approach of Paraconsistent Logic
Annotated Evidential E (E Logic), a technique that is gaining space and consideration
in the various fields of research. The main advantages of the use of E Logic derived from one of the input parameters
are set by the thought of evaluator’s structure, consolidating a collective logic translated
into mathematical terms. De Bes e Kotler [1] claim that "without innovation companies end up." Innovation
is the development and introduction of a new idea, making it a process, product or ser-
vice. O'Sullivan [2], states that: "Innovation is the process by which the productive
resources are developed and used to generate higher quality and / or lower cost products
compared to the current available". According to Tidd e Bessant [3], innovation "is
more than just having good ideas is the process of growing them". HAL Id: hal-01615717
https://inria.hal.science/hal-01615717v1
Submitted on 12 Oct 2017 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Nélio F. dos Reis1,2, Priscila Facciolli S. L. Tavares1, Cristina Oliveira1, Jair Minoro
Abe1 Nélio F. dos Reis1,2, Priscila Facciolli S. L. Tavares1, Cristina Oliveira1, Jair Minoro
Abe1 1Paulista University, São Paulo, Brazil
2 Federal Institute of Education, Science and Technology of São Paulo
neliojundiai@ig.com.br; pri1979@gmail.com 1Paulista University, São Paulo, Brazil
2 Federal Institute of Education, Science and Technology of São Paulo
neliojundiai@ig.com.br; pri1979@gmail.com Abstract. This paper is intended to serve as a support for decision in innovation
management from the E Logic. We intend to propose a new method for innova-
tion management, based on technical and operational criteria in order to make
decisions can be not only reliable but also operationally efficient. This research
presents results that can serve to innovation managers. Keywords: Innovation · Management · Paraconsistent Annotated Logic adfa, p. 1, 2011.
© Springer-Verlag Berlin Heidelberg 2011 Logical Decision-Making Method Relating to Innovation
Management Nélio F. dos Reis1,2, Priscila Facciolli S. L. Tavares1, Cristina Oliveira1, Jair Minoro
Abe1 1.1
Innovation Sinsit [4], he distinguishes invention of innovation as follows: "invention is the creation
of a new idea or concept. While innovation transforms the new concept in commercial
success or general use. Ronan [5], explains innovation as "profitable implementation of strategic creativity and which comprises four main components (1) creativity, (2)
strategy (3) implementation and (4) profit". Innovation is both a strategic factor for companies willing to remain competitive in
the long term [6] and [7], as one of the least known aspects of business as states
Takeuchi e Nonaka [8]. Due to increased competition, changes in demand and customer
tastes, it seems extremely important for companies to manage innovation in a fast and
flexible way in order to beat the competition and achieve a sustainable competitive
advantage. The scope of the innovation can be incremental or disruption [9]: "Incremental: typ-
ically performed to improve a product or service with a new feature that is easily inte-
grated; and radical: usually associated with disruption for new steps and unpredictable
uses for existing technology." Still according to Vacek [9], the common places of in-
novation are product, process, structure and market. 1.2
Innovation Models Many companies claim to be innovative, creative and be ahead of their competitors,
despite the fact that they are not using appropriate metrics and methods to innovative
processes and results. "Innovation is a disorderly process: difficult to measure and dif-
ficult to manage [1]." However, states that innovation is an art, not a science and it is therefore not possible
to predict the success of an innovation until it is accepted by the respective market [10]. Since the 1950s there has been a proliferation of innovation models, each with the pur-
pose of guiding the innovation process within companies, as summarized in Tab 1. Table 1. Development of innovation models (Adapted from Hobday [11])
Model
Generation
Feature
Technological
1ª (1950-1960)
Emphasis on R&D. Innovation R&D. Market
2ª (1960-1970)
Market is the source of new ideas for R&D
Coupled
3ª (1970-1980)
Feedback link between market and R&D. Interactive
4ª (1980-1990)
Emphasis in marketing and R&D. Network
5ª (1990-2000)
Knowledge and systems integration. Open
6ª (2000-current)
Combined ideas for innovation. Table 1. Development of innovation models (Adapted from Hobday [11]) For each step, there are several tools and techniques that can be selected based on
company structure, the target market or type of products or services. The decision-mak-
ing method in Innovation Management is established not as a process of innovation,
but through the interaction between six distinct phases: identification, intelligence, ide-
alization, instrumentation, implementation and indicators. These phrases have been
identified in the A-F models [1], who claim: "This model results from analysis of vari-
ous companies that are consider innovative and have good results concerning the time
and resources invested in innovation: Apple, Google, Netflix, 3M, Procter & Gamble, General Electric, BMW, Frito Lay, IBM, Toyota, Southwest Airlines, Starbucks, Mi-
crosoft, Tesco, Royal Dutch / Shell, Walmart, Exxon, Ikea, Ericsson, Nokia and Corn-
ing were the main companies studied". General Electric, BMW, Frito Lay, IBM, Toyota, Southwest Airlines, Starbucks, Mi-
crosoft, Tesco, Royal Dutch / Shell, Walmart, Exxon, Ikea, Ericsson, Nokia and Corn-
ing were the main companies studied".
E Logical The E logical allows to treat subjective data from the real worldinto accurate data with
numerical outputs [12]. One of its advantages is to perform the translation of natural
language (linguistic terms) used in daily communication in mathematical expressions. This is achieved by means of crosslinked annotation properties. De Carvalho e Abe [13], states that: “In Elogical logicalevery p prepositio associ-
ates , n commom sense, a pair (), represented by Greek letters mi () and lambda
(),depicted in the following manner: p(). nd varies in the real closed interval [0,
1]. Therefore, the pair () belongs to the Cartesian product [0, 1] x [0, 1]. Intuitively,
represents the degree of favorable evidence expressed in p, nd the degree of con-
trary evidence expressed by p. The pair ()It is called annotation constant or simply
note. The atomic propositions of E logical are p() type”. The advantages in the development of paraconsistent systems are: how quickly the
building of system is carried out in relation to models based on logic "fuzzy" (common
or Boolean) and make it unnecessary knowledge or the development of a mathematical
model [13]. Innovation is important, but that executives lack confidence in their deci-
sions [1]. The decision-making method in Innovation Management is established through six
distinct phases, as follows [1]: (i) Identification: Expressed by X percentage of the pop-
ulation on the whole; (ii) Intelligence: Translated globally in size of the impact on the
innovation site; (iii) Idealization: The Y amount, of ideas attributes, can be presented
as a comparison measurement with the average M attributes in available similar. (iv)
Instrumentation: Can be represented by the average expenditure G function compared
to similar available S; (v) Implementation: In period P to development and implantation
measured as a function of C lifecycle; and (vi) Indicators: Investment I measured by
the R expected return on the life cycle of innovation. 2
Methods The methodology used was as follows: 1) Problem in the form of question: - How to
make innovation management with contradictory data? 2) Elaborated hypothesis: - You
can take decision in innovation management with logical data. 3) Literature review: -
Theoretical research on innovation and E Logic. 4) Data collection: - Expert systems,
divided into three groups: - A: 03 (three) innovation experts; - B: 03 (three) manage-
ment experts; and - C: 02 (two) risk investment experts. 5) Study object: - Extensive
direct observation of experts. The experts evaluated the Six I's factors for innovation in product, process and mar-
ket. Initially the experts understood the model. After that, brainstorm sessions were conducted to identify the factors and building the logical proposition. The Six I's and
the propositions are presented in the table below. the propositions are presented in the table below. Table 2. Six I’s and Propositions
Factors
Sections
Propositions
Description
Identifica-
tion
I1
S1
X >70%
The population benefited X is greater than 70% of the
whole. S2
30% ≤ X ≤ 70%
The population benefited X is greater than or equal to 30%
and less than or equal to 70% of the whole. S3
X < 30%
The population benefited X is less than 30% of the whole. Intelligence
I2
S1
Large
Major positive impact at the place of innovation
S2
Medium
Medium positive impact on the local of innovation
S3
Small
Small positive impact on the local of innovation
Idealization
I3
S1
Y > 1,5M
Y innovation attributes are 1.5 times higher than the aver-
age similar M. S2
0,5M ≤ Y ≤
1,5M
Y attributes of innovation are greater than or equal to 0.5
times and less than or equal to 1.5 times the similar aver-
age. S3
Y < 0,5M
Y attributes of innovation are greater than or equal to 0.5
times and less than or equal to 1.5 times the similar aver-
age. Instrumen-
talization
I4
S1
G < 40%S
Average spending G innovation is less than 40% of similar
S. S2
70%S ≤ G ≤
40%S
Average spent G of innovation is greater than or equal to
70% and less than or equal to 40% similar to S. S3
G > 70%S
Average spent G of innovation is greater than 70% similar
to S. 3
Aplication Regarding the factors, one should reason that they are independent of each other. Thus,
the experts allocated their degree of favorable evidence () and contrary evidence ()
in each proposition, as shown in Figure 1. Fig. 1. Database of evidence from experts Fig. 1. Database of evidence from experts With the Table 1 database, it emerges clearly the evidence of experts on the Six I's
factors. They are shown in Table 3 with use of Maximum and Minimum rules. 2
Methods Implemen-
tation
I5
S1
P < 30%C
P deadline of development and deployment of innovation
is less than 30% of the current life cycle. S2
30%C≤P ≤
70%C
P deadline of development and deployment of innovation
is greater than or equal to 30% and less than or equal to
70% of the current life cycle. S3
P > 70%C
P deadline of development and deployment of innovation
is greater than 70% of the current life cycle. Indicators
I6
S1
I < 30%R
Investment in innovation is less than 30% of the expected
return in the life cycle. S2
30%R≤ I ≤
70%R
Investment in innovation is greater than or equal to 30%
and less than or equal to 70% of the expected return in the
life cycle. S3
I > 70%R
Investment in innovation is greater than 70% of the ex-
pected return in the life cycle. Were executed two (02) Delphi rounds that sought to: identify the certainty of the
expert regarding the decision of each factor and the possible contradiction in relation
to expert. The consultation was conducted by form via email. The biggest difference between
them is that in the second round, the answers to each of the first round proposals of all
the experts had the result informed, giving the respondent an opportunity to review its
earlier evidence, if desired. 3.1
Maximization (Max) and Minimization (Mini) rules The next step is to apply the maximization (Max) and minimizing (Min) rule to the
evidence of the experts for each innovation factors. Apply to the rules of maximization of favorable evidence within groups being con-
nective (Max) the favorable evidence and connective (Min) in contrary evidence within each group and the minimization rule of favorable evidence among the groups, the con-
nective (min) the favorable evidence, and connective (Max) in contrary evidence to the
results obtained in the three groups (between groups), grouped as shown in Figure 2: Fig. 2. Application of the scheme of MAX and MIN operators. Fig. 2. Application of the scheme of MAX and MIN operators. 3.2
Analysis There is favorable or contrary evidence of innovation acceptance, if there is a degree
of certainty in magnitude equal to or greater than 0.6. This level of demand may change
depending on the focus of innovation. However, in this study, the value after discus-
sions with experts are as follows. The Certainty is defined as follows: Gcert = Summarizing, the division criterion is the following: Summarizing, the division criterion is the following: a) Gcert 0,6 True (T), ie, INNOVATE; a) Gcert 0,6 True (T), ie, INNOVATE; a) Gcert 0,6 True (T), ie, INNOVATE; b) Gcert -0,6 False (F), ie, NOT INNOVATE; and b) Gcert -0,6 False (F), ie, NOT INNOVATE; and c) -0,6 < Gcert < 0,6 Area between Truth and False is DOUBT. The database was treated with connective Max and Min and the results of the three
themes are shown in Figure 3. The database was treated with connective Max and Min and the results of the three
themes are shown in Figure 3. Fig. 3. Evidence degrees resulting from the application of Max and Mini rules Fig. 3. Evidence degrees resulting from the application of Max and Mini rules Observing the degree of favorable and contrary evidence resulting from the applica-
tion of maximizing rules (OR) and minimizing (AND) the evidence of experts, we note
that the degree of certainty (Gcert) to S1 is above 0.6 as established the criteria for
certainty in relation to innovation. For example, although experts E3, E4 and E7 in I1
S1 have given evidence ) which is a statement of (F) Falsehood, so there's
sure the statement is false and should not innovate, to take into account other evidence
of other experts is the result (V) True, that is, this proposition is a condition for inno-
vation. Anyway, you can tell which region he sure is. As shown in the figure is plotted
on I1S1 factor () in the region of OPJC = (V) true. I1S2 already has its col-
lective evidence in the area of () paracompleteza and is plotted in () in the
central region, highlighting questions regarding innovate. I1S3 already has its collec-
tive evidence in the area of (F) is plotted on falsehood (), ie, the evidence of
the expert is not innovate given this proposition. 3.2
Analysis The I2S1 factor is plotted below the
square unit of the Cartesian plane - QUPC, adapted from studies of De Carvalho and
Abe [13]. The other results coincide with I1S1, I1S2, I1S3 and I2S1 as plotted in Figure
4. Fig. 4. Application of para-analizer device at USCP. Fig. 4. Application of para-analizer device at USCP. If there is need for a more stringent criterion for decision making, safer decision,
more reliable, it is necessary to increase the level of demand, that is, one should ap-
proximate the PQ and TU lines of C and D, respectively, and also may use a larger
number of experts, or even consider evidence assigned depending on the weight of each
expert. You can still adjust and calibrate the data of propositions for a better result in
decision-making. 4
Conclusion If there is need for a more stringent criterion, ie, more safe and reliable decision, it is
necessary to increase the level of requirement, or use a larger number of experts, or
even consider the evidence given depending on the weight of each expert. A major advantage of this method is its versatility. Experts can be influenced, but in general are
not the same for everyone. Surely, at a time of latent conflict, the expert tends to dis-
credit innovation more than believing and the opposite can happen in times of elation,
joy, but hardly all specialists will be with the same feeling. Finally, virtually all prob-
lems in which uncertainty, ambiguity or natural language of human beings is relevant
present favorable situations the application of E Logic. References 1. De Bes, F.T.; Kotler, P. A.: Biblía da Inovação: Princípios Fundamentais para Levar a Cul-
tura da Inovação Contínua às Organizaçoes. São Paulo, Leya (2011) 2. O’Sullivan M.: The Innovative Enterprise and Corporate Governance. Cambridge Journal
of Economics, 24(4), 393-416 (2000) 3. Tidd, J., Bessant J.: Managing Innovation: Integrating Technological, Market and Organi-
zational Change. Wiley Press (2009) 4. Sinsit, Z.T., Vayvay, O., Ozturk, O. An Outline of Innovation Management Process: Build-
ing a Framework for Managers to Implement Innovation. Procedia - Social and Behavioral
Sciences, 150, 690 – 699 (2014) 5. Ronan, D.: A Study of Innovation Measurement and Innovation Management at Irish Med-
ical Device SME’s”. National University of Ireland (2009) 6. Prahalad C.K., Hamel G.: The Core Competence of the Corporation. Harward Business Re-
view, 68(3), 79-91 (1990) 7. Gourville J.T.: The Curse of Innovation: A Theory of Why Innovative New Products Fail in
the Marketplace. Marketing Research Papers, 05-06 (2005) 8. Takeuchi H., Nonaka I.: The New Product Development Game. Harvard Business Review,
137-146 (1986) 9. Vacek, J.: Innovation Management. University Washington Bothell (2009) 10. Poots A. J., Woodcock T.: Statistical Process Control for Data Without Inherent Order. BMC
Medical Informatics and Decision Making, 12(1), 86 (2012) 11. Hobday, M.: Firm-level Innovation Models, Technology Analysis & Strategic Management,
17(2), 121-145 (2005) 12. Reis, N.F.: Método Paraconsistente de Cenários Prospectivos. Universidade Paulista (2014) 13. De Carvalho, F.R.; Abe, J.M.: Tomadas de Decisão com Ferramentas da Lógica Anotada. São Paulo, Blucher (2011)
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Plant roots increase both decomposition and stable organic matter formation in boreal forest soil
|
Nature communications
| 2,019
|
cc-by
| 10,706
|
ARTICLE Plant roots increase both decomposition and stable
organic matter formation in boreal forest soil The mechanisms behind accumu-
lation and stabilization of SOM in boreal forest soils are poorly
understood but essential for predicting C stocks in a future cli-
mate2. The boreal forest C storage is tightly linked to the nitrogen
(N) cycle3, which is characterized by the binding or complexation
of a large fraction of soil N to other soil compounds such as
minerals and polyphenols, resulting in low N availability4–8. Thus
forest soil N is to a large extent present in chemically stable form9,
with the remainder in labile form (easily available N forms, dis-
solved inorganic and organic N) or retained in living organisms,
e.g. mycorrhizal fungi10. B three growing seasons. Three mesh sizes were compared that
excluded the ingrowth of plant roots as well as mycorrhizal
hyphae (1 µm mesh size treatment), permitted the ingrowth of
hyphae but not of roots (50 µm mesh size treatment) or permitted
the ingrowth of both hyphae and roots (1000 µm mesh size
treatment). We compare SOM loss and stable SOM-N formation,
soil chemistry and fauna, microbial community structure and
activities (enzymatic activity and microbial N transformations)
between the mesh size treatments, focussing specifically on N
pools and fluxes that may be tightly linked to C storage in boreal
forests27. Based on our field experiment, we provide a framework
on the role of plant roots in SOM decomposition and stabiliza-
tion. Plant roots not only accelerate SOM decomposition but also
increase the chemically stable N pool. On the other hand,
exclusion of plant roots leads to increased levels of inorganic N
(IN) and reduced build-up of stable soil N, as well as decreased
decomposition rates due to a lack of rhizosphere priming support
from plants. Thus our field study sheds light on the mechanisms
behind plant root-driven SOM transformations, increasing the
systems understanding needed for improving forest soil man-
agement to enhance SOM accumulation. Transformations of boreal forest SOM are driven by a complex
network of interactions among soil microorganisms, including
ectomycorrhizal and ericoid mycorrhizal (EEM) fungi, sapro-
trophic fungi, bacteria11 and plant roots12. Plant roots support
microorganisms in the rhizosphere, i.e. in the narrow soil zone
surrounding roots11, with easily available C, which may stimulate
microbial activity and thereby increase SOM decomposition
(referred to as the rhizosphere priming effect)13–15. Plant roots increase both decomposition and stable
organic matter formation in boreal forest soil Boreal forests are ecosystems with low nitrogen (N) availability that store globally significant
amounts of carbon (C), mainly in plant biomass and soil organic matter (SOM). Although
crucial for future climate change predictions, the mechanisms controlling boreal C and N
pools are not well understood. Here, using a three-year field experiment, we compare SOM
decomposition and stabilization in the presence of roots, with exclusion of roots but presence
of fungal hyphae and with exclusion of both roots and fungal hyphae. Roots accelerate SOM
decomposition compared to the root exclusion treatments, but also promote a different soil N
economy with higher concentrations of organic soil N compared to inorganic soil N
accompanied with the build-up of stable SOM-N. In contrast, root exclusion leads to an
inorganic soil N economy (i.e., high level of inorganic N) with reduced stable SOM-N build-
up. Based on our findings, we provide a framework on how plant roots affect SOM decom-
position and stabilization. 1 Department of Agricultural Sciences, University of Helsinki, PO Box 66Helsinki, Finland. 2 Institute for Atmospheric and Earth System Research (INAR),
University of Helsinki, Helsinki, Finland. 3 Department of Microbiology, University of Helsinki, PO Box 66Helsinki, Finland. 4 Natural Resources Institute
Finland, PL 2, 00791 Helsinki, Finland. 5 Department of Forest Sciences, University of Helsinki, PO Box 27Helsinki, Finland. 6 Department of Microbiology and
Ecosystem Science, University of Vienna, Althanstr. 14, 1090 Wien, Austria. 7 Department of Earth Sciences, University of Gothenburg, Gothenburg, Sweden. 8 Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden. 9 Bolin Centre for Climate Research, Stockholm
University, Stockholm, Sweden. 10 Natural Resources Institute Finland, Tietotie 2, 31600 Jokioinen, Finland. 11 Viikki Plant Science Centre (ViPS), University of
Helsinki, Helsinki, Finland. 12 Finnish Meteorological Institute, Climate System Research, Helsinki, Finland. Correspondence and requests for materials should
be addressed to B.A. (email: bartosz.adamczyk@luke.fi) 1 NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 B
oreal forests are key components of the global carbon (C)
cycle due to their high C storage and enormous potential
for C sequestration into soil organic matter (SOM)1. Nevertheless, with climate change SOM decomposition might
increase shifting boreal forests from C sinks to C sources, thereby
accelerating global warming1. Plant roots increase both decomposition and stable
organic matter formation in boreal forest soil Interactions
between different microbial guilds can further influence SOM
decomposition rates; for instance, EEM fungi may suppress
fungal saprotrophs, thereby decreasing SOM decomposition and
carbon dioxide (CO2) production (a phenomenon known as the
Gadgil effect)16,17. In addition to soil microorganisms, soil fauna
may enhance the SOM decomposition by fragmentation and
processing dead organic matter into a more available form for
microbes18. An emerging view underlines the role of micro-
organisms not only in SOM decomposition but also in SOM
stabilization19–21, i.e. the transformation of SOM into more stable
forms. According to the microbial carbon pump concept,
microorganisms metabolically process plant residues and gen-
erate biomass, and microbial residues are stabilized in soil (in vivo
turnover)19, via multiple mechanisms including reactions with
minerals and soil aggregates22. Alternatively, microbial enzymes
may stabilize plant residues by transforming them into less-
available forms (ex vivo modification)19. Overall, EEM plants
may thus accelerate SOM decomposition through the rhizosphere
priming effect or decelerate decomposition (e.g. via the Gadgil
effect) and increase the formation of stable SOM12,23,24 (e.g. via
the microbial C pump19). The balance between microbial priming
and soil C increase has been proposed to regulate the stable soil C
pool19,25. Although crucial for modelling responses of SOM
stocks to land use and climate change26, an in-depth mechanistic
understanding of SOM transformations is still lacking. Results and discussion Decomposition and microbial decomposers. In our 3-year
experiment, chemical and biological soil properties (Fig. 1) were
significantly affected by both time and mesh size (P < 0.001, R2 =
0.490 overall effect for different years and P < 0.05, R2 = 0.328
overall effect for different mesh sizes, with permutational multi-
variate analysis of variance (PERMANOVA)). The 1000 µm −4
−3
−2
−1
0
1
2
−3
−2
−1
0
1
2
CCA1 (28.4 %)
CCA2 (6.9 %)
Chitin
CT
stableN
NH4
pH
totalAA
NperSOM
CperSOM
Mass loss
PLFA_B
PLFA_F
1 µm
50 µm
1000 µm
Year
Fig. 1 Effect of the treatments and time on chemical and biological soil
properties. Partial constrained correspondence analysis (pCCA) illustrates
the effect of the three treatments and experimental time (year) on the
chemical and biological compositions of the mesh bags. The CCA1 axis
explains 28.4% (P ≤0.001, F = 31.36) of the data variability, and the CCA2
axis explains 6.8% (P ≤0.001, F = 7.59) of the data. The different mesh
treatments are represented with a green triangle (1000 µm mesh),
magenta circle (50 µm mesh) and black square (1 µm mesh). The variables
responsible for the separation of the different samples in the analysis have
been marked on the diagram with the following abbreviations: total AAs
total free amino acids, CT condensed tannins, stableN stable SOM-nitrogen
(N) pool, SOM soil organic matter, NH4 ammonium, PLFA_B bacterial
phospholipid fatty acids (PLFA), PLFA_F fungal PLFA −4
−3
−2
−1
0
1
2
−3
−2
−1
0
1
2
CCA1 (28.4 %)
CCA2 (6.9 %)
Chitin
CT
stableN
NH4
pH
totalAA
NperSOM
CperSOM
Mass loss
PLFA_B
PLFA_F
1 µm
50 µm
1000 µm
Year g
g
This study aims to elucidate the mechanisms underlying SOM
transformations and improve our understanding of its controls,
especially those related to N pools. The classic hypothesis is
formed that roots and associated microorganisms would stimu-
late microbial biomass growth and activity (priming) and con-
sequently SOM decomposition rates including stable SOM to
obtain N for plants and vice versa the lack of roots would result in
lower decomposition rates (hypothesis 1). Alternatively, this root-
induced priming effect is at some point exceeded by the build-up
of stable SOM-N from decomposition-released organic C and N
inputs (hypothesis 2). This would lead to stable soil C formation
especially in treatment with roots providing significant amounts
of C and N inputs. Fig. Results and discussion 1 Effect of the treatments and time on chemical and biological soil
properties. Partial constrained correspondence analysis (pCCA) illustrates
the effect of the three treatments and experimental time (year) on the
chemical and biological compositions of the mesh bags. The CCA1 axis
explains 28.4% (P ≤0.001, F = 31.36) of the data variability, and the CCA2
axis explains 6.8% (P ≤0.001, F = 7.59) of the data. The different mesh
treatments are represented with a green triangle (1000 µm mesh),
magenta circle (50 µm mesh) and black square (1 µm mesh). The variables
responsible for the separation of the different samples in the analysis have
been marked on the diagram with the following abbreviations: total AAs
total free amino acids, CT condensed tannins, stableN stable SOM-nitrogen
(N) pool, SOM soil organic matter, NH4 ammonium, PLFA_B bacterial
phospholipid fatty acids (PLFA), PLFA_F fungal PLFA Fig. 1 Effect of the treatments and time on chemical and biological soil Fig. 1 Effect of the treatments and time on chemical and biological soil
properties. Partial constrained correspondence analysis (pCCA) illustrates
the effect of the three treatments and experimental time (year) on the
chemical and biological compositions of the mesh bags. The CCA1 axis
explains 28.4% (P ≤0.001, F = 31.36) of the data variability, and the CCA2
axis explains 6.8% (P ≤0.001, F = 7.59) of the data. The different mesh
treatments are represented with a green triangle (1000 µm mesh),
magenta circle (50 µm mesh) and black square (1 µm mesh). Results and discussion These results confirm our first hypothesis that
microorganisms supported by plant C increase SOM decom-
position, compared to microorganisms lacking plant C input
(1 µm treatment). The positive effect of plant roots on SOM
decomposition rate falls in line with previous studies on rhizo-
sphere priming effect13,14,28, as well as with a meta-analysis where
decomposition was significantly higher in the rhizosphere than in
the bulk soil15. Phospholipid fatty acid (PLFA) analysis showed
that microbial community composition was significantly affected
by time since exposure (P < 0.001 and R2 = 0.516 with PERMA-
NOVA) and by treatment (P < 0.05 and R2 = 0.077 when the
sampling year was set as grouping factor with PERMANOVA;
Supplementary Fig. 1). Fungal PLFA markers became more
dominant with time in the 1000 µm treatment comparing to the
other treatments in the same year (Fig. 2c), whereas bacteria
showed higher abundance in 1000 µm treatment only during the
second year (P < 0.05) (Fig. 2d). The highest SOM decomposition
in the 1000 µm treatment may therefore be ascribed to increased
activity of fungal decomposers supported by plant-derived carbon
input (i.e. rhizosphere priming15). In line with that, combined
fungal DNA and ergosterol results after 3 years showed increased
colonization in 1000 and 50 µm bags by ectomycorrhizal and
saprotrophic fungi compared to 1 µm bags (Supplementary Fig. 2). Similarly, also root ingrowth into 1000 µm increased with time
of incubation (Supplementary Table 1). We did not find major
differences in soil mesofauna and microfauna between treatments
(Supplementary Table 2) and this suggests that the effect of plant
roots and associated microorganisms explains most of the
changes in SOM decomposition. Formation of stable SOM. Although SOM loss was the highest in
the 1000 µm treatment, also here the stable SOM-N pool showed
the strongest increase among the three treatments, by 23% after 3
years (P < 0.01, Fig. 3a). This result contradicts hypothesis 1 and
suggests that the presence of plant roots did not only enhance net
SOM decomposition but also simultaneously the formation of
stable SOM-N. Characterization of soil from mesh bags with
Fourier-transform infrared spectroscopy (FTIR) revealed differ-
ences between treatments mainly in polyphenols and polypep-
tides (Supplementary Figs. 6 and 7). The concentrations of
condensed tannins (CTs) and FTIR absorbance values repre-
senting polyphenols were greatest in the 1000 µm treatment
(Fig. Results and discussion Source data are provided as a Source
Data file 24
a
18
12
aC
aB
bB bB
aA
abA
bA
bC
bC
Mass loss
(%SOM)
6
0
1 year
2 years
3 years b
17
14
11
8
5
aB
bC
bB
aA
bB bA
aA aA
bA
N loss
(% of initial N)
1 year
2 years
3 years b a c
aAB
aA aA
aA
bB bB
aB
bB bB
Fungal PLFA
(nmol g–1 DW)
1 year
2 years
3 years
60
45
30
15
0 d d
800
600
400
200
0
aA aA aA
aA
bB
bB
aB aB aC
Bacterial PLFA
(nmol g–1 DW)
1 year
2 years
3 years d Fig. 2 Soil decomposition and microbiology: a Soil organic matter (SOM) loss, b N loss, c PLFA fungal biomarkers, d PLFA bacterial biomarkers. The
different mesh sizes are represented with different colours (from left to right): 1000 µm (green), 50 µm (magenta), and 1 µm (black). The given values are
the means of 24 replicates. Significant differences (P < 0.05) between treatments within 1 year are indicated by different letters, and the differences
between different years for the same treatment are indicated by capital letters. The error bars represent ±s.e.m. Source data are provided as a Source
Data file the other treatments, we did not find higher soil CO2 production
or soil enzymatic activity. Soil respiration was only marginally
higher in the 1000 µm treatment compared to other treatments,
and enzymatic activity was largely at the same level, with
significantly higher activities only for the 50 µm treatment after 2
years (P < 0.01; Supplementary Figs. 3 and 4). We additionally
conducted a follow-up experiment to test for treatment effects on
microbial N transformation activities. After harvesting the mesh
bags from the soil, roots were removed and the soil homogenized,
and gross rates of protein depolymerization, N mineralization as
well as microbial amino acid and ammonium uptake were
quantified using 15N-pool dilution assays29. We did not detect
consistent differences between the treatments (Supplementary
Fig. 5), contradicting hypothesis 1, as microbial activities should
be enhanced owing to priming. Since some of the above-
mentioned results (i.e. soil respiration, enzyme activities) are
derived from single point measurements after the autumn
harvest, and are most sensitive to moisture changes, these results
should be, however, treated with caution. In contrast, the general
soil chemical and microbial indicators (e.g. Results and discussion The variables
responsible for the separation of the different samples in the analysis have
been marked on the diagram with the following abbreviations: total AAs
total free amino acids, CT condensed tannins, stableN stable SOM-nitrogen
(N) pool, SOM soil organic matter, NH4 ammonium, PLFA_B bacterial
phospholipid fatty acids (PLFA), PLFA_F fungal PLFA Here we test these hypotheses in a field experiment where mesh
bags filled with unsterilized, homogenized soil organic layer with
the natural microbial community were returned into the organic
layer of the boreal pine forest soil and collected after one, two and TURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 24
a
b
c
d
17
14
11
8
5
800
600
400
200
0
18
12
aC
aB
bB bB
aA
abA
bA
aB
bC
bB
aA
bB bA
aA aA
bA
bC
aAB
aA aA
aA
bB bB
aB
bB bB
aA aA aA
aA
bB
bB
aB aB aC
bC
Mass loss
(%SOM)
N loss
(% of initial N)
Fungal PLFA
(nmol g–1 DW)
Bacterial PLFA
(nmol g–1 DW)
6
0
1 year
2 years
3 years
1 year
2 years
3 years
1 year
2 years
3 years
1 year
2 years
3 years
60
45
30
15
0
Fig. 2 Soil decomposition and microbiology: a Soil organic matter (SOM) loss, b N loss, c PLFA fungal biomarkers, d PLFA bacterial biomarkers. The
different mesh sizes are represented with different colours (from left to right): 1000 µm (green), 50 µm (magenta), and 1 µm (black). The given values are
the means of 24 replicates. Significant differences (P < 0.05) between treatments within 1 year are indicated by different letters, and the differences
between different years for the same treatment are indicated by capital letters. The error bars represent ±s.e.m. Results and discussion SOM loss, PLFA)
better reflect changes over the entire incubation period, giving
more reliable long-term estimates of belowground processes. treatment that permitted root ingrowth showed the highest SOM
and total N loss throughout all years (P < 0.01; Fig. 2a, b). In the
last year, the fungal hyphal treatment (50 µm) also tended to
accelerate SOM and N losses (P < 0.09 and P < 0.05, respectively,
Fig. 2a, b). These results confirm our first hypothesis that
microorganisms supported by plant C increase SOM decom-
position, compared to microorganisms lacking plant C input
(1 µm treatment). The positive effect of plant roots on SOM
decomposition rate falls in line with previous studies on rhizo-
sphere priming effect13,14,28, as well as with a meta-analysis where
decomposition was significantly higher in the rhizosphere than in
the bulk soil15. Phospholipid fatty acid (PLFA) analysis showed
that microbial community composition was significantly affected
by time since exposure (P < 0.001 and R2 = 0.516 with PERMA-
NOVA) and by treatment (P < 0.05 and R2 = 0.077 when the
sampling year was set as grouping factor with PERMANOVA;
Supplementary Fig. 1). Fungal PLFA markers became more
dominant with time in the 1000 µm treatment comparing to the
other treatments in the same year (Fig. 2c), whereas bacteria
showed higher abundance in 1000 µm treatment only during the
second year (P < 0.05) (Fig. 2d). The highest SOM decomposition
in the 1000 µm treatment may therefore be ascribed to increased
activity of fungal decomposers supported by plant-derived carbon
input (i.e. rhizosphere priming15). In line with that, combined
fungal DNA and ergosterol results after 3 years showed increased
colonization in 1000 and 50 µm bags by ectomycorrhizal and
saprotrophic fungi compared to 1 µm bags (Supplementary Fig. 2). Similarly, also root ingrowth into 1000 µm increased with time
of incubation (Supplementary Table 1). We did not find major
differences in soil mesofauna and microfauna between treatments
(Supplementary Table 2) and this suggests that the effect of plant
roots and associated microorganisms explains most of the
changes in SOM decomposition. treatment that permitted root ingrowth showed the highest SOM
and total N loss throughout all years (P < 0.01; Fig. 2a, b). In the
last year, the fungal hyphal treatment (50 µm) also tended to
accelerate SOM and N losses (P < 0.09 and P < 0.05, respectively,
Fig. 2a, b). NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications Results and discussion More results characterizing the soil
are given in Supplementary Figs. 3–6. Significant differences (P < 0.05) between treatments within 1 year are indicated by different letters, and the
differences between different years for the same treatment are indicated by capital letters. The error bars represent ±s.e.m. Source data are provided as a
Source Data file 12
a
b
2.0
1.5
1.0
0.5
0
11
Stable SOM-N
(mg N g–1 SOM)
Condensed tannins
(mg g–1 SOM)
10
aB
aB
aA
aA
bA bA
aA
aB
aB
aB aA
aA
bA bA
bA bA
aA
aA
9
8
1 year
2 years
3 years
1 year
2 years
3 years 12
a
11
Stable SOM-N
(mg N g–1 SOM)
10
aB
aB
aA
aA
bA bA
aA
aB
aB
9
8
1 year
2 years
3 years b
2.0
1.5
1.0
0.5
0
Condensed tannins
(mg g–1 SOM)
aB aA
aA
bA bA
bA bA
aA
aA
1 year
2 years
3 years a c
FTIR-polyphyenolics
(absorbance unit, second derivative)
aA aA aA
aA
bA
bA
aA
abA
bA
1 year
2 years
3 years
–0.0004
–0.0003
–0.0002
–0.0001
0 d
14
12
10
8
6
1.0
1.5
2.0
2.5
Stable SOM-N
(mg N g–1 SOM)
Condensed tannins, mg g–1 SOM d e
f
0.20
0.15
0.10
0.05
0
Chitin
(mg N g–1 SOM)
FTIR-polypeptides
(absorbance unit, second derivative)
aA
aA
aA aA
aA
bB bB
aA
aA
aA aA
bA
cB
aA
bA
bA
aA
aB
Condensed tannins, mg g–1 SOM
1 year
2 years
3 years
1 year
2 years
3 years
–0.0016
–0.0012
–0.0008
–0.0004
0 f
0.20
0.15
0.10
0.05
0
Chitin
(mg N g–1 SOM)
aA aA
aA
bB bB
aA
aA
aA aA
1 year
2 years
3 years f g
log DON/IN
(µg N g–1 DW)
aB aB aB
aAB
bB
aA
bA bA
bAB
1 year
2 years
3 years
4
3
2
1
0 g 3 years Fig. 3 Soil chemistry and correlations with condensed tannins: a stable SOM-N pool, b condensed tannins, c Fourier-transform infrared (FTIR)
polyphenolics (sums of 1512, 1421 and 1388 infrared (IR) bands), d correlation between stable SOM-N vs CT plotted for the whole set of data, e FTIR
polypeptides (sums of 1668 and 1543 IR bands) (see Supplementary Figs. 5 and 6), e chitin concentration, f chitin concentration, g dissolved organic N/
inorganic N (DON/IN) ratio (logarithmic values). Results and discussion 3b, c), and CTs were significantly correlated with the stable
SOM-N (r = 0.680, P < 0.01, Pearson correlation, Fig. 3d). In
addition, FTIR absorbance values representing polypeptides as In accordance with the higher SOM decomposition rate in the
1000 µm treatment, we expected to see enhanced microbial
activity and a decrease in the stable SOM-N (the chemically stable
N pool), which would be in agreement with the N mining
hypothesis
of
rhizosphere
priming13,14
and
hypothesis
1. Although fungal abundance was higher in the 1000 µm than in TURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 12
a
b
c
d
e
f
g
2.0
1.5
1.0
0.5
0
14
12
10
8
6
1.0
1.5
2.0
2.5
0.20
0.15
0.10
0.05
0
11
Stable SOM-N
(mg N g–1 SOM)
Stable SOM-N
(mg N g–1 SOM)
Chitin
(mg N g–1 SOM)
Condensed tannins
(mg g–1 SOM)
log DON/IN
(µg N g–1 DW)
FTIR-polyphyenolics
(absorbance unit, second derivative)
FTIR-polypeptides
(absorbance unit, second derivative)
10
aB
aB
aA
aA
aA
aA
aA aA
aA
bB bB
aA
aA
aA aA
bA
cB
aA
bA
bA
aA
aB
aB aB aB
aAB
bB
aA
bA bA
bAB
aA aA
aA
bA
bA
aA
abA
bA
aA
bA bA
aA
aB
aB
aB aA
aA
bA bA
bA bA
aA
aA
9
8
1 year
2 years
3 years
Condensed tannins, mg g–1 SOM
1 year
2 years
3 years
1 year
2 years
3 years
1 year
2 years
3 years
1 year
2 years
3 years
1 year
2 years
3 years
–0.0004
–0.0003
–0.0002
–0.0001
0
–0.0016
–0.0012
–0.0008
–0.0004
0
4
3
2
1
0
Fig. 3 Soil chemistry and correlations with condensed tannins: a stable SOM-N pool, b condensed tannins, c Fourier-transform infrared (FTIR)
polyphenolics (sums of 1512, 1421 and 1388 infrared (IR) bands), d correlation between stable SOM-N vs CT plotted for the whole set of data, e FTIR
polypeptides (sums of 1668 and 1543 IR bands) (see Supplementary Figs. 5 and 6), e chitin concentration, f chitin concentration, g dissolved organic N/
inorganic N (DON/IN) ratio (logarithmic values). The different mesh sizes are represented with different colours (from left to right): 1000 µm (green),
50 µm (magenta) and 1 µm (black). The given values are the means of the 24 replicates (except 9 for FTIR and chitin). Results and discussion The different mesh sizes are represented with different colours (from left to right): 1000 µm (green),
50 µm (magenta) and 1 µm (black). The given values are the means of the 24 replicates (except 9 for FTIR and chitin). More results characterizing the soil
are given in Supplementary Figs. 3–6. Significant differences (P < 0.05) between treatments within 1 year are indicated by different letters, and the
differences between different years for the same treatment are indicated by capital letters. The error bars represent ±s.e.m. Source data are provided as a
Source Data file with CTs is responsible for stabile SOM-N formation and
knowing that C:N ratio of complexes is about 6:137, we can
extrapolate it into 30 mg of C stabilized after 3 years. Taking into
account that fine root turnover may be higher than previously
assumed38 and that fine roots provide significant amount of CTs
to the soil35, this mechanism might play a significant role in SOM
stabilization in boreal forest soils. In addition, increased fungal
biomass could directly decrease N availability through N immo-
bilization23 and increase stabilization of SOM via recalcitrant
melanin production39,40. Therefore, those potential mechanisms
of SOM build-up could explain how ectomycorrhizal trees
decrease N availability in boreal forest ecosystems and drive soil C
accumulation. well as concentrations of chitin were also greatest in the 1000 µm
treatment (Fig. 3 e, f) and correlated with CTs (for FTIR poly-
peptides r = 0.698, P < 0.036, chitin for third year only r = 0.750,
P < 0.001, Pearson correlation). We here propose that this sig-
nificant increase in the stable SOM-N resulted from the forma-
tion of complexes with root-derived CTs, i.e. polyphenols able to
form complexes with proteins30–32, and other organic N com-
pounds, e.g. chitin4,5. These results are in line with our earlier
paper, in which we showed that CTs stabilize fungal necromass in
boreal forest soil33,34. Thus we suggest the formation of organic
N–CT complexes as one of the mechanisms behind the stabili-
zation of SOM-N in the presence of plant roots, which is in
agreement with build-up of SOM from root- and fungal-derived
biomass20,27,35,36, and supports our hypothesis 2. In our experi-
ment, formation of stabile SOM-N reached 5 mg N g−1 SOM
after 3 years. Results and discussion Assuming that complexation of proteins and chitin Going further, it is important to consider the role of stable
SOM-N formation through reactions with CTs in the N cycle. The complexation of proteins by tannins has been proposed to NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 mycorrhizal component can play a crucial role, as it was shown
that soil in ecosystems with ecto- and ericoid mycorrhiza (like
boreal forests) contains 70% more C per unit N than soil in
ecosystems dominated by arbuscular mycorrhizal plants24,
suggesting different mechanisms of soil C storage. induce a shift from mineral- to organic-dominated N cycling, i.e. that an increasing fraction of dissolved N released from litter is in
organic rather in than mineral forms7. In line with this concept,
we observed the highest dissolved organic N (DON)/inorganic N
(IN) ratio in the 1000 µm treatment (P < 0.05; Fig. 3g), which
reflects natural forest conditions. Lower concentrations of IN in
this treatment may emerge not only from lower N mineralization
but also from higher IN uptake by plants roots. Previous work at
our study site in Hyytiälä also demonstrated a tight N cycle that
was dominated by organic N and proposed that most of the N
assimilated into tree biomass originated from SOM decomposi-
tion, with negligible amounts of IN in the soil41. We have to be aware that the mesh bag-based experimental
design may suffer from some caveats. For example, in addition
to the loss of SOM from mesh bags due to decomposition and
uptake, C loss may partially emerge from leaching of dissolved
organic carbon (DOC) into deeper soil horizons42. The
translocation of organic C to mineral soils may also stabilize
organic matter, which is responsible for the formation of
decadal and centurial old C pools in boreal forest soils43. The
DOC pool may also be susceptible to leaching from the soil to
surface and ground waters, although as the DOC outflow from
undisturbed forest soils is generally low44, we assumed that also
in our experiment DOC leaching is of minor effect. Similarly to
the trenching method45, mesh bag manipulations can also
potentially be affected by differences in moisture and limitation
of soil fauna entrance. However, in our study the moisture
content and soil fauna abundance did not show major
differences
between
treatments
(Supplementary
Tables
1
and 2). Results and discussion All in all, these caveats did not have substantial effect
on our results. Soil nitrogen economy. The combined results suggest that plant
roots with associated microorganisms (1000 µm) promoted an
organic soil N economy, i.e. the domination of organic N forms
and stable SOM-N build-up (Fig. 4). In contrast, the artificial
treatments where ingrowth of roots (50 µm) and roots as well as
fungal hyphae (1 µm) were excluded showed an inorganic soil
N economy with the accumulation of IN (Fig. 4), potentially
promoting N losses by leaching and denitrification. This N
economy observed for boreal forests cannot be simply extra-
polated to temperate and tropical forests where soil N stock per
area (kg N m−2) is about three times lower than in boreal for-
ests24. Moreover, formation of stable SOM-N may be sig-
nificantly affected by quantity and quality of soil components. For instance, the clay content of boreal forest soils is almost
three times lower than that of temperate forests and up to ten
times lower than that of tropical forests24. Similarly, also Implications for biogeochemical predictions. We provide a
framework on the role of plant roots in SOM decomposition and
stabilization based on a field experiment in boreal forest soils
(Fig. 4). Plant roots promote organic N economy not only with 1000 µµm
50 µm
1 µm
SOM-N
Plant C-flow
Plant C-flow
Plant C-flow
SOM-N
SOM-N
+CT
+CT
+CT
HO
HO
HO
HO
HO
OH
O
HO
HO
HO
HO
HO
OH
O
HO
HO
HO
HO
HO
OH
O
Labile
SOM-N
DON/IN
DON/IN
DON/IN
Stabile
SOM-N
Labile
SOM-N
Stabile
SOM-N
Labile
SOM-N
Stabile
SOM-N
Fig. 4 Framework of SOM-N transformations: organic N economy under natural conditions (1000 µm), inorganic N economy for exclusion of roots
(50 µm), as well as roots and fungal hyphae (1 µm). Organic N economy (i.e. the domination of organic N) includes also build-up of stable SOM due to
stabilization of organic N compounds with plant-derived tannins. Exclusion treatments (50 and 1 µm) led to an inorganic N economy (i.e. increased
concentration of inorganic N forms) with reduced SOM build-up. The arrow thickness is proportional to the magnitude of the net flux. CT condensed
tannins (shown as a monomer), SOM soil organic matter, DON dissolved organic N, IN inorganic N. Methods SOM content was measured as
loss on ignition at +550 °C and soil pH in a soil–water suspension of 1:2.5. Concentration of CTs. CTs were extracted from 0.2 g ground soil with 70%
aqueous acetone, dried, dissolved in water and exposed to a modified acid-butanol
assay (proanthocyanidin assay)60 after extraction from soil with acetone:water
(7:3). The method involves the HCl-catalysed depolymerization of CT in butanol to
yield a pink-red anthocyanidin product, which was measured spectro-
photometrically. As a standard, we used CT extracted from Norway spruce needles,
which was profoundly characterized before (see ref. 61 for details). Phospholipid fatty acids. Phospholipid extraction and PLFA analysis were
carried out as described earlier49. Briefly, between 1 and 1.5 g of fresh soil was
freeze-dried and extracted with a chloroform:methanol:citrate buffer mixture
(1:2:0.8) and the lipids were separated into neutral lipids, glycolipids and
phospholipids on a silicic acid column. The phospholipids were subjected to
mild alkaline methanolysis, and the fatty acid methyl esters were detected by gas
chromatography (flame ionization detector) using a 50-m HP-5 (phenylmethyl
silicone) capillary column. Helium was used as a carrier gas. Oven temperature
was 50 °C and raised at a rate of 30 °C min−1 to 160 °C, then at a rate of 2 °C
min−1 to 270 °C and finally kept constant for 5 min at 270 °C. Peak areas were
quantified by adding methyl nonadecanoate fatty acid (19:0) as an internal
standard before methanolysis. The sum of the following PLFAs was considered
to be predominantly of bacterial origin and chosen as an index of bacterial
biomass (PLFA, bacterial biomarkers): i15:0, a15:0, 15:0, i16:0, 16:1ω9, 16:1ω7t,
i17:0, a17:0, 17:0, cy17:0, 18:1ω7, and cy19:0. The quantity of 18:2ω6 was used
as an indicator of fungal biomass (PLFA, fungal biomarker)49,50. Concentration of chitin. Chitin was measured using HPLC as described by ref. 62. Briefly, 5 mg of dried and grounded soil was treated with 0.2 N NaOH to remove
proteins and amino acids. The soil was hydrolysed with 5 ml 6 N HCl for 7 h at
105 °C. After centrifugation (5000 × g, 10 min), 500 µl of hydrolysate was evapo-
rated to dryness under nitrogen to remove HCl, redissolved in 1 ml of water,
evaporated and redissolved again in water and centrifuged (5000 × g, 10 min). The
supernatant was then used to measure the glucosamine content (product of chitin
hydrolysis in HCl), after derivatization with 9-flourenylmethylchloroformate. Results and discussion On the other hand, exclusion of plant roots leads to
increased levels of IN (IN economy) and reduced build-up of
stable soil N, as well as decreased SOM decomposition rates due
to a lack of rhizosphere priming support from plants. The fra-
mework provided by our study may facilitate an improved
understanding of boreal forest soil responses to global change and
improve predictions of biogeochemical effects of possible shifts in
SOM accumulation and decomposition. The incorporation of this
framework into other forest ecosystems appears to be the next
step to understand mechanisms controlling the soil C seques-
tration. However, extrapolation to other ecosystems than boreal
forests may require significant modifications according to dif-
ferences in plant CT contents and soil processes that are affected
by, for example, mycorrhizal type (ectomycorrhiza vs arbuscular
mycorrhiza) and its effect on soil carbon content24 or differences
in the C cost of N acquisition46. Finally, additional studies are
required to test how major global environmental changes (e.g. N
deposition, climate warming) alter interactions between EEM
fungi and free-living microbial decomposers24 and, consequently,
change soil C storage. identification, fungal sequences were aligned against UNITE database and the
obtained fungal taxa were assigned to functional guilds according to the FUN-
Guild database54. Fungal biomass. Fungal biomass was measured through its biomarker, ergos-
terol, with the classic high-performance liquid chromatography (HPLC)
method50. Briefly, ergosterol was extracted from 0.25 g soil samples by adding
1 ml cyclohexane and 4 ml 10% KOH in methanol. After 15 min ultrasonic
treatment, the samples were incubated at 70 °C for 60 min. After the heat
treatment, 1 ml distilled water and 2 ml cyclohexane were added. The tubes were
shaken and the top phase was moved to another test tube. The procedure with
water and cyclohexane was repeated and the combined extracts were evaporated
under N2 gas at 40 °C. The samples were re-dissolved in methanol by heating at
40 °C for 15 min and filtered (0.2-μm PTFE filter). The amount of ergosterol was
measured with HPLC (HP Agilent 1100, Hewlett Packard, USA) using a C18
100A reverse-phase column. Ergosterol was analysed using a 10-μl sample
injection and isocratic separation with methanol (1 ml min−1) and detected at
282 nm. Soil fauna. Results and discussion Soil mesofauna and microfauna were measured using modified sugar
flotation method, where soil (2 g) was first sieved through 1-mm mesh, diluted to
100 ml sugar:water suspension (1:1) and then let to settle 2 h before extraction
(25-µm sieve) of soil organisms from the suspension55. Ammonium-N, nitrate-N and total free amino acid content. Fresh soils (5 g)
from mesh bags were extracted with 20 ml of 1 M KCl for 80 min on a reciprocal
shaker (120 rpm). Extracts were centrifuged, filtered (0.45-µm filters, PALL Cor-
poration) and frozen at −20 °C until analysis. IN (NO3-N and NH4-N) was
measured according to ref. 56. The assay for ammonium-N was based on a mod-
ified indophenol method and for NO3-N on the reduction of nitrate by vanadium
(III). Soil total free amino acids were measured from KCl extracts using a
fluorometric method with o-phthaldialdehyde and β-mercaptoethanol, modified as
in ref. 57. Absorbance and fluorescence were measured with a microplate reader
(Infinite M200, Tecan, Switzerland). Methods Experimental design. Soil for this study was collected in the vicinity of the
SMEAR II station of Helsinki University at Hyytiälä (61°84’N, 24°26’E) in southern
Finland (see ref. 47 for site details). The soil was a haplic Podzol, and Scots pine
(Pinus sylvestris L.) was the dominating tree species. The soil was taken from the
organic layer (Ofh), visible roots were removed and the soil was homogenized and
sieved through a 4-mm mesh. Such prepared soil was placed in mesh bags (cor-
responding to 14.2 g dry weight per bag) with different mesh size to form 3 types of
treatment: mesh size 1000 µm did not limit fine root and hyphal in-growth, 50 µm
mesh excluded roots but not fungal hyphae, and 1 µm excluded root and also
fungal penetration48. Mesh bags in 24 replicates per treatment (1, 50, 1000 µm)
were placed on topmost mineral horizons in the soil organic layer relative to
horizon position at Hyytiälä station in May 2013. Mesh bags were collected in 3
September campaigns in 2013, 2014 and 2015. Samples were transported to the
laboratory at +4 °C and after removal of roots divided for different measurements
(see below). Enzyme and DNA analyses, 15N pool-dilution and KCl extractions
were conducted using fresh soil, the rest of the samples was frozen at −20 °C, and
later freeze-dried and ground to a fine powder using a ball-grinder (2000–230
Geno/Grinder, Spex Sample Pred, US). Chemically stable and degradable N pools. We separated soil N pools into a
hydrolysable N pool (hereafter called degradable N pool) and non- hydrolysable N
pool (hereafter called stable N pool) using acid hydrolysis with methyl sulfonic acid
(MSA)58,59. Briefly, dried and ground soil was hydrolysed with 4 M MSA in an
autoclave (136 °C, 134 kPa; 90 min) in the presence of tryptamine to avoid amino
acid degradation. This hydrolysis released the degradable N pool but not the
recalcitrant N pool. Extracts were neutralized with KOH and stored at −20 °C
before analysis for total dissolved N content (TOC-Vcph/cpn TNM-1, Shimadzu,
Japan), which consequently reflects the degradable N pool. The chemically stable
pool was calculated as the difference between total soil N content and the
degradable N pool. Total C, N, SOM and pH. Total soil C and N contents were determined with an
elemental CN analyser (LECO, Michigan, USA). Results and discussion The sizes of DON and IN are proportional to their
concentrations 1 µm
Plant C-flow
SOM-N
+CT
HO
HO
HO
HO
HO
OH
O
DON/IN
Labile
SOM-N
Stabile
SOM-N 50 µm Fig. 4 Framework of SOM-N transformations: organic N economy under natural conditions (1000 µm), inorganic N economy for exclusion of roots
(50 µm), as well as roots and fungal hyphae (1 µm). Organic N economy (i.e. the domination of organic N) includes also build-up of stable SOM due to
stabilization of organic N compounds with plant-derived tannins. Exclusion treatments (50 and 1 µm) led to an inorganic N economy (i.e. increased
concentration of inorganic N forms) with reduced SOM build-up. The arrow thickness is proportional to the magnitude of the net flux. CT condensed
tannins (shown as a monomer), SOM soil organic matter, DON dissolved organic N, IN inorganic N. The sizes of DON and IN are proportional to their
concentrations TURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 accelerated SOM decomposition but also with stabilization of
SOM-N. On the other hand, exclusion of plant roots leads to
increased levels of IN (IN economy) and reduced build-up of
stable soil N, as well as decreased SOM decomposition rates due
to a lack of rhizosphere priming support from plants. The fra-
mework provided by our study may facilitate an improved
understanding of boreal forest soil responses to global change and
improve predictions of biogeochemical effects of possible shifts in
SOM accumulation and decomposition. The incorporation of this
framework into other forest ecosystems appears to be the next
step to understand mechanisms controlling the soil C seques-
tration. However, extrapolation to other ecosystems than boreal
forests may require significant modifications according to dif-
ferences in plant CT contents and soil processes that are affected
by, for example, mycorrhizal type (ectomycorrhiza vs arbuscular
mycorrhiza) and its effect on soil carbon content24 or differences
in the C cost of N acquisition46. Finally, additional studies are
required to test how major global environmental changes (e.g. N
deposition, climate warming) alter interactions between EEM
fungi and free-living microbial decomposers24 and, consequently,
change soil C storage. accelerated SOM decomposition but also with stabilization of
SOM-N. Methods Samples were mixed with homocysteic acid (5 µM) as internal standard. Separation
and detection were carried out on a Waters HPLC with a 250 × 5 mm2 (I.D.) ODS-
hypersil (5 μm) column. The column effluent was directed to a fluorescence
detector (excitation 330 nm and emission 445 nm). Results are presented as mg
glucosamine per g−1 SOM. Fungal DNA analysis. Total DNA was extracted from 50 mg of soil using the
NucleoSpin® Soil DNA Extraction Kit (Macherey-Nagel GmbH&Co). All the
extracted DNA samples were further purified with the PowerClean® Pro DNA
Clean Up Kit (MO BIO Laboratories, USA) following the manufacturer’s
instructions. From the extracted total DNA, fungal ITS2 regions were sequenced
using Illumina® MiSeq at the Institute of Biotechnology (University of Helsinki). The general read quality of raw ITS2 reads was checked with the FastQC soft-
ware (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adapter
and barcode sequences were trimmed away with the Cutadapt software51. The
sequence data were further filtered and clustered to operational taxonomic units
using mothur52 and following the workflow described in ref. 53. For Fungal DNA analysis. Total DNA was extracted from 50 mg of soil using the
NucleoSpin® Soil DNA Extraction Kit (Macherey-Nagel GmbH&Co). All the
extracted DNA samples were further purified with the PowerClean® Pro DNA
Clean Up Kit (MO BIO Laboratories, USA) following the manufacturer’s
instructions. From the extracted total DNA, fungal ITS2 regions were sequenced
using Illumina® MiSeq at the Institute of Biotechnology (University of Helsinki). The general read quality of raw ITS2 reads was checked with the FastQC soft-
ware (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adapter
and barcode sequences were trimmed away with the Cutadapt software51. The
sequence data were further filtered and clustered to operational taxonomic units
using mothur52 and following the workflow described in ref. 53. For 15N pool dilution assays. Gross rates of protein depolymerization, microbial
amino acid uptake, N mineralization and microbial ammonium uptake were
measured using 15N pool dilution assays, as described in principle by Kirkham and
Bartholomew63. For gross rates of protein depolymerization and microbial amino
acid uptake, we followed the assay of Wanek et al29., with slight modifications. Duplicates of fresh soil (ca. 1 g) were amended with a mixture of 15N-enriched
amino acids (18.75 ng µl−1, 500 µl, ≥96 at% 15N) and incubated for 10 and 30 min,
respectively. References Plant Soil 338, 143–158
(2011). 9. Bingham, A. H. & Cotrufo, M. F. Organic nitrogen storage in mineral soil:
implications for policy and management. Sci. Total Environ. 551–552,
116–126 (2016). 10. Lindahl, B. D. et al. Spatial separation of litter decomposition and mycorrhizal
nitrogen uptake in a boreal forest. New Phytol. 173, 611–620 (2007). Statistical analysis. To determine the differences between treatment means, a
two-way analysis of variance (ANOVA) test was used with time (year) and mesh
size as fixed factors. Where necessary, data were transformed (log10 or arcsin for
percentage data) to fulfil the assumptions of the ANOVA. The interactions between
time (year) and treatment (mesh size) were all significant. The effect of factors
(time or mesh size) were determined separately with post hoc Tukey tests. In the
figures, significant differences (P < 0.05) between treatments within 1 year are
indicated by different letters, and the differences between different years for the
same treatment are indicated by capital letters. PERMANOVA test was conducted
with the adonis function from the vegan package in the R program (R core team)68
using 999 permutations. In the PERMANOVA, the duration of the experiment was
used as explanatory variable. When testing the effect of mesh size with PERMA-
NOVA, duration of the experiment was used as grouping factor. A partially con-
strained correspondence analysis (pCCA) was performed in the R program with
the function cca from the vegan package68. Prior to pCCA, cross-correlations
between variables were determined with the corvif function, and only variables not
significantly correlated with each other were selected for the analysis. All variables
were normalized between 0 and 1 with the ‘range’ option in the decostand function
in the vegan package. In the CCA, the effect of the experimental site was partialized
by setting it as ‘Condition’ in the CCA. The statistical significances of the CCA axes
and explanatory variables were determined with the anova.cca function. The pCCA
ordination was visualized with the plot.cca function and scaled for site scores
(scaling = 1). 11. Wardle, D. A., Yeates, G. W., Williamson, W. M., Bonner, K. I. & Barker, G. M. Linking aboveground and belowground communities: the indirect
influence of aphid species identity and diversity on a three trophic level soil
food web. Oikos 107, 283–294 (2004). 12. Moore, J. A. M. et al. Interactions among roots, mycorrhizas and free-living
microbial communities differentially impact soil carbon processes. J. Ecol. References 1. Crowther, T. W. et al. Quantifying global soil carbon losses in response to
warming. Nature 540, 104–108 (2016). 2. Arneth, A. et al. Historical carbon dioxide emissions caused by land-
use changes are possibly larger than assumed. Nat. Geosci. 10, 79–84
(2017). Enzyme measurements. Extracellular enzymes were recovered from the fresh soil
using the filter centrifugation method66. Activities of acid phosphatase (EC 3.1.3.2),
chitinase (EC 3.2.1.14), β-glucosidase (EC 3.2.1.21), β-glucuronidase (EC 3.2.1.31),
β-xylosidase (EC 3.2.1.37), cellobiohydrolase (EC 3.2.1.91) and leucine amino-
peptidase (EC 3.4.11.1) were measured using fluorometric substrates (Sigma)67. Fluorescence was measured with a Wallac Victor2 (Perkin Elmer, Inc., USA) plate
reader using excitation at 355 nm and emission at 460 nm. 3. Tateno, M. & Chapin, F. S. The logic of carbon and nitrogen interactions in
terrestrial ecosystems. Am. Nat. 149, 723–744 (1997). 4. Adamczyk, B., Adamczyk, S., Smolander, A. & Kitunen, V. Tannic acid and
Norway spruce condensed tannins can precipitate various organic nitrogen
compounds. Soil Biol. Biochem. 43, 628–637 (2011). 5. Adamczyk, B., Kitunen, V. & Smolander, A. Response of soil C and N
transformations to condensed tannins and different organic N-condensed
tannin complexes. Appl. Soil Ecol. 64, 163–170 (2013). Fourier transform infrared spectroscopy. Soil infrared spectra were recorded with
a FTIR spectrometer (Shimadzu IRPrestige-21; Shimadzu Corporation, Japan)
equipped with a high-sensitivity DLATGS detector with Germanium-coated KBr
beam splitter and signal/noise ratio 40,000:1. We used the IRsolution 1.40 software
(Shimadzu Corporation). The spectral data within the band range of 4000–400 cm−1
were recorded, and 24 scans were averaged for each spectrum with a spectral
resolution of 4 cm−1. Three milligrams of dry and finely grounded soil from mesh
bags were mixed with 300 mg of KBr and pressed into discs (13 mm in diameter) by
applying 112 bars pressure for 2 min (hydraulic press, Perkin Elmer). A blank KBr
beam splitter was used to adjust to the baseline level before measurements. All
analyses were performed in nine replicates. 6. Smolander, A., Kanerva, S., Adamczyk, B. & Kitunen, V. Nitrogen
transformations in boreal forest soils-does composition of plant secondary
compounds give any explanations? Plant Soil 350, 1–26 (2011). compounds give any explanations? Plant Soil 350, 1–26 (201 7. Northup, R. R. et al. Polyphenol control of nitrogen release from pine litter. Nature 377, 227–229 (1995). 8. Rumpel, C. & Kögel-Knabner, I. Deep soil organic matter-a key but
poorly understood component of terrestrial C cycle. Methods Samples were then extracted with 20 ml 10 mM CaSO4 that contained
3.7% formaldehyde to stop microbial activity, filtered and loaded on pre-cleaned 15N pool dilution assays. Gross rates of protein depolymerization, microbial
amino acid uptake, N mineralization and microbial ammonium uptake were
measured using 15N pool dilution assays, as described in principle by Kirkham and
Bartholomew63. For gross rates of protein depolymerization and microbial amino
acid uptake, we followed the assay of Wanek et al29., with slight modifications. Duplicates of fresh soil (ca. 1 g) were amended with a mixture of 15N-enriched
amino acids (18.75 ng µl−1, 500 µl, ≥96 at% 15N) and incubated for 10 and 30 min,
respectively. Samples were then extracted with 20 ml 10 mM CaSO4 that contained
3.7% formaldehyde to stop microbial activity, filtered and loaded on pre-cleaned NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 cation exchange cartridges. Cartridges were eluted with 3 M NH3, and samples
were amended with internal standards (nor-valine, nor-leucine, para-chloro-phe-
nylalanine, 1 µg each per sample), dried, derivatized with ethyl-chloroformate29
and analysed with gas chromatography–mass spectrometry (Thermo TriPlus
Autosampler, Trace GC Ultra and ISQ mass spectrometer, Agilent DB-5 column). Concentrations of alanine, glycine, isoleucine, leucine, phenylalanine, proline,
valine, aspartate+asparagine and glutamate+glutamine were quantified against
external standard curves, and the corresponding 15N contents were calculated from
peak areas of light- and heavy-mass fragments29. Aspartate and asparagine, as well
as glutamate and glutamine, were not distinguished since formaldehyde causes the
deamination of asparagine to aspartate and of glutamine to glutamate29. We cor-
rected for background concentrations of amino acids and incomplete recovery
from the cation exchange cartridges using blanks and a second set of standards that
were processed together with the samples throughout the procedure. Infrared spectra were transformed by the second derivative (i.e. the
Savitzky–Golay method) using Unscrambler (CAMO, Norway) to remove the
baseline shift and enhance the spectral information69. A principal component (PC)
analysis was performed with Canoco 5 (Microcomputer Power, USA). Wavenumbers that were most affected by treatment (best fit in PC ordination
space) were selected and assigned, as in a previous study70. A sum of absorbance
values at 1668 and 1543 cm−1 was used as a representative of polypeptides, and a
sum of absorbance values at 1641, 1512, 1421 and 1388 cm−1 was used as a
representative of polyphenolics. cation exchange cartridges. Methods Cartridges were eluted with 3 M NH3, and samples
were amended with internal standards (nor-valine, nor-leucine, para-chloro-phe-
nylalanine, 1 µg each per sample), dried, derivatized with ethyl-chloroformate29
and analysed with gas chromatography–mass spectrometry (Thermo TriPlus
Autosampler, Trace GC Ultra and ISQ mass spectrometer, Agilent DB-5 column). Concentrations of alanine, glycine, isoleucine, leucine, phenylalanine, proline,
valine, aspartate+asparagine and glutamate+glutamine were quantified against
external standard curves, and the corresponding 15N contents were calculated from
peak areas of light- and heavy-mass fragments29. Aspartate and asparagine, as well
as glutamate and glutamine, were not distinguished since formaldehyde causes the
deamination of asparagine to aspartate and of glutamine to glutamate29. We cor-
rected for background concentrations of amino acids and incomplete recovery
from the cation exchange cartridges using blanks and a second set of standards that
were processed together with the samples throughout the procedure. Reporting summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article. Data availability
h d
h
h For gross rates of N mineralization and microbial ammonium uptake,
duplicates of fresh soil (ca. 1.5 g) were amended with 15N-enriched (NH4)2SO4
(0.125 mM, 500 µl, 10 at% 15N), and incubated for 4 and 24 h, respectively. Samples
were then extracted with 15 ml 2 M KCl and filtered through ash-free filter paper. Ammonium was isolated into acid traps64, and the amount and isotopic
composition were measured with elemental analysis–isotope ratio mass
spectrometry (CE Instrument EA 1110 Elemental Analyzer, Finnigan MAT ConFlo
II Interface, Finnigan MAT DeltaPlus IRMS). We corrected for background
concentrations of ammonium using blanks that were processed together with the
samples throughout the procedure. The data that support the findings of this study are available on request from the
corresponding author (B.A.). The source data underlying Figs. 1–3 and Supplementary
Figs. 1–7 are provided as a Source Data file. Received: 10 February 2019 Accepted: 12 August 2019 Gross rates of protein depolymerization, microbial amino acid uptake, N
mineralization and microbial ammonium uptake were calculated using equations
modified after Kirkham and Bartholomew63. Microbial N use efficiency was
calculated as the proportion of N taken up in the form of amino acids that was
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(2015). 43. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11993-1 21. Kyaschenko, J., Clemmensen, K. E., Karltun, E. & Lindahl, B. D. Below-
ground organic matter accumulation along a boreal forest fertility gradient
relates to guild interaction within fungal communities. Ecol. Lett. 20,
1546–1555 (2017). 48. Wallander, H., Nilsson, L. O., Hagerberg, D. & Baath, E. Estimation of the
biomass and seasonal growth of external mycelium of ectomycorrhizal fungi
in the field. New Phytol. 151, 753–760 (2001). y
49. Peltoniemi, K. et al. Responses of methanogenic and methanotrophic
communities to warming in varying moisture regimes of two boreal fens. Soil
Biol. Biochem. 97, 144–156 (2016). 22. Mikutta, R., Kleber, M., Torn, M. S. & Jahn, R. Stabilization of soil organic
matter: association with minerals or chemical recalcitrance? Biogeochemistry
77, 25–56 (2006). 50. Frostegård, A. & Bååth, E. The use of phospholipid fatty acid analysis to
estimate bacterial and fungal biomass in soil. Biol. Fertil. Soils 22, 59–65
(1996). 23. Franklin, O., Näsholm, T., Högberg, P. & Högberg, M. N. Forests trapped in
nitrogen limitation - an ecological market perspective on ectomycorrhizal
symbiosis. New Phytol. 203, 657–666 (2014). (
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51. Martin, M. Cutadapt removes adapter sequences from high-throughput
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24. Averill, C., Turner, B. L. & Finzi, A. C. Mycorrhiza-mediated competition
between plants and decomposers drives soil carbon storage. Nature 505,
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community-supported software for describing and comparing microbial
communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009). 25. Kögel-Knabner, I. The macromolecular organic composition of plant and
microbial residues as inputs to soil organic matter: fourteen years on. Soil Biol. Biochem. 105, A3–A8 (2017). pp
53. Sietiö, O.-M. et al. Ericoid plant species and Pinus sylvestris shape fungal
communities in their roots and surrounding soil. New Phytol. 218, 738–751
(2018). 26. Kallenbach, C. M., Frey, S. D. & Grandy, A. S. Direct evidence for microbial-
derived soil organic matter formation and its ecophysiological controls. Nat. Commun. 7, 13630 (2016). 54. Nguyen, N. H. et al. FUNGuild: an open annotation tool for parsing fungal
community datasets by ecological guild. Fungal Ecol. 20, 241–248 (2016). 27. Kyaschenko, J. et al. Soil fertility in boreal forest relates to root-driven
nitrogen retention and carbon sequestration in the mor layer. New Phytol. 221, 1492–1502 (2019). 55. Southwood, R. Ecological Methods, with Particular Reference to the Study of
Insect Populations (Methuen, 1978). 56. Acknowledgements 44. Pumpanen, J. et al. Precipitation and net ecosystem exchange are the most
important drivers of DOC flux in upland boreal catchments. J. Geophys. Res. Biogeosciences 119, 1861–1878 (2014). This research was supported by grants (263858 and 292669) from the Academy of
Finland, two grants from the Finnish Cultural Foundation (granted to B.A. and O-M.S.),
the Niemi Foundation and the University of Helsinki Doctoral Program of Microbiology
and Biotechnology (MBDP; to O.-M.S.). Miikka Olin is acknowledged for help with the
chitin measurements. 45. Savage, K. E., Davidson, E. A., Abramoff, R. Z., Finzi, A. C. & Giasson, M.-A. Partitioning soil respiration: quantifying the artifacts of the trenching method. Biogeochemistry 140, 53–63 (2018). and Biotechnology (MBDP; to O.-M.S.). Miikka Olin is acknowledged for help with the
chitin measurements. 46. Gill, A. L. & Finzi, A. C. Belowground carbon flux links biogeochemical cycles
and resource-use efficiency at the global scale. Ecol. Lett. 19, 1419–1428
(2016). ARTICLE Liski, J. & Westman, C. J. Carbon storage in forest soil of Finland. 2. Size and
regional pattern. Biogeochemistry 36, 261–274 (1997). References 103,
1442–1453 (2015). 13. Cheng, W. et al. Synthesis and modeling perspectives of rhizosphere priming. New Phytol. 201, 31–44 (2013). 14. Huo, C., Luo, Y. & Cheng, W. Rhizosphere priming effect: a meta-analysis. Soil Biol. Biochem. 111, 78–84 (2017). 15. Finzi, A. C. et al. Rhizosphere processes are quantitatively important
components of terrestrial carbon and nutrient cycles. Glob. Chang. Biol. 21,
2082–2094 (2015). 16. Gadgil, R. L. & Gadgil, P. D. Mycorrhiza and litter decomposition. Nature 233,
133–133 (1971). 17. Averill, C. & Hawkes, C. V. Ectomycorrhizal fungi slow soil carbon cycling. Ecol. Lett. 19, 937–947 (2016). 18. Setälä, H., Marshall, V. G. & Trofymow, J. A. Influence of body size of soil
fauna on litter decomposition and 15N uptake by poplar in a pot trial. Soil
Biol. Biochem. 28, 1661–1675 (1996). For the analysis of PLFA patterns, a linear discriminant analysis (LDA) was
performed with the MASS package in the R software. Since the sampling year had
the most significant effect on the PLFA patterns and values, the heterogeneity
between years was normalized between 0 and 1 with the decostand function from
vegan68 prior to performing the LDA, in order to visualize the differences caused
by the mesh treatments. 19. Liang, C., Schimel, J. P. & Jastrow, J. D. The importance of anabolism
in microbial control over soil carbon storage. Nat. Microbiol. 2, 17105
(2017). 20. Clemmensen, K. E. et al. Roots and associated fungi drive long-term carbon
sequestration in boreal forest. Science 340, 1615–1618 (2013). 7 TURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications Author contributions B.A., J.H. and A.R. planned and designed the research; B.A., J.H., A.R., O.-M.S., P.S.,
M.P., M.H., J.P., B.W. and H.F. performed the experiments and data analysis. B.A. wrote
the manuscript with help from other co-authors. 47. Hari, P. et al. Station for measuring ecosystem-atmosphere relations: SMEAR. Phys. Physiol. Ecol. Forest 10, 471–487 (2013). 8 NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications Additional information Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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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/. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
019-11993-1. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ Peer review information: Nature Communications thanks Ivika Ostonen, Karin Pritsch
and other anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. © The Author(s) 2019 NATURE COMMUNICATIONS | (2019) 10:3982 | https://doi.org/10.1038/s41467-019-11993-1 | www.nature.com/naturecommunications
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To enhance pupils’ and students’ spatial imagination and logical thinking skills
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1 Teacher of the Faculty of Art History, Navoi State Pedagogical Institute. ABSTRACT Every day we are faced with many tasks, the solution of
which requires us to think logically. The ability to think and
logic as a cause takes us in many cases, from sequentially and
consistently finishing judgement interlocutors and shoppers,
starting with complex technical and business tasks. But, despite
the great need for this skill, we often make logical mistakes
without knowing it. In fact, among many people the right
mindset can be based on experience and so-called common
sense, not on the idea, using laws and special methods, said “the
official logic”. To perform simple logic operations, the initial
proposal and statements of simple conclusions can lead to often
errors in the general sense of closeness and common sense, and
if you know or understand something more complex. In this
article, ideas and feedback are made about how to increase the
spatial imagination and logical thinking ability of students and
students. Article history:
Received February 2021
Received in revised form
28 March 2022
Accepted 20 April 2022
Available online
15 May 2022 Keywords:
pupils and students,
spatial imagination,
logical thinking,
ability,
development,
importance of education. 2181-1415/© 2022 in Science LLC. DOI: https://doi.org/10.47689/2181-1415-vol3-iss4/S-pp222-226
This is an open access article under the Attribution 4.0 International
(CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 2181-1415/© 2022 in Science LLC. DOI: https://doi.org/10.47689/2181-1415-vol3-iss4/S-pp222-226
This is an open access article under the Attribution 4.0 International
(CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) To enhance pupils’ and students’ spatial imagination and
logical thinking skills Shavkat JALOLOV1 Navoi State Pedagogical Institute Kalit so‘zlar: Har kuni biz ko‘plab vazifalarga duch kelamiz, ularni hal
qilish bizdan mantiqiy fikrlashni talab qiladi. Sabab sifatida
fikrlash va mantiq qobiliyati bizni ko‘p hollarda murakkab
texnik va biznes vazifalaridan boshlab, suhbatdoshlar va
xaridorlarning xulosalarini ketma-ket va izchil yakunlashdan
tortib oladi. Ammo bu mahoratga katta ehtiyoj borligiga
qaramay, biz, ko‘pincha, bilmasdan mantiqiy xatolarga yo‘l Kalit so‘zlar:
o‘quvchilar va talabalar,
fazoviy tasavvur,
mantiqiy fikrlash,
qobiliyat,
rivojlanish,
ta’limning ahamiyati. rivojlanish, Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 qo‘yamiz. Aslida, ko‘p odamlar orasida to‘g‘ri fikrlash qonunlar
va maxsus usullar yordamida g‘oya emas, balki tajriba va deb
atalmish sog‘lom fikr asoslangan bo‘lishi mumkin, deydi “rasmiy
mantiq”. Oddiy mantiqiy operatsiyalarni bajarish uchun oddiy
xulosalarning
dastlabki
bayonotlari,
ko‘pincha,
umumiy
ma’noda xatolarga olib kelishi mumkin. Ushbu maqolada
o‘quvchi va talabalarning fazoviy tasavvurini va mantiqiy
fikrlash qobiliyatini qanday oshirish haqida fikr va mulohazalar
bildirilgan. АННОТАЦИЯ Ключевые слова:
учащиеся и студенты,
пространственное
воображение,
логическое мышление,
способности,
развитие,
значение образования. Каждый день перед нами стоит множество задач,
решение которых требует от нас логического мышления. Способность мыслить и логика как причина уводит нас во
многих случаях, от последовательно и последовательно
заканчивая суждениями собеседников и покупателей,
начиная со сложных технических и деловых задач. Но,
несмотря на большую потребность в этом умении, мы
часто совершаем логические ошибки, сами того не
подозревая. Ведь у многих правильное мышление может
быть основано на опыте и так называемом здравом смысле,
а не на идее, с использованием законов и специальных
методов, говорит «официальная логика». Для выполнения
простых
логических
операций
первоначальное
предложение и формулировка простых выводов могут
привести к часто ошибкам в общем смысле близости и
здравого смысла, а если вы знаете или понимаете что-то
более сложное. В этой статье представлены идеи и отзывы
о том, как повысить пространственное воображение и
способность
к
логическому
мышлению
учащихся
и
школьников. “Only a few people think logically. In most cases, we express a wrong opinion, we are
smeared with a wrong conclusion, doubt, fear, pride and envy”. Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 In our opinion, a person’s logical thinking and ability to feel should be developed
on a regular basis and on the basis of special terminology and logical means. In the online
training class you will learn the techniques of self-education for the development of
logical thinking, learn the main categories, characteristics of principles and laws of logic,
but also learn the examples and exercises of the application of acquired knowledge and
skills, for example, what is logical thinking, spatial imagination and how can you develop
them? It is known that modeling is one of the most effective forms of classroom work that
develops the spatial imagination of students and students. The didactic advantage of such
exercises is that they not only make it easier to imagine about the spatial properties and
relationships of the described object, but also help to better understand all the remaining
information on the basis of the drawing. The essence of this method is that it is the
formation of mental activity inherent in the process of imagination. Practical changes in
appearance in the modeling process will be the basis for the formation of the necessary
mental activity. In the process of modeling, the student not only perceives the image of
the body, but also through the skin sensation is engaged in such activities as the
construction of the organism, logical analysis, distribution. Well logical thinking? To explain what “logical thinking” is, we divide this concept
into two parts: thinking and logic. Logical thinking-a thought process in which a person
whose goal is to judge, and characteristic, uses logical concepts and design of evidence,
whose purpose is to get the sound coming out of the existing premises. Let’s define each
of these components. Thinking of a person is a mental process of processing information
and establishing relationships between objects, their properties or phenomena of the
surrounding world. Thinking allows a person to find the connection between events of
reality, but communication, of course, thought, in other words, found that the object
properly reflect the actual state of affairs or should be, it is logical that logic is subject to
the laws. Dale Carnegie The causes of these errors are based on the principles of the formation and
formation of the basis of logical thinking of people, which are established in childhood. Education is not targeted at logical thinking and is being determined by the lessons of
mathematics (for children or university students in school), as well as by the decision and
the passage of a variety of games, tests, tasks and puzzles. But such actions help develop
only a small part of logical thinking processes. It is also primitive enough to explain to us
the principles of solving assignments. Develop verbal-logical thinking (or verbal-logical
thinking) the ability to perform mental operations correctly, consistently draw
conclusions, and then we will not be taught this. Therefore, the level of development of
logical thinking of people is not enough. 223 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 “imagination”, which allows you to accurately and accurately create a variety of different
real properties of an object or phenomenon. This kind of mental activity of a person is
formed in childhood, approximately from the age of 1,5 years. Verbal logical thinking (verbal-logical thinking) is one of the types of logical
thinking, the use of which is language objects and speech structures. Such thinking
involves not only the skillful application of thought processes, but also the skillful
mastery of speech. For us, verbal-logical thinking is necessary public speaking, writing
texts, conducting discussions, and in other cases, it is necessary to express our thoughts
with the help of language. It’s no secret that many students do not have a very developed spatial imagination. The problem is old, but relevant. If the teacher heads the junior and middle classes, it will
be a few years later that the lessons with the same students after a few years will lose
their multiplicity. Everything is psychological processes, development and improvement
of spatial imagination among other things. This activity should be encouraged and sent. You need a system of physical exercises. Over the years at school, I came to the
conclusion that the spatial imagination of students should be developed from the first
lessons of Mathematics from the fifth grade. At the moment, there are various systems of
developing spatial imagination high school students, including computer. For several
years, I call the course “geometry”, which is designed for training in 5-6 classes. Its
purpose is to prepare students for mastering regular geometrics. When determining the
composition of the “administration”, it was necessary to regularly understand what
exactly is the most difficult for children. This course is a faith. Almost motivation, its logic
is hidden from children. In fact, it starts with points and straight, the corners go, then
triangles, etc. But the students do not know what will happen before, I do not know about
cylinders or pyramids. The interruption of the ministry and very harmful for business. Pupils are suppressed by spatial imagination. In translation from the Greek language of logic, there are several meanings:
“correct thinking”, “the art of thinking”, “speech”, “thinking” and even “thinking”. In our
case, we act as a normative science of the forms, methods and laws of human intellectual
activity, one of the most logical definitions. The logic is not an emotional experiment, but
a study of ways of achieving truth in the educational process through mediation, but it is
also taken forward, to take on knowledge so it can be defined as the science of how to get
out of knowledge. One of the main tasks of logic – to determine how to get accurate
knowledge about the subject of reflection in order to understand the nuances of the
relationship with the object of thought and other aspects of the phenomenon, to conclude
existing premises and well-studied. What is spatial imagination and how to develop it? One of the best ways to develop
spatial imagery in students and students is through the use of modeling and cutting-edge
technology. The reason for the development of this spatial imagination in students and
students should be preliminary, theoretical information about space, the universe and its
structure in them earlier in the beginning. Only then it will be possible to develop in them
such thinking. Fine-logical thinking (English. symbolic thinking) – the situation involves the
processing of visuals and images of its structural objects, the so-called “shaped” problem-
solving, different thought processes. Visual-figurative thinking is a synonym for the word 224 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 • Able to quickly and efficiently solve complex logical and spatial problems (both
educational and practical). • Able to quickly and efficiently solve complex logical and spatial problems (both
educational and practical). The conclusion is the third form of thinking, in which one, two or more debts are
called parcels, a new proposal is formed, so-called conclusions or conclusions. The
indicators are divided into three types: deductive, inductive and output by analogy. If the
deduction is preferential (preferential), a general conclusion is drawn from the general
rule for a particular case. Induction is the consequence of which the general rule arises
from several separate cases. In analogies with hysteria, based on the similarity of things
in certain signs, the conclusion is drawn about their similarity in other signs. In this
lesson, you will get acquainted with all kinds of conclusions, learn how to create any
cause-effect relationships. The term “space” refers to an area that is devoid of
boundaries. Millions of stars, celestial bodies, planets, the solar system, meteorites, and
natural and satellite satellites can all be found there. To communicate this enigmatic
Infinity to readers and to feel this dynamic environment, the same compositional style is
applied. Additionally, the utilization of information technology and media sources, such
as video, photos, and audio, will aid in the development of spatial imagination in
students’ minds. Complex procedures should be taught to pupils, who should be able to
explain and apply them in simple terms. This necessitates the use of an instructor with
specialized abilities. It’s also crucial to consider how students and students differ in
terms of their knowledge and thinking. Several composing strategies are frequently
utilized in this process. The most important thing is that the development of spatial
thinking is based on the content of knowledge, vision and hearing and perception. y
Ennis R.H. 1993 Critical thinking assessment Theory Pract. 32. – PP. 179–186 The latest edition of the textbook
“geometry”, 10 – 11 classes of the authors of “Lsato Syan” attempts to stop the transition
to a colored container, but the transition of students to the use of the textbook, this
attempt is over. The image of the form on the laptop becomes colorless, and students will
have difficulty reading and describing such pictures. Engage in the use of logical means in almost all spheres of human activity,
including concrete and humanitarian, economics and business, rhetoric and lectures,
creative processes and inventions. In some cases, strict and formalized logic is used, for
example, in mathematics, philosophy, technology. In other cases, logic provides a useful
technique to draw conclusions only in economic, historical or simple “living” conditions. Often the use of logical reasoning is associated with the rapid solution of logical
tasks and the passage of tests to determine the level. Intellectual development (IQ). But
this direction is a huge linkage to the extent that it leads to the automation of thinking
operations, which is a very small part of the usefulness of logic for a person. Logical
thinking and spatial imagination combine many skills in the application of different
thinking processes and include the following: • Knowledge of logic and spatial theoretical framework. Ability to correctly
perform intelligent operations, such as classification, clarification, generalization,
comparison, comparison, etc. • Knowledge of logic and spatial theoretical framework. Ability to correctly
perform intelligent operations, such as classification, clarification, generalization,
comparison, comparison, etc. • Reliable use of the main forms of thinking: concentration, judgment, exodus • Logic and the ability to argue thoughts in accordance with the laws of space. 225 Жамият ва инновациялар – Общество и инновации – Society and innovations
Special Issue – 04 (2022) / ISSN 2181-1415 REFERENCES: 1. Gal H. and Linchevski L. 2010 To see or not to see: analyzing difficulties in
geometry from the perspective of visual perception Educ. Stud. Math. 74. – PP. 163–183. g
g
3. Trilling B and Fadel C 2009 21st Century Skills: Learning for Life in Our Times
(San Francisco: Jossey-Bass). 3. Trilling B and Fadel C 2009 21st Century Skills: Learning for Life in Our Times
(San Francisco: Jossey-Bass). 4. Hadi S., Retnawati H., Munadi S., Apino E. and Wulandari N.F. 2018 The
difficulties of high school students in solving higher-order thinking skills problems Probl. Educ. 21st Century 76. – PP. 520–532. y
5. Ennis R.H. 1993 Critical thinking assessment Theory Pract. 32. – PP. 179–186. 5. Ennis R.H. 1993 Critical thinking assessment Theory Pract. 32. – PP. 179–186. 6. Hapsari S. 2016 A descriptive study of the critical thinking skills of social science
at junior high school J. Educ. Learn. 10. – PP. 228–234. 6. Hapsari S. 2016 A descriptive study of the critical thinking skills of social science
at junior high school J. Educ. Learn. 10. – PP. 228–234. j
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7. https://www.researchgate.net/publication/343045221_How_to_improve_critical_t
hinking_skills_and_spatial_reasoning_with_augmented_reality_in_mathematics_learning. 7. https://www.researchgate.net/publication/343045221_How_to_improve_critical_t
hinking_skills_and_spatial_reasoning_with_augmented_reality_in_mathematics_learning. 226
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Relation between Temperature and Mortality in Thirteen Spanish Cities
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International journal of environmental research and public health/International journal of environmental research and public health
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Int. J. Environ. Res. Public Health 2010, 7, 3196-3210; doi:10.3390/ijerph7083196
International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph
Article
Relation between Temperature and Mortality in Thirteen
Spanish Cities
Carmen Iñiguez 1,2,3, Ferran Ballester 1,2,3,4,*, Juan Ferrandiz 4,†, Santiago Pérez-Hoyos 2,3,5,
Marc Sáez 2,6, Antonio López 4 and TEMPRO-EMECAS ‡
1 Center for Public Health Research (CSISP), Avda Catalunya 21, 46020, Valencia, Spain;
E-Mail: inyigez_car@gva.es (C.I.)
2 Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP),
Doctor Aiguader, 88 1ª Planta, 8003 Barcelona, Spain
3 Valencian School for Studies on Health (EVES), Juan de Garay 21, 46017, Valencia, Spain
4 University of Valencia, Jaume Roig s/n, 46010, Valencia, Spain;
E-Mail: Antonio.Lopez@uv.es (A.L.)
5 Research Institute, Hospital Vall d‘Hebrón, Passeig Vall d‘Hebron, 119-129 08035 Barcelona,
Spain; E-Mail: santi.perezhoyos@gmail.com (S.P.-H.)
6 Departament d‘Economia, University of Girona, Campus de Montilivi 17071 Girona, Spain;
E-Mail: marc.saez@udg.edu (M.S.)
† In memoriam
‡ See the complete list of participants in the EMECAS project at the end of the manuscript.
* Author to whom correspondence should be addressed; E-Mail: ballester_fer@gva.es;
Tel.: +34-961-92-57-79 (direct); +34-961-92-57-00 (Central); Fax: +34-961-92-57-03.
Received: 26 June 2010; in revised form: 28 July 2010 / Accepted: 6 August 2010 /
Published: 11 August 2010
OPEN ACCESS Article Relation between Temperature and Mortality in Thirteen
Spanish Cities Carmen Iñiguez 1,2,3, Ferran Ballester 1,2,3,4,*, Juan Ferrandiz 4,†, Santiago Pérez-Hoyos 2,3,5,
Marc Sáez 2,6, Antonio López 4 and TEMPRO-EMECAS ‡ 1 Center for Public Health Research (CSISP), Avda Catalunya 21, 46020, Valencia, Spain;
E-Mail: inyigez_car@gva.es (C.I.) 2 Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP),
Doctor Aiguader, 88 1ª Planta, 8003 Barcelona, Spain 3 Valencian School for Studies on Health (EVES), Juan de Garay 21, 46017, Valencia, Spain
4 University of Valencia, Jaume Roig s/n, 46010, Valencia, Spain;
E-Mail: Antonio.Lopez@uv.es (A.L.) 4 University of Valencia, Jaume Roig s/n, 46010, Valencia, Spain;
E-Mail: Antonio.Lopez@uv.es (A.L.) 5 Research Institute, Hospital Vall d‘Hebrón, Passeig Vall d‘Hebron, 119-129 08035 Barcelona,
Spain; E-Mail: santi.perezhoyos@gmail.com (S.P.-H.) 6 Departament d‘Economia, University of Girona, Campus de Montilivi 17071 Girona, Spain;
E-Mail: marc.saez@udg.edu (M.S.) † In memoriam † In memoriam
‡ Int. J. Environ. Res. Public Health 2010, 7, 3196-3210; doi:10.3390/ijerph7083196 Int. J. Environ. Res. Public Health 2010, 7, 3196-3210; doi:10.3390/ijerph7083196 † In memoriam
‡ ‡ See the complete list of participants in the EMECAS project at the end of the manuscript. * Author to whom correspondence should be addressed; E-Mail: ballester_fer@gva.es;
Tel.: +34-961-92-57-79 (direct); +34-961-92-57-00 (Central); Fax: +34-961-92-57-03. Received: 26 June 2010; in revised form: 28 July 2010 / Accepted: 6 August 2010 /
Published: 11 August 2010 Abstract: In this study we examined the shape of the association between temperature and
mortality in 13 Spanish cities representing a wide range of climatic and socio-demographic
conditions. The temperature value linked with minimum mortality (MMT) and the slopes
before and after the turning point (MMT) were calculated. Most cities showed a V-shaped
temperature-mortality relationship. MMTs were generally higher in cities with warmer
climates. Cold and heat effects also depended on climate: effects were greater in hotter
cities but lesser in cities with higher variability. The effect of heat was greater than the 3197 Int. J. Environ. Res. Public Health 2010, 7 effect of cold. The effect of cold and MMT was, in general, greater for cardio-respiratory
mortality than for total mortality, while the effect of heat was, in general, greater among
the elderly. effect of cold. The effect of cold and MMT was, in general, greater for cardio-respiratory
mortality than for total mortality, while the effect of heat was, in general, greater among
the elderly. Keywords: temperature; mortality; Spain 1. Introduction For many years, the effect of temperature on mortality has been the subject of numerous studies,
mostly examining the impact of extreme weather events [1,2]. These studies have shown the existence
of an association between temperature and mortality, but some characteristics such as the shape of the
association remain less clear. The relationship between temperature and mortality usually displays a V
or U-shaped pattern [3,4] (i.e., populations experience increases in mortality due to both high and low
temperatures), but other shapes like W [5] or J [6] have also been described. The shape of the
association may vary widely from city to city because it depends on many factors such as
climate (range of temperatures and atmospheric conditions to which the population has adapted),
socio-economical level and age. Two conclusions may be drawn from this: on the one hand, studies
incorporating information from several locations would be useful [6,7] and on the other, all available
factors that might confound the relationship should be considered. In recent years there has been growing concern about the possible effects of climatic changes
including their effect on health [8,9]. Long term weather forecasts show that, if the tendency towards
global warming continues, extreme meteorological phenomena such as heat waves or floods are likely
to occur with higher intensity and frequency. These phenomena could have a greater impact on human
health than that caused by the generalised temperature increase itself [10,11]. It is, therefore, of major
interest to conduct studies which help to understand the complex relationships between meteorological
factors and also contribute to the evaluation of surveillance and prevention measures [12]. We investigated the shape of the relationship between temperature and mortality in 13 Spanish
cities representing a wide range of climatic, socio-demographic and environmental conditions. Our
aims were: To determine the temperature at which mortality is lowest, to evaluate the impact of
temperature changes below and above such value, to detect if there is any specificity of the effect for
groups of causes or ages, and to identify possible common patterns among cities. 2. Material and Methods In this study, we used data from the EMECAM project (Multicenter Study on Short-Term Effects of
Air Pollution on Mortality) [13] including daily information on 13 Spanish cities: Barcelona, Bilbao,
Cartagena, Castellón, Gijon, Huelva, Madrid, Oviedo, Seville, Valencia, Vigo, Vitoria and Zaragoza
(Figure 1). The series covered variable study periods between 1990 and 1996, and always included at
least three consecutive years. The data were obtained from public institution registers by the researcher
responsible for each city, following a standardised protocol. 3198 Int. J. Environ. Res. Public Health 2010, 7 Figure 1. Spanish cities participating in the study. Figure 1. Spanish cities participating in the study. Mortality was computed as the daily number of deaths occurring in each city. All natural deaths
(ICD-9: 0–799), deaths due to cardio-respiratory causes (ICD-9: 390–519), and deaths in people aged
over 70 years were analysed. Daily mean temperature (average of the minimum and maximum values
of current day) and daily mean humidity (average of the values at 0, 7, 13 and 18 hours in the current
day) were obtained from the airport meteorological station located closest to the city centre and were
provided by the Meteorological Service in each city (Table 1). Figure 1. Spanish cities participating in the study. Figure 1. Spanish cities participating in the study Mortality was computed as the daily number of deaths occurring in each city. All natural deaths
(ICD-9: 0–799), deaths due to cardio-respiratory causes (ICD-9: 390–519), and deaths in people aged
over 70 years were analysed. Daily mean temperature (average of the minimum and maximum values
of current day) and daily mean humidity (average of the values at 0, 7, 13 and 18 hours in the current
day) were obtained from the airport meteorological station located closest to the city centre and were
provided by the Meteorological Service in each city (Table 1). Mortality was computed as the daily number of deaths occurring in each city. All natural deaths
(ICD-9: 0–799), deaths due to cardio-respiratory causes (ICD-9: 390–519), and deaths in people aged
over 70 years were analysed. Daily mean temperature (average of the minimum and maximum values
of current day) and daily mean humidity (average of the values at 0, 7, 13 and 18 hours in the current
day) were obtained from the airport meteorological station located closest to the city centre and were
provided by the Meteorological Service in each city (Table 1). Table 1. 2. Material and Methods Characteristics of cities. Table 1. Characteristics of cities. Table 1. Characteristics of cities. City
Population
na
Total
mortalityb
Mortality
among 70+b
Cardio-
respiratory
mortality b
Temperature
mean
CV
Vitoria
214,148
1,826
3.53(1.9)
2.38(1.6)
1.58(1.3)
11.59
0.53
Oviedo
198,050
1,461
4.53(2.2)
3.25(1.9)
2.01(1.5)
13.19
0.34
Vigo
274,574
1,461
5.31(2.4)
3.49(2.0)
2.51(1.6)
13.49
0.35
Gijon
261,724
1,096
6.34(2.7)
1.77(1.4)
1.30(1.2)
13.83
0.30
Madrid
2,940,896
1,461
60.82(11.1)
40.47(8.7)
27.83(7.7)
14.43
0.53
Bilbao
667,034
1,461
13.62(4.0)
8.89(3.3)
5.97(2.6)
15.28
0.31
Zaragoza
572,212
1,826
12.49(3.9)
8.94(3.2)
5.80(2.7)
15.45
0.48
Barcelona
1,643,545
1,826
43.62(8.6)
31.04(7.2)
21.23(6.1)
16.49
0.35
Castellón
134,213
1,826
2.9(1.8)
2.15(1.5)
1.58(1.3)
17.23
0.33
Huelva
142,547
1,097
2.59(1.7)
1.77(1.4)
1.30(1.2)
18.53
0.30
Valencia
749,796
1,096
16.09(4.6)
11.1(3.7)
7.73(3.1)
18.61
0.29
Seville
683,028
1,380
13.53(4.3)
8.92(3.4)
6.75(3.0)
19.00
0.33
Cartagena
168,023
1,827
3.47(2.0)
2.47(1.6)
1.77(1.4)
19.07
0.27
anumber of days in the series; bMean (Standard Deviation). The following variables were considered as potential confounders: air pollution (level of suspended
particulates measured as particulates of less than 10 g in diameter (PM10) or, in its absence, black
smoke (BS) or, in its absence, total suspended particulates (TSP)), daily incidence of influenza Int. J. Environ. Res. Public Health 2010, 7 Int. J. Environ. Res. Public Health 2010, 7 3199 obtained for each day as 1/7 of the weekly value declared to the Compulsory Notifiable Disease
Registry, and other calendar variables such as the day of the week, bank holiday and unusual
events indicators. The statistical model used to describe the relationship was the Poisson generalised additive
model (GAM). This model was chosen as being the most suitable to explore the shape of a
relationship. Its usefulness lies in the possibility of incorporating variables in a non-parametric way
using smooth functions such as loess or spline, therefore avoiding the need to presuppose the shape of
the relationship and later trying to reproduce it by means of an approximate functional expression. All
analyses were carried out in S-Plus with stricter convergence criteria than default (tolerance = 10–8 and
maximum number of iterations = 1,000) [14-16]. The models were constructed as follows: first, time since the beginning of the study smoothed by
loess was introduced to control for secular trends and seasonality. The number between 90 and 360
(30 by 30) days that minimised the sum of the residual‘s partial autocorrelation function (PACF) was
chosen as span. 2. Material and Methods After, a cubic smoothing spline of influenza cases previously smoothed by loess (with
a 30 day span) was introduced. The number of degrees of freedom of this spline was 2 or 3 according
to the minimum Akaike criterion [17]. Temperature and humidity were incorporated, each by means of
three cubic smoothing splines with four degrees of freedom each. The first was the spline of current
mean daily values, the second the spline of the linear regression residuals of the current values over
lags from 1 to 3, and the third the spline of the linear regression residuals of the current values over
lags from 4 to 10. This was done in an attempt to account for the lagged effect of both temperature and
humidity, avoiding collinearity as far as possible [6]. Day of the week, air pollution (lags 0 and 1
average), holidays and special events, in this order, were linearly incorporated into the model if the
likelihood ratio test was significant (p < 0.2). To account for serial correlation in the residuals where it
remained in the final model, autoregressive terms were added into the model as appropriate. Following this scheme, explicative models for each outcome and city were constructed. The
significance of temperature was evaluated using the likelihood ratio test. The relationship was
graphically represented and the current day temperature at which the curve achieved its minimum,
the minimum mortality temperature (MMT), was obtained. Slopes below (cold slope) and above
(heat slope) the MMT were estimated by linear regression of predicted mortality over temperature. The
impact of cold and heat was expressed as the percentage change in mortality for a temperature change
of 1 ºC. Int. J. Environ. Res. Public Health 2010, 7 3200 Int. J. Environ. Res. Public Health 2010, 7 Focusing on significant associations, the relationship between temperature and mortality was V or
U-shaped (Figure 2), with largest effects (steeper slopes) for cardio-respiratory deaths. This shape does
not suggest an acute effect of extreme temperatures but a rise in the death rate when the weather gets
colder or hotter than the comfort band. Figure 2. Curves of significant associations between temperature and mortality for: total
mortality (Alltm) in the first row, cardio-respiratory mortality (Rcvm) in the second row,
and mortality among people 70 years old or over (A70tm) in the third. Cities are grouped
according to their mean temperature into ―cold‖ cities (Vitoria, Oviedo, Vigo, Gijon),
―mild‖ cities (Madrid, Bilbao, Zaragoza, Barcelona) and ―warm‖ cities (Castellón,
Valencia, Huelva, Seville, Cartagena). The rest of the curves are drawn in light gray. Int. J. Environ. Res. Public Health 2010, 7 Alltm vs Temperature in “warm” cities
Alltm vs Temperature in “warm” cities Alltm vs Temperature in “cold” cities
Alltm vs Temperature in “cold” cities Valencia
Castellón
Sevilla
Alltm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Valencia
Castellón
Sevilla
Alltm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 Alltm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Gijón
Vigo
Alltm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Gijón
Vigo Alltm vs Temperature in”mild” cities
Alltm vs Temperature in”mild” cities Alltm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Gijón
Vigo
Alltm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Gijón
Vigo
Alltm vs Temperature in”mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Alltm vs Temperature in”mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona
Valencia
Castellón
Sevilla
Alltm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Valencia
Castellón
Sevilla
Alltm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vitoria
Rcvm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vitoria
Oviedo
Vitoria
Rcvm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Rcvm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona
Rcvm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Rcvm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Valencia
Castellón
Sevilla
A70tm vs Temperature in “cold” cities”
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vigo
A70tm vs Temperature in “cold” cities”
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vigo
Oviedo
Vigo
A70tm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Madrid
Zaragoza
Barcelona
A70tm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Madrid
Zaragoza
Barcelona
Madrid
Zaragoza
Barcelona
A70tm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Sevilla
A70tm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Sevilla
The only exception was the linear shape with a negative slope found in the two coldest cities
(Vitoria and Oviedo). Int. J. Environ. Res. Public Health 2010, 7 This shape could be explained by the narrow range of temperatures in these
series and the lack of sufficient high temperatures to reveal the potential effect of heat on mortality due
to circulatory causes: in Vitoria the temperature remained below 23 ºC for 95 % of the days of the
series and in Oviedo below 20 ºC. Furthermore, both cities are small and reported a low number of
deaths which could result in somewhat imprecise estimates. Int. J. Environ. Res. Public Health 2010, 7 In fact, from a statistical point of view, Alltm vs Temperature in”mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Alltm vs Temperature in”mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona Rcvm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vitoria
Rcvm vs Temperature in “cold” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vitoria
Oviedo
Vitoria Rcvm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Rcvm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Bilbao
Madrid
Zaragoza
Barcelona
Bilbao
Madrid
Zaragoza
Barcelona Rcvm vs Temperature in “cold” cities
Rcvm vs Temperature in “cold” cities Rcvm vs Temperature in “mild” cities
Rcvm vs Temperature in “mild” cities Rcvm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Rcvm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Castellón
Sevilla
Valencia
Castellón
Sevilla Rcvm vs Temperature in “warm” cities
Rcvm vs Temperature in “warm” cities Oviedo
Vitoria
Oviedo
Vitoria
Oviedo
Vitoria A70tm vs Temperature in “cold” cities”
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vigo
A70tm vs Temperature in “cold” cities”
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Oviedo
Vigo
Oviedo
Vigo A70tm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Madrid
Zaragoza
Barcelona
A70tm vs Temperature in “mild” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Madrid
Zaragoza
Barcelona
Madrid
Zaragoza
Barcelona A70tm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Sevilla
A70tm vs Temperature in “warm” cities
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Valencia
Sevilla A70tm vs Temperature in “warm” cities
A70tm vs Temperature in “warm” cities A70tm vs Temperature in “cold” cities”
A70tm vs Temperature in “cold” cities” A70tm vs Temperature in “mild” cities
A70tm vs Temperature in “mild” cities The only exception was the linear shape with a negative slope found in the two coldest cities
(Vitoria and Oviedo). 3.1. Shape of the Relationship between Temperature and Mortality The relationship between temperature and total mortality was significant in nine of the 13 cities,
including the most populated. In the case of mortality due to specific causes and mortality in the
elderly, the relationship was also significant in Madrid, Barcelona, Valencia, Seville and Zaragoza
(five of the six most populated cities), and non-significant in Huelva and Cartagena (two of the three
least populated cities). Int. J. Environ. Res. Public Health 2010, 7 This shape could be explained by the narrow range of temperatures in these
series and the lack of sufficient high temperatures to reveal the potential effect of heat on mortality due
to circulatory causes: in Vitoria the temperature remained below 23 ºC for 95 % of the days of the
series and in Oviedo below 20 ºC. Furthermore, both cities are small and reported a low number of
deaths which could result in somewhat imprecise estimates. In fact, from a statistical point of view, Int. J. Environ. Res. Public Health 2010, 7 3201 one of the limitations of our study may be the low power when analysing series with a scarce number
of events [18]. Grouping the cities with a significant relationship into three categories, ―cold cities‖, ―mild cities‖
and ―warm cities‖, according to the daily mean temperature, allows for an appreciation of patterns
between the curves. A relevant feature is the MMT deviation towards the right of each curve. Madrid
and Zaragoza, classified as ―mild cities‖, showed a higher smoothening in both slopes. The other two
―mild‖ cities: Bilbao and Barcelona are alike in their shape and different from the two previous cities. This coincides with the similarities in climate and temperature range between them: Madrid and
Zaragoza have a continental climate, with a mean temperature very close to that of Barcelona and
Bilbao, but with a much wider range. This indicates the existence of latent variables relating to the
location, which could play a relevant role in the shape of the relationship between temperature and
mortality. On the other hand, when cities were divided into ―low‖, ―medium‖ and ―high-variability‖
cities on the basis of their coefficient of variation, Madrid and Zaragoza, both classified as
―high-variability‖ cities, showed practically the same pattern marked by a lower risk of mortality. 3.2. Temperature Value Associated with Minimum Mortality (MMT) The temperature associated with minimum mortality (MMT) for total mortality varied from city to
city (14 ºC in Vigo to 23 ºC in Seville) and tended to increase with the mean temperature (Figure 3). The MMT was slightly higher for cardio-respiratory deaths, and the greatest difference with total
mortality was around 2 ºC. The graphs in Figure 4 also show that the MMT obtained with these
adjusted models remained above the mean temperature. In the three outcomes, the percentage of days
with a temperature below the MMT exceeded 50%, and in the case of total mortality this percentage
ranged between 60% and 84%. The MMT varied between 13.90 ºC (Vigo) and 22.75 ºC (Seville). We
observed a shift of MMT towards higher values as the mean temperature of the cities rises. Figure 3. Curves of significant associations between temperature and mortality for: total
mortality (Alltm) in the first row, cardio-respiratory mortality (Rcvm) in the second row,
and mortality among people 70 years old or over (A70tm) in the third. Cities are grouped
according to their Coefficient of Variation into ―high variability‖ (Vitoria, Madrid,
Zaragoza), ―medium variability‖ (Oviedo, Vigo, Barcelona, Castellón, Seville) and ―low
variability‖ cities (Gijon, Bilbao, Valencia, Huelva, Cartagena). The rest of the curves are
drawn in light gray. Alltm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Alltm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Alltm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
R
hi h
i bilit
R
di
i bilit
R
l
i bilit
Alltm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Alltm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Alltm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
R
hi h
i bilit
R
di
i bilit
R
l
i bilit Int. J. Environ. Res. Public Health 2010, 7
0.0
0.2
0.0
0.2
0.0
0.2
0.0
0.2 3202 Figure 3. Cont. 0
10
20
30
-0. 0
10
20
30
-0. 0
10
20
30
-0. 3.2. Temperature Value Associated with Minimum Mortality (MMT) 10
20
10
20 0
10
20
30
0
10
20
30
0
10
20
30
Rcvm; high variability
0
10
20
30
Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
0
10
20
30
0
10
20
30
0
10
20
30
Rcvm; high variability
0
10
20
30
Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 Rcvm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 Rcvm; medium variability
Rcvm; medium variability Rcvm; high variability
Rcvm; high variability Rcvm; high variability
Rcvm; high variability Rcvm; low variability
Rcvm; low variability A70tm; high variability
A70tm; high variability A70tm; low variability
A70tm; low variability A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 A70tm; medium variability
A70tm; medium variability A70tm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8 ure 4. This table includes the temperature associated with minimum mortality (MMT) Figure 4. This table includes the temperature associated with minimum mortality (MMT)
in each city for total mortality (alltm), mortality due to cardio-respiratory causes (rcvm)
and mortality among people 70 years old or over (a70tm). 3.2. Temperature Value Associated with Minimum Mortality (MMT) Rcvm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
0
10
20
30
-0. 0
10
20
30
-0. 0
10
20
30
-0. Rcvm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Rcvm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; high variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; medium variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
A70tm; low variability
0
10
20
30
-0.2
0.0
0.2
0.4
0.6
0.8
Figure 4. This table includes the temperature associated with minimum mortality (MMT)
in each city for total mortality (alltm), mortality due to cardio-respiratory causes (rcvm)
and mortality among people 70 years old or over (a70tm). The graphs show: MMT (black
segment), the temperature range where the predicted mortality is not statistically different
from the minimum mortality (black brackets) and the mean temperature of each city (change
from empty to solid box). Red is used for significant associations and blue for
non-significant associations. Figure 3. Cont. 3.2. Temperature Value Associated with Minimum Mortality (MMT) The graphs show: MMT (black
segment), the temperature range where the predicted mortality is not statistically different
from the minimum mortality (black brackets) and the mean temperature of each city (change
from empty to solid box). Red is used for significant associations and blue for
non-significant associations. Figure 4. This table includes the temperature associated with minimum mortality (MMT)
in each city for total mortality (alltm), mortality due to cardio-respiratory causes (rcvm)
and mortality among people 70 years old or over (a70tm). The graphs show: MMT (black
segment), the temperature range where the predicted mortality is not statistically different
from the minimum mortality (black brackets) and the mean temperature of each city (change
from empty to solid box). Red is used for significant associations and blue for
non-significant associations. City
MMT alltm
MMT rcvm
MMT a70tm
Vitoria
18.40
26.90*
19.20
Oviedo
24.70
24.70**
21.50**
Vigo
13.90**
17.25
13.90
Gijón
15.90**
17.10
16.60**
Madrid
17.60**
19.00**
18.60**
Bilbao
17.58**
19.14*
15.68
Zaragoza
18.80**
20.55**
19.00**
Barcelona
20.32**
19.87**
19.94**
Castellón
19.90**
19.40**
18.30
Huelva
21.80
22.10
19.75
Valencia
20.00**
22.00**
19.4**
Sevilla
22.75**
23.25**
22.50**
Cartagena
23.30
22.40
23.40 City
MMT alltm
MMT rcvm
MMT a70tm
Vitoria
18.40
26.90*
19.20
Oviedo
24.70
24.70**
21.50**
Vigo
13.90**
17.25
13.90
Gijón
15.90**
17.10
16.60**
Madrid
17.60**
19.00**
18.60**
Bilbao
17.58**
19.14*
15.68
Zaragoza
18.80**
20.55**
19.00**
Barcelona
20.32**
19.87**
19.94**
Castellón
19.90**
19.40**
18.30
Huelva
21.80
22.10
19.75
Valencia
20.00**
22.00**
19.4**
Sevilla
22.75**
23.25**
22.50**
Cartagena
23.30
22.40
23.40 Int. J. Environ. Res. Public Health 2010, 7 3203 Figure 4. Cont. (*) indicates 10% statistically significant associations; (**) indicates 5% statistically significant associations. Figure 4. Cont. (*) indicates 10% statistically significant associations; (**) indicates 5% statistically significant associations. In other countries and cities, variable results have been obtained, depending on the climate. For
instance, MMTs of around 14 ºC in the south of Finland, 16.5 ºC in Holland, 18ºC in England, 21 ºC in
Boston, 27 ºC in Florida and 28 ºC in Taiwan have been reported [3,6,19-22].Different studies have
shown that the variation in mortality related to temperature is higher in warm southern countries than
in northern ones [23,24]. Even within a country important differences have been found: in England and
Wales variations of up to 41% in mortality due to ischemic illness were registered among cities,
depending on their different ambient temperature, rainfall and socio-economical differences [25]. 3.2. Temperature Value Associated with Minimum Mortality (MMT) In our
study, a higher impact was seen in hotter cities In order to explain these phenomena, several hypotheses have been put forward. On the one hand,
there could be a physical process which allows people to adapt to the most habitual temperatures in an
area [26]. On the other hand, inhabitants of cities may sometimes adopt, in a secular way, preventive
measures to mitigate the impact of temperature variations. These measures are related to the type of
housing, the clothes used, or the activities performed in different seasons and at different times during
the day [27]. In all cities, the thermal optimum or MMT was found to coincide with a warm temperature for the
city, which is broadly consistent with the results found in earlier studies conducted in cities around the
world [28-30]. Finally, the comparison with results obtained using a simple model (including only
temperature as a predictor) showed that the comfort point moves to the left after controlling for the
confounders taken into account. This fact corroborates the importance of considering all the variables
which could play a role in the relationship. 3.3. Impact of Cold and Heat on Mortality In general, the effect of heat exceeded the effect of cold. The only exceptions were Valencia for
cardio-respiratory mortality and Oviedo for mortality among the population aged under 70 years (Table 2). The effect of cold was greater in hotter cities, showing a correlation coefficient of around 40% with the
mean temperature of the cities. The heat effect was also positively correlated with the temperature of
cities for specific causes of mortality and mortality in the older population. Int. J. Environ. Res. Public Health 2010, 7 3204 Int. J. Environ. Res. Public Health 2010, 7 Looking at the relationship between the gradient of association and the variability of climate, in
general, slopes were negatively correlated with the coefficient of variation, suggesting that the risks are
lower in cities with higher variance, in line with the pattern observed in Figure 3. Looking at the relationship between the gradient of association and the variability of climate, in
general, slopes were negatively correlated with the coefficient of variation, suggesting that the risks are
lower in cities with higher variance, in line with the pattern observed in Figure 3. Finally, there was no significant effect of temperature in the analysis of mortality in people younger
than 70 years. In fact, only in two cities, Madrid and Valencia, did the relationship remain significant. In the analysis of mortality due to other causes (non cardio-respiratory ones), there was a significant
effect of temperature in six cities. In all of them the impact of cold was lower compared to the impact
of cold for cardio-respiratory causes (results not shown). Table 2. Percentage increase in mortality (100 × [exp(b) − 1]) associated with 1 ºC
decrease (cold) and 1 ºC increase (heat) in temperature from the MMT for each outcome:
total mortality (Alltm), mortality due to cardio-respiratory causes (Rcvm), and total
mortality in people 70 years old or over (A70tm). R mean: Pearson correlation coefficient
between slopes and mean temperature of cities. R CV: Pearson correlation coefficient
between slopes and the Coefficient of Variation of cities. city
% Alltm
% Rcvm
% A70tm
cold
heat
cold
heat
cold
heat
Vitoria
1.12
1.52
1.86*
1.44
0.99
Oviedo
0.96
2.15*
0.68*
0.46*
Vigo
1.19*
1.93*
1.55
3.66
1.58
1.37
Gijón
0.57*
2.88*
1.31
1.33
2.43*
6.50*
Madrid
0.27*
0.93*
1.31*
2.40*
0.38*
2.43*
Bilbao
0.85*
1.59*
1.48*
3.74*
1.32
1.86
Zaragoza
0.14*
1.39*
0.76*
1.74*
0.06*
1.79*
Barcelona
1.41*
2.52*
2.23*
3.42*
1.21*
2.82*
Castellón
1.74*
2.60*
3.28*
3.59*
3.95
4.46
Huelva
1.11
2.10
2.95
4.17
3.07
4.83
Valencia
1.29*
1.44*
1.90*
0.87*
1.44*
1.45*
Sevilla
0.88*
2.00*
3.16*
7.72*
0.85*
2.87*
Cartagena
1.45
2.16
3.40
4.70
1.32
2.23
R mean
0.43
0.05
0.46
0.40
0.37
0.48
R CV
−0.28
−0.40
−0.52
−0.28
−0.48
0.01
(*) indicates a statistically significant association. 3.4. Differences in the Impact on Specific Causes or Age Groups 3.4. Differences in the Impact on Specific Causes or Age Groups 3.4. Int. J. Environ. Res. Public Health 2010, 7 Differences in the Impact on Specific Causes or Age Groups 3.4. Differences in the Impact on Specific Causes or Age Groups A greater effect of temperature, in particular the impact of cold, was found on mortality due to
cardio-respiratory causes than on total mortality. The MMTs themselves were higher for specific
causes than for total mortality. These results are consistent with those of most published studies. Different hypotheses have been suggested to explain high mortality due to cardiovascular problems in
cold months. Among the most plausible ones we find the association between exposure to cold
temperatures and a physical reaction resulting in a decrease in blood irrigation to the skin in an attempt
to prevent heat loss. This implies an increase in blood volume in the central organs, with a subsequent Int. J. Environ. Res. Public Health 2010, 7 3205 cardiac overload and an increased blood concentration with higher blood viscosity [31,32]. To some
extent, the increase in mortality due to respiratory causes could be due to the impact of certain
infectious diseases such as influenza or pneumonia, which present a higher incidence during the cold
months [24], and to the increase in fibrinogen concentration related to respiratory infections [32]. As
for the increase in cardiovascular mortality due to high temperatures, this has been demonstrated in
epidemiological studies [27,33] and related to arterial thrombosis [32]. In the case of mortality in people 70 years old or over, results were very similar to those for total
mortality, with a slight increase in the heat effect. This similarity was to be expected given the degree
of ageing of the Spanish population: in our series, mortality among elderly people accounts for
approximately 70% of total mortality. Our results show great variability as far as the temperature associated with minimum mortality
is concerned. The MMT increases as the mean temperature of each city rises. Classifying the
cities into three groups according to their mean temperature—―cold cities‖, ―mild cities‖ and ―warm
cities‖—allows for an appreciation of patterns between the curves. A relevant feature is the MMT
displacement towards the right (heat) of each curve. 3.5. Comparison with Other Studies Whilst many studies have investigated the relationship between changes in temperature and
mortality [5,22,27,34], few have undertaken an analysis similar to the one presented here [6,7,35-38]. Among them only two have been performed in Europe. The first one was conducted in The
Netherlands, several years ago [35]. More recently, results from a European multicenter study,
including Barcelona and Valencia (the PHEWE Project) [38] have been published [39,40]. These
results also showed a U shape in most of the cities. As in our study, greater heat effects were observed
in the Mediterranean cities than in the Northern European ones, both for respiratory causes and the
elderly [39]. The cold effect was also greater in mild cities and for respiratory causes [40]. The impact of temperature variations on mortality is not negligible. In fact, the effect of a 1 ºC
displacement from the MMT is equal to or greater than that associated with a 10 g/m3 increase in the
levels of PM10. An increase in temperature above the MMT has, in general, a greater effect than a
decrease of the same magnitude. For instance, an increase of 5 ºC above the comfort point in
Barcelona (20.3 ºC) would be associated with a response of 12.6%, meaning five more deaths a day,
while a decrease of the same magnitude would be associated with a response of 7%, or three more
deaths a day. 4. Conclusions To conclude, variations in temperature are strongly associated with mortality. Our study provides
results which may help when proposing preventive measures. In any case, further investigation is
needed to identify confounders or effect modifiers of the illnesses related to temperature. In this sense,
we consider characterisation of the lagged effect, the stratified by seasons study (cold, hot), and the use
of combined meteorological variables as a comfort indicator with respect to climatic conditions, i.e., an
indicator of the thermal feeling perceived by an individual, to be of great interest for future studies. Lastly, studies which monitor variations in meteorological variables and their relationship with health
indicators in places with different climatic, socio-sanitary and ambient features should be undertaken
with adequate methods in order to answer specific questions concerning this relationship and to help
take preventive actions to minimise the impact of changes in the climatic situation in the short,
medium and long term. Int. J. Environ. Res. Public Health 2010, 7 Int. J. Environ. Res. Public Health 2010, 7 3206 countries. What does seem to be foreseeable is that the increase in number and intensity of unusual
episodes (both of heat and of cold), which will presumably accompany global warming, will have a
major impact on health. Furthermore, the steeper slope found for changes in heat compared with those
in cold make it plausible to expect total increases in mortality as mean temperatures rise. The range of
values of MMT found in our study, together with the displacement towards lower values after
controlling for confounding, indicates that an increase in temperature, even without reaching extreme
situations, may cause an effect on health, as already described in Barcelona [34] and in Valencia [5]. From the point of view of prevention, a series of measures aimed at avoiding or diminishing the
effects of temperature variations have been proposed. Such measures comprise short term surveillance
actions, such as the implementation of alert systems [43-45] or health education, aimed at health
professionals [1], risk groups such as the elderly, children and people with chronic pathologies so that
they are informed and able to take adequate personal action in case of extreme temperatures [43]. Ambient corrective actions have also been proposed, such as the adaptation of houses (including
heating and air conditioning systems) [46,47]. However, air conditioning should not be the main
protective action as it is energy consuming itself, thus contributing even more to the .increase in local
and global emissions. Long term climate friendly actions should be preferred as urban planning to
mitigate the urban heat island (UHI) phenomenon [48] (temperature increase in cities due to heat
emission and obstruction of air circulation caused by buildings) as it could have an important impact
on human health. Recent research in Shanghai (China) found heat related mortality was higher in the
city centre than in extra-urban locations [49] indicating that UHI may enhance the intensity of heat
waves and extreme temperatures, thereby affecting human health due to increased exposure to extreme
thermal conditions. Urban planners and local officials should be aware of the increased thermal loads
experienced in urban regions and take appropriate action to help reduce the impact of heat on
the population. 3.6. Implications for Public Health With the evidence currently available, it is difficult to predict how global warming will affect the
balance of heat and cold health effects. Some authors consider that there may be a positive effect,
consisting in a reduction in the number of deaths in winter that outweighs the extra deaths attributable
to the generalised increase in temperatures [19,41]. Other authors provide either positive or negative
global estimations, depending on the assumptions they make [42]. Finally, the 4th IPPC Report
considered than increase in the number of deaths related to an increase in temperature should outweigh
the reduction in deaths expected due to exposure to the cold. However, it is difficult to generalise to all Acknowledgements The authors are grateful to all the participants in the EMECAS study. The EMECAS group consists
of: F. Ballester, C. Iñíguez, P. Rodríguez, S. Pérez-Hoyos, J.M. Tenías, F. Gómez, R. Molina, The authors are grateful to all the participants in the EMECAS study. The EMECAS group consists
of: F. Ballester, C. Iñíguez, P. Rodríguez, S. Pérez-Hoyos, J.M. Tenías, F. Gómez, R. Molina, Int. J. Environ. Res. Public Health 2010, 7 3207 J. González-Aracil, S. Medina, J.A. Patiño, J.L. Bosch, S. Peiró: Center for Public Health Research
(CSISP) and Escuela Valenciana de Estudios para la Salud (EVES). Conselleria de Sanidad. Generalitat Valenciana; M. Saez, M.A. Barceló, C. Saurina, A. Lertxundi, L. Artacoz, J. Sunyer, A. Plasència, A. Tobías: Grup de Recerca en Estadística, Economia Aplicada i Salut (GRECS),
Departament d’Economia, Universitat de Girona; A. Daponte, J.L. Gurucelain, P. Gutiérrez, J.A. Maldonado, J.M. Mayoral, R. Ocaña, E. Caro, P. Lopez, S. Toro, I. Aguilera: Escuela Andaluza de
Salud Pública (EASP), Granada; J.M. Ordóñez, E. Aránguez, I. Galán, A.M. Gandarillas, L. López, N. Aragonés, M. Martinez, R. Fernández, P. Arias, A. Brezmes, F.J. Mantero: Dirección General de
Salud Pública. Comunidad de Madrid; K. Cambra, E. Alonso, F.B. Cirarda, T. Martínez-Rueda:
Dirección General de Sanidad del Gobierno Vasco, Vitoria; M. Taracido, A. Figueiras, M.R. Aguiar,
C. Sixto, M.C. Sánchez: Departamento de Medicina Preventiva, Universidad de Santiago de
Compostela; F. Arribas, M. Navarro, C. Martos, M.J. Rabanaque, E. Muniesa, J.T. Alcalá, J.I. Urraca,
M. Loarte: Departamento de Sanidad, Bienestar y Trabajo, Diputación General de Aragón, Zaragoza;
J.B. Bellido, A. Arnedo, F. González, C. Felis, C. Herrero: Centro de Salud Pública de Castellón. Generalitat Valenciana; J.J. Guillén, L. Cirera, L. García, E. Jiménez, M.J. Martínez, S. Moreno,
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